U.S. patent application number 14/112769 was filed with the patent office on 2014-09-25 for compositions and methods for stabilization of active agents.
This patent application is currently assigned to TRUSTEES OF TUFTS COLLEGE. The applicant listed for this patent is David L. Kaplan, Fiorenzo Omenetto. Invention is credited to David L. Kaplan, Fiorenzo Omenetto.
Application Number | 20140287043 14/112769 |
Document ID | / |
Family ID | 47041958 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140287043 |
Kind Code |
A1 |
Kaplan; David L. ; et
al. |
September 25, 2014 |
COMPOSITIONS AND METHODS FOR STABILIZATION OF ACTIVE AGENTS
Abstract
Provided herein are methods and compositions for stabilization
of active agents. The active agents are distributed, mixed or
embedded in a silk fibroin matrix, thereby retaining the
bioactivity of the active agents upon storage and/or
transportation. In some embodiments, the storage-stable
vaccine-silk compositions are also provided herein.
Inventors: |
Kaplan; David L.; (Concord,
MA) ; Omenetto; Fiorenzo; (Wakefield, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kaplan; David L.
Omenetto; Fiorenzo |
Concord
Wakefield |
MA
MA |
US
US |
|
|
Assignee: |
TRUSTEES OF TUFTS COLLEGE
Medford
MA
|
Family ID: |
47041958 |
Appl. No.: |
14/112769 |
Filed: |
April 23, 2012 |
PCT Filed: |
April 23, 2012 |
PCT NO: |
PCT/US12/34643 |
371 Date: |
June 12, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61477737 |
Apr 21, 2011 |
|
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|
Current U.S.
Class: |
424/489 ;
424/202.1; 424/204.1; 424/209.1; 424/215.1; 424/224.1; 424/225.1;
424/227.1; 424/230.1; 424/234.1; 424/239.1; 424/244.1; 424/248.1;
424/250.1; 424/254.1; 424/256.1 |
Current CPC
Class: |
A61K 9/06 20130101; A61K
39/099 20130101; A61K 9/5026 20130101; C12N 2760/18434 20130101;
Y02A 50/30 20180101; A61K 9/7007 20130101; A61K 9/19 20130101; A61K
39/0015 20130101; A61K 39/165 20130101; A61K 2039/622 20130101;
A61P 31/16 20180101; A61P 31/22 20180101; A61K 39/08 20130101; A61K
39/12 20130101; A61K 2039/64 20130101; A61K 39/04 20130101; A61K
47/46 20130101; A61K 47/42 20130101; A61K 2039/70 20130101; A61K
9/5063 20130101; A61K 39/02 20130101; A61K 39/20 20130101; C12N
2760/18734 20130101; A61K 9/0019 20130101; A61K 2039/5252 20130101;
A61K 2039/5254 20130101; A61K 2039/6031 20130101; C12N 2770/36234
20130101; A61P 37/04 20180101; A61P 43/00 20180101; B82Y 5/00
20130101; A61K 9/146 20130101; A61K 38/00 20130101; A61P 31/04
20180101; A61P 31/20 20180101; A61P 31/14 20180101; A61K 39/095
20130101; A61K 39/092 20130101 |
Class at
Publication: |
424/489 ;
424/202.1; 424/227.1; 424/256.1; 424/204.1; 424/250.1; 424/209.1;
424/230.1; 424/239.1; 424/215.1; 424/224.1; 424/225.1; 424/234.1;
424/248.1; 424/254.1; 424/244.1 |
International
Class: |
A61K 39/20 20060101
A61K039/20; A61K 39/08 20060101 A61K039/08; A61K 39/02 20060101
A61K039/02; A61K 47/42 20060101 A61K047/42; A61K 39/09 20060101
A61K039/09; A61K 39/165 20060101 A61K039/165; A61K 9/19 20060101
A61K009/19; A61K 9/14 20060101 A61K009/14; A61K 39/095 20060101
A61K039/095; A61K 39/04 20060101 A61K039/04 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under grants
EB002520 awarded by the National Institutes of Health and
FA9550-07-1-0079 awarded by the US Air Force. The Government has
certain rights in the invention.
Claims
1.-238. (canceled)
239. A storage-stable composition comprising a silk fibroin matrix
and an active agent distributed therein, wherein the active agent
is an immunogen selected from the group consisting of a live,
attenuated virus; an inactivated virus; a viral subunit; a viral
vector; a live, attenuated bacterium; a killed bacterium; an
inactivated bacterium; a bacterial subunit; and an inactivated
toxin from a pathogen; and the active agent retains at least about
30% of its original bioactivity when the composition is (a)
subjected to at least one freeze-thaw cycle, or (b) maintained for
at least about 24 hours at a temperature above 0.degree. C., or (c)
both (a) and (b).
240. The composition of claim 239, wherein the silk fibroin matrix
is a solution.
241. The composition of claim 239, wherein the silk fibroin matrix
is a solid-state form.
242. The composition of claim 241, wherein the solid-state form is
a film, a fiber, a particle, a gel, a hydrogel, or a composite
thereof.
243. The composition of claim 239, wherein the composition is
lyophilized.
244. The composition of claim 239, wherein the composition is
micronized.
245. The composition of claim 244, wherein the micronized
composition comprises nanoparticles or microparticles.
246. The composition of claim 245, wherein the nanoparticles or
microparticles have a size of about 10 nm to about 1000 .mu.m.
247. The composition of claim 239, further comprising an additive
distributed in the silk fibroin matrix.
248. The composition of claim 247, wherein the additive is selected
from a stabilizing agent, a pharmaceutically acceptable carrier, or
any combinations thereof.
249. The composition of claim 248, wherein the stabilizing agent is
selected from the group consisting of a saccharide, a sugar
alcohol, an ion, a surfactant, and any combinations thereof.
250. The composition of claim 249, wherein the saccharide is
sucrose.
251. The composition of claim 239, wherein the immunogen is derived
from hepatitis B virus, Haemophilus influenzae Type B, poliovirus,
Neisseria meningitides C, influenza, Varicella, or Mycobacteria
tuberculosis bacille Calmette-Guerin, tetanus toxoid, diphtheria
toxoid, and Bordetella pertussis.
252. The composition of claim 239, wherein the immunogen is a
combination immunogen selected from the group consisting of DTaP,
DTwP, DTwP hepB, DTP hep B Hib, DTaP hep B Hib IPV, and any
combinations thereof.
253. The composition of claim 239, wherein the immunogen is a live,
attenuated virus.
254. The composition of claim 253, wherein the live, attenuated
virus is an enveloped virus.
255. The composition of claim 254, wherein the enveloped virus is
selected from the group consisting of Paramyxoviridae, Togaviridae,
Orthomyxoviridae, Flaviviridae, Herpesviridae, Rhabdovirus,
Retroviridae, and any combinations thereof.
256. The composition of claim 253, wherein the virus is
varicella.
257. The composition of claim 253, wherein the virus is
influenza.
258. The composition of claim 253, wherein the live, attenuated
virus causes measles, mumps, or rubella.
259. The composition of claim 239, wherein the live, attenuated
virus or inactivated virus is an enveloped virus selected from the
group consisting of varicella, measles virus, mumps virus, German
measles virus, respiratory syncytial virus, yellow fever virus, and
influenza virus.
260. The composition of claim 239, wherein the immunogen is a live,
attenuated, non-enveloped virus.
261. The composition of claim 260, wherein the non-enveloped virus
is rotavirus, reovirus, hepatitis virus, rabies virus or
poliovirus.
262. The composition of claim 239, wherein the immunogen is a live,
attenuated bacterium; a killed bacterium; or an inactivated
bacterium.
263. The composition of claim 262, wherein the bacterium is
Mycobacteria tuberculosis bacilli Calmette-Guerin or Bordetella
pertussis.
264. The composition of claim 239, wherein the immunogen is a
bacterial subunit.
265. The composition of claim 264, wherein the bacterial subunit is
derived from Neisseria meningitides type C, Haemophilus influenzae
type B, Streptococcus pneumoniae, or Group B streptococcus.
266. The composition of claim 264, wherein the bacterial subunit is
a polysaccharide.
267. The composition of claim 239, wherein the immunogen is a viral
subunit.
268. The composition of claim 267, wherein the viral subunit is
derived from Hepatitis B virus or Human Papillomavirus.
269. The composition of claim 239, wherein the immunogen is
recombinant.
270. The composition of claim 239, wherein the immunogen is a
vaccine product selected from the group consisting of Anthrax
vaccine (BioThrax); BCG (Bacillus Calmette-Guerin) (Tice, Mycobax);
DTaP (Daptacel); DTaP (Infanrix); DTaP (Tripedia); DTaP/Hib
(TriHIBit); DTaP-IPV (Kinrix); DTaP-HepB-IPV (Pediarix);
DtaP-IPV/Hib (Pentacel); DT (diphtheria vaccine plus tetanus
vaccine) (Sanofi); Hib vaccine (ACTHib); DT (Massachusetts); Hib
(PedvaxHib); Hib/Hep B (Comvax); Hep A (Havrix), Hepatitis A
vaccine; Hep A (Vaqta), Hepatitis A vaccine; Hep B (Engerix-B),
Hepatitis B vaccine; Hep B (Recombivax), Hepatitis B vaccine;
HepA/HepB vaccine (Twinrix); Human Papillomavirus (HPV) (Gardasil);
Influenza vaccine (Afluria); Influenza vaccine (Fluarix); Influenza
vaccine (Flulaval); Influenza vaccine (Fluvirin); Influenza vaccine
(Fluzone); Influenza vaccine (FluMist); IPV (Ipol), Polio vaccine;
Japanese encephalitis vaccine (JE-Vax); Japanese encephalitis
vaccine (Ixiaro); Meningococcal vaccine (Menactra); MMR vaccine
(MMR-11); MMRV vaccine (ProQuad); Pneumococcal vaccine (Pneumovax);
Pneumococcal vaccine (Prevnar); Poliovirus inactivated (Poliovax),
Polio vaccine; Rabies vaccine (Imovax); Rabies vaccine (RabAvert);
Rotavirus vaccine (RotaTeq); Rotavirus vaccine (Rotarix); Td
vaccine (Decavac); Td vaccine (Massachusetts); Tdap vaccine
(Adacel); Tdap vaccine (Boostrix); Typhoid (inactivated--Typhim
Vi), Typhus vaccine; Typhoid (oralTy21a), Typhus vaccine; Vaccinia
(ACAM2000); Varicella vaccine (Varivax); Yellow fever vaccine
(YF-Vax); Zoster vaccine (Zostavax); and any combinations
thereof.
271. A method for preparing a storage-stable composition of claim
241, the method comprising the steps of: a. providing a silk
fibroin solution comprising at least one active agent; and b.
drying the silk fibroin solution of step (a) to form a solid-state
silk fibroin, thereby obtaining a composition in which the at least
one active agent retains at least about 30% of its original
bioactivity upon storage.
272. The method of claim 271, further comprising lyophilizing the
solid-state silk fibroin from step (b).
273. The method of claim 271, further comprising post-treatment of
the composition.
274. The method of claim 273, wherein the post-treatment alters the
crystallinity of the composition.
275. The method of claim 273, wherein the post-treatment comprises
contacting the composition with methanol or ethanol, subjecting the
composition to shear stress, subjecting the composition to an
electric field, subjecting the composition to pressure, or
contacting the composition with salt.
276. The method of claim 271, further comprising reducing the
solid-state silk fibroin of step (b) by a mechanical means to
obtain micronized particles.
277. A method of stabilizing an immunogen comprising mixing an
immunogen and a silk fibroin solution, wherein the immunogen is
selected from the group consisting of a live, attenuated virus; an
inactivated virus; a viral subunit; a viral vector; a live,
attenuated bacterium; a killed bacterium; an inactivated bacterium;
a bacterial subunit; and an inactivated toxin from a pathogen.
278. The method of claim 277, further comprising forming a
solid-state form from the silk fibroin solution.
279. The method of claim 278, wherein the solid-state form is
selected from the group consisting of a film, a fiber, a hydrogel,
a scaffold, a mat, a particle, a lyophilized matrix, a needle, and
a composite thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C
.sctn.119(e) of U.S. Provisional Application No. 61/477,737, filed
Apr. 21, 2011, which is herein incorporated by reference in its
entirety.
TECHNICAL FIELD
[0003] The present invention generally relates to methods and
compositions for stabilization of active agents.
BACKGROUND
[0004] Stabilization of active agents is a critical feature of many
applications, because the active agents are usually labile and
sensitive to changes in surrounding conditions, e.g., temperature,
humidity and/or light. Even if an active agent is identified to be
useful for a given reaction, its application is often hampered by a
lack of long-term stability under process conditions.
[0005] Various modes to stabilize active agents, e.g., enzymes and
therapeutic proteins, have been studied, from lyophilization to
covalent immobilization, for different applications. In general,
many immobilized active agents demonstrate improved stability,
likely due to reduce mobility to prevent changes in hydrophobic
hydration and thus aggregation and loss of activity. Techniques for
immobilization of active agents, e.g., enzymes, usually fall into
four categories: (1) noncovalent adsorption of enzymes to carrier
material surfaces; (2) covalent attachment to material surfaces;
(3) physical entrapment into a material matrix; and (4)
crosslinking of an enzyme to "lock" the structure. All these
approaches are a compromise between maintaining high catalytic
activity while achieving the advantages listed above. The lack of
materials that provide specific surface binding sites or relative
hydrophilic/hydrophobic microenvironments for the retention of high
loading and activity of active agents limits the application of the
above carrier-based immobilization approaches. Further, for many
applications, carrier materials need to be biodegradable and
biocompatible for biomedical applications, which rules out the use
of most synthetic polymer materials.
[0006] Recently, new immobilization approaches have been developed
to improve stability and activity of active agent, e.g., enzymes.
For example, the microenvironment of the carrier material may be
engineered by using blocking agents to reduce non-specific binding
sites. Alternatively, hydrophilic macromolecules may be introduced
proximal to the active agent, or hydrophilic spacers used between
the active agent and the material surface. Further, sol-gel
materials have been used for immobilization and found to enhance
the activity of enzymes, e.g., lipases, up to 100-fold due to the
effects of microenvironmental confinement.
[0007] Further, enzyme cross-linking methods have been combined
with protein crystallization to generate cross-linked enzyme
crystals (CLECs) with increased enzyme stability and selectivity
when compared to the native enzyme. While this method has been used
by pharmaceutical companies to formulate therapeutic protein drugs,
protein crystallization is complicated and often unpredictable.
Cross-linked enzyme aggregates (CLEAs) can be obtained by
precipitation of proteins followed by cross-linking with
glutaraldehyde. The CLEA from penicillin acylase had the same
activity as a CLEC in the synthesis of ampicillin. Magnetic
nanoparticles have also been used for covalent immobilization of
enzymes and thus enhancement of enzyme stability. Yet none of these
immobilization methods are biocompatible/biodegradable or simple to
use while providing stability under ambient storage conditions
(e.g., room temperature) for long periods of time.
[0008] In particular, vaccine stabilization has been a long-lasting
challenge and large amounts of vaccines have been wasted due to
improper storage. Although global immunization currently saves the
lives of 2-3 million children every year, of the 10.5 million child
deaths that occur annually, 2.5 million are due to diseases that
are preventable by vaccines. Measles, mumps, and rubella are three
common childhood diseases, caused by measles virus, mumps virus
(paramyxoviruses), and rubella virus (togavirus), respectively,
that may be associated with serious complications and/or death. For
example, pneumonia and encephalitis are caused by measles. Mumps is
associated with aseptic meningitis, deafness and orchitis; and
rubella during pregnancy may cause congenital rubella syndrome in
the infants of infected mothers. The impact of measles, mumps, and
rubella vaccination on the natural history of each disease in the
U.S. can be quantified by comparing the maximum number of measles,
mumps, and rubella cases reported in a given year prior to vaccine
use to the number of cases of each disease reported in 1995. For
measles, 894,134 cases reported in 1941 compared to 288 cases
reported in 1995 resulted in a 99.97% decrease in reported cases;
for mumps, 152,209 cases reported in 1968 compared to 840 cases
reported in 1995 resulted in a 99.45% decrease in reported cases;
and for rubella, 57,686 cases reported in 1969 compared to 200
cases reported in 1995 resulted in a 99.65% decrease. Monthly
Immunization Table, 45 MMWR 24 (1996).
[0009] Vaccines are biological substances that may lose their
effectiveness quickly if they become too hot or too cold,
especially during transport and storage. Inadvertent freezing,
heating above 8.degree. C. or other breaks in the cold chain may
result in either failure of efficacy or vaccine wastage. According
to the WHO, between 2006-2015, the U.S. alone will have contributed
$35 billion for global vaccination programs. About one third will
be spent on vaccines and remainder will be spent on vaccine
delivery systems. It is clear that even 1% vaccine wastage because
of cold chain failure is a considerable sum. Indeed, for five U.S.
states, the average wastage of 1% to 5% cost approximately $6-$31
million. In other parts of the world, vaccine wastage can reach
10%. The two most common forms of wastage relate to heat stability
and shelf life, with inadvertent freezing remaining another key
problem. Hence, there is a great need for storage-stable active
agents, e.g., storage-stable vaccines, with longer shelf life that
can maintain efficacy under various robust environmental
conditions, e.g., without requiring cold chain compliance.
SUMMARY
[0010] Various embodiments described herein provide for a
storage-stable composition comprising a silk fibroin matrix and an
active agent distributed therein, wherein the active agent retains
at least about 30% of its original bioactivity when the composition
is subjected to at least one state-changing cycle, and/or is
maintained for a period of time under a specified condition. In one
embodiment, the state-changing cycle is a freeze-thaw cycle. In one
embodiment, the period of time for maintaining the active agent is
at least about 24 hours. In some embodiments, the specified
condition can be an environmental condition under which an active
agent is stored and/or transported. Non-limiting examples of
environmental conditions include temperatures, air pressures,
humidity, and light exposure. In some embodiments, the active agent
is an immunogen. In some embodiments, the active agent is a
vaccine.
[0011] Kits and delivery devices, e.g., useful in biomedical
fields, are also provided herein. Exemplary delivery devices
include, but are not limited to, syringes, dry powder injectors,
nasal sprays, nebulizers, and implants. Such kits and devices
comprise a storage-stable composition described herein, and
optionally a pharmaceutically acceptable solution. In one
embodiment, the kit further includes at least one delivery device
for administering to a subject a storage-stable composition
described herein, and/or a disinfectant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows the linear relationship between the log.sub.10
dilution of the vaccine sample and the Ct values for measles, mumps
and rubella. N=3, error bars represent standard deviations.
[0013] FIG. 2 shows results of reconstituted MMR vaccine at 24, 18,
12 and 6 hours in water prior to cell inoculation stored at
25.degree. C. protected from light. N=3, error bars represent
standard deviations.
[0014] FIG. 3 shows the stability of measles, mumps and rubella
virus stored in 9% (w/v) silk films over 3 months. N=3, error bars
represent standard deviations.
[0015] FIG. 4 is a bar graph comparing initial recovered potency of
measles, mumps, and rubella virus in silk films with the addition
of the stabilizing additives MgCl.sub.2, MgSO.sub.4 and sucrose.
N=3, error bars represent standard deviations.
[0016] FIG. 5 shows reconstituted MMR vaccine at 24, 18, 12 and 6
hours in 70% sucrose prior to cell inoculation stored at 25.degree.
C. protected from light. N=3, error bars represent standard
deviations.
[0017] FIGS. 6A-6D present comparisons of residual potency of
reconstituted MMR vaccine in water at 4.degree. C. (FIG. 6A) or
37.degree. C. (FIG. 6C), or 70% sucrose at 4.degree. C. (FIG. 6B)
or 37.degree. C. (FIG. 6D). Vaccines were reconstituted at 24, 18,
12 and 6 hours in water or 70% sucrose prior to cell inoculation
stored at 25.degree. C. protected from light. N=3, error bars
represent standard deviations.
[0018] FIG. 7 shows a schematic of vaccine-encapsulated silk film
fabrication and infectivity assay. (1) Lyophilized vaccine powder
is reconstituted in sterilized aqueous silk solution. (2a)
Vaccine-encapsulated silk films were prepared by casting an aliquot
of the vaccine-silk mixture onto a Teflon-coated surface and
allowed to dry in a sterile hood for 12 hours at room temperature,
protected from light. (3a) Individual dried films were stored in
Eppendorf tubes at the appropriate temperatures for stability
studies. (2b) After lyophilized vaccine powder was reconstituted in
sterilized aqueous silk solution, lyophilized vaccine-encapsulated
silk films were prepared by casting an aliquot of vaccine-silk
mixture into a 96-well plate and lyophilized. (3b) Individual
lyophilized films were removed from the well plate and transferred
to glass serum vials, capped with a lyophilization stopper and alum
seal under nitrogen and vacuum conditions. (4) For infectivity
studies, the films were redissolved in nuclease-free sterile water
and the solution was added directly to Vero cells grown in M199
media cultured in a 24-well plate. The cells were incubated for 3
days to allow the virus to replicate, then the RNA was isolated,
converted to cDNA and quantitated using real-time PCR. (5) RNA was
isolated from the Vero cells using TRIzol/chloroform, the RNA was
purified and reverse transcription was carried out to synthesize
cDNA for real-time RT-PCR.
[0019] FIGS. 8A-8C show graphs of residual potency of measles,
mumps and rubella components of the lyophilized vaccine
reconstituted in water for different times, stored at 4.degree. C.
(FIG. 8A), 25.degree. C. (FIG. 8B) and 37.degree. C. (FIG. 8C)
(.diamond-solid.) measles, (.largecircle.) mumps, (.box-solid.)
rubella. N=3, error bars represent standard deviations.
[0020] FIGS. 9A-9D show graphs of the stability of the measles
virus component of the MMR vaccine stored in 9% (w/v) silk films
over 6 months at 4.degree. C. (FIG. 9A), 25.degree. C. (FIG. 9B),
37.degree. C. (FIG. 9C), and 45.degree. C. (FIG. 9D).
(.diamond-solid.) MMR-silk films. (.quadrature.) MMR powder. N=3,
error bars represent standard deviations.
[0021] FIGS. 10A-10D show graphs of the stability of the mumps
virus component of the MMR vaccine stored in 9% (w/v) silk films
over 6 months at 4.degree. C. (FIG. 10A), 25.degree. C. (FIG. 10B),
37.degree. C. (FIG. 10C), and 45.degree. C. (FIG. 10D).
(.diamond-solid.) MMR-silk films. (.quadrature.) MMR powder. N=3,
error bars represent standard deviations.
[0022] FIGS. 11A to 11D shows graphs of the stability of the
rubella virus component of the MMR vaccine stored in 9% (w/v) silk
films over 6 months at 4.degree. C. (FIG. 11A), 25.degree. C. (FIG.
11B), 37.degree. C. (FIG. 11C), and 45.degree. C. (FIG. 11D).
(.diamond-solid.) MMR-silk films. (.quadrature.) MMR powder. N=3,
error bars represent standard deviations.
[0023] FIGS. 12A to 12D show graphs of the stability of the measles
virus component of the MMR vaccine stored in 9% (w/v) lyophilized
silk films over 6 months at 4.degree. C. (FIG. 12A), 25.degree. C.
(FIG. 12B), 37.degree. C. (FIG. 12C), and 45.degree. C. (FIG. 12D).
(.diamond-solid.) MMR-silk lyophilized films. (.quadrature.) MMR
powder. N=3, error bars represent standard deviations.
[0024] FIGS. 13A-13D show graphs of the stability of the mumps
virus component of the MMR vaccine stored in 9% (w/v) lyophilized
silk films over 6 months at 4.degree. C. (FIG. 13A), 25.degree. C.
(FIG. 13B), 37.degree. C. (FIG. 13C), and 45.degree. C. (FIG. 13D).
(.diamond-solid.) MMR-silk lyophilized films. (.quadrature.) MMR
powder. N=3, error bars represent standard deviations.
[0025] FIGS. 14A-14D show graphs of the stability of the rubella
virus component of the MMR vaccine stored in 9% (w/v) lyophilized
silk films over 6 months at 4.degree. C. (FIG. 14A), 25.degree. C.
(FIG. 14B), 37.degree. C. (FIG. 14C), and 45.degree. C. (FIG. 14D).
(.diamond-solid.) MMR-silk lyophilized films. (.quadrature.) MMR
powder. N=3, error bars represent standard deviations.
[0026] FIGS. 15A-15C show Arrhenius plots of the degradation rates
of the measles (FIG. 15A), mumps (FIG. 15B) and rubella (FIG. 15C)
components of the vaccine as a function of the inverse of the
absolute temperature. (.diamond-solid.) lyophilized silk films,
(.largecircle.) silk films, (.box-solid.) powder.
[0027] FIGS. 16A-16C show graphs of the predicted half-lives of the
measles (FIG. 16A), mumps (FIG. 16B) and rubella (FIG. 16C) viral
components as a function of temperature and the corresponding upper
and lower limits of the half-life. The predicted half-lives
represent the estimated time required for the viral component to
degrade to 50% of the initial value. (.diamond-solid.) lyophilized
silk films, (.largecircle.) silk films, (.box-solid.) powder
[0028] FIG. 17 shows a graph of differential scanning calorimetry,
solid-state DSC. Solid-state DSC of a lyophilized silk film shows
glass transition (Tg) at 178.degree. C. The Tg of the manufacturer
provided MMR vaccine powder (containing a wide variety of
excipients and stabilizers) was 68.9.degree. C. The lyophilized
MMR-silk films showed a Tg at 89.2.degree. C., indicating the
addition of silk to the MMR powder increased stability of the
vaccine reflected in the increased Tg. The MMR-silk lyophilized
film curve, however, showed two peaks at 116.6.degree. C. and
164.8.degree. C. which could indicate a Tm and Td, describing the
unfolding or degradation of vaccine components.
[0029] FIG. 18 shows a graph of nano differential scanning
calorimetry, nano-DSC. The Tm of purified viral particles appears
around 16.8.degree. C. The presence of silk increases the Tm of the
viral particles to 68.3.degree. C. The sharp drop following the Tm
is an exothermic event most likely due to aggregation as a result
of the protein unfolding at the Tm. The Tg of silk was around
178.degree. C. so the elevated Tg values were due to the effect
silk has on the encapsulated viral proteins.
[0030] FIG. 19 shows a graph indicating comparison of dynamic light
scattering of purified viral particles in water and purified viral
particles in silk solution. The average mean effective diameter of
MMR viral particles was about 250 nm. The mean effective diameter
of the purified MMR solution began increasing at around 16.degree.
C., indicating aggregation of viral particles due to the increased
thermal input. The MMR-silk solution did not show signs of
aggregation until 70.degree. C., indicating silk provided the
structural stability to prevent the aggregation of the viral
proteins.
[0031] FIGS. 20A-20B show graphs of MMR release from (FIG. 20A)
silk films and (FIG. 20B) lyophilized silk films. N=3, error bars
represent standard deviations.
[0032] FIGS. 21A-21B show graphs of MMR release (FIG. 21A) silk
hydrogels and (FIG. 21B) silk microspheres. N=3, error bars
represent standard deviations.
[0033] FIGS. 22A-22D show schematic diagrams. FIG. 22A, measles and
mumps belong to the Paramyxoviridae family and their structures
consist of single-stranded, negative-sense RNA enclosed in
nucleocapsids within a lipid bilayer. The viral envelop is formed
by the matrix protein (M), haemaglutinin protein (H) and fusion
protein (F). Structurally intact H and F proteins are responsible
for binding and fusion of the viral particles to the animal cells.
FIG. 22B, by a combination of hydrophobic interaction and limited
chain mobility, silk-entrapped viral particles maintain structural
activity at elevated temperatures. FIG. 22C, the F and H proteins
binding to the receptors CD46 and CD150 (collectively known as
SLAM) to gain entry into the cell to initiate viral replication.
FIG. 22D, denaturation of the surface proteins can cause
aggregation of the viral particles. The perturbation of the
proteins can cause it to be unrecognized by the cell and denied
entry.
DETAILED DESCRIPTION
[0034] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0035] As used herein and in the claims, the singular forms include
the plural reference and vice versa unless the context clearly
indicates otherwise. Other than in the operating examples, or where
otherwise indicated, all numbers expressing quantities of
ingredients or reaction conditions used herein should be understood
as modified in all instances by the term "about."
[0036] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0037] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as those commonly understood to
one of ordinary skill in the art to which this invention pertains.
Although any known methods, devices, and materials may be used in
the practice or testing of the invention, the methods, devices, and
materials in this regard are described herein.
[0038] One aspect provided herein relates to methods and
compositions of maintaining or stabilizing the bioactivity of an
active agent. The method includes maintaining a composition,
wherein the composition comprises a silk fibroin matrix and at
least one active agent distributed, mixed, or embedded therein, and
wherein the at least one active agent retains or stabilizes at
least about 30% of its original bioactivity when the composition is
subjected to a specified condition, which inhibits or reduces the
bioactivity of the active agent, for a period of time. Such
conditions can include, but are not limited to, a state-changing
cycle, temperatures, air pressures, humidity, and light exposure.
In one embodiment, the state-changing cycle is a freeze-thaw
cycle.
[0039] Embodiments of various aspects described herein provide for
stabilized active agents, in which stabilization of an active agent
is achieved by distributing, mixing, or embedding an active agent
in a silk fibroin matrix. The silk fibroin matrix can be a silk
fibroin solution or a solid-state silk fibroin matrix. This
approach provides for the active agent to retain bioactivity
regardless of the cold chain and/or environmental conditions under
which the active agent is stored and/or transported. Exemplary
environmental conditions include, but are not limited to,
temperatures, air pressures, humidity, and light exposure. For
example, the cold chain is a standard practice for stabilizing
active agents in the pharmaceutical industry: maintaining the cold
chain ensures that active agents are transported and stored
according to the manufacturer's recommended temp range (e.g.,
2.degree. C. to 8.degree. C. or sub-zero temperatures) until time
of use.
[0040] In certain embodiments, the active agents described herein
are immunogens. In one embodiment, the immunogen is a vaccine. Most
vaccines are sensitive to environmental conditions under which they
are stored and/or transported. For example, freezing may increase
reactogenicity (e.g., capability of causing an immunological
reaction) and/or loss of potency for some vaccines (e.g., HepB, and
DTaP/IPV/HIB), or cause hairline cracks in the container, leading
to contamination. Further, some vaccines (e.g., BCG, Varicella, and
MMR) are sensitive to heat. Many vaccines (e.g., BCG, MMR,
Varicella, Meningococcal C Conjugate, and most DTaP-containing
vaccines) are light-sensitive. See, e.g., Galazka et al.,
Thermostability of vaccines, in Global Programme for Vaccines &
Immunization (World Health Organization, Geneva, 1998); Peetermans
et al., Stability of freeze-dried rubella virus vaccine (Cendehill
strain) at various temperatures, 1 J. Biological Standardization
179 (1973). Thus, the compositions and methods described herein
also provide for stabilization of vaccines regardless of the cold
chain and/or other environmental conditions.
Stabilization of Active Agents
[0041] The terms "stabilizing," "stabilize," "stability," and
"stabilization," are used herein in reference to maintaining or
retaining bioactivity of at least one active agent in a silk
fibroin matrix. The phrase "stabilization of active agents" as used
herein means that one or more active agents distributed, mixed or
embedded in a silk fibroin matrix retain at least about 30% of its
original bioactivity, including at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90% of its original bioactivity or higher. The terms
"stabilize" and "retain" in reference to bioactivity of active
agents are used herein interchangeably.
[0042] As used herein, the terms "maintaining," "maintain," and
"maintenance," when referring to compositions or active agents mean
keeping, sustaining, or retaining the bioactivity of at least one
active agent in a silk fibroin matrix, when the active agent is
subjected to certain conditions. In some embodiments, one or more
active agents distributed in a silk fibroin matrix retains at least
about 30% of its original bioactivity, including at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90% of its original bioactivity or
higher.
[0043] The term "bioactivity," as used herein in reference to an
active agent, generally refers to the ability of an active agent to
interact with a biological target and/or to produce an effect on a
biological target. For example, bioactivity can include, without
limitation, elicitation of a stimulatory, inhibitory, regulatory,
toxic or lethal response in a biological target. The biological
target can be a molecule or a cell. For example, a bioactivity can
refer to the ability of an active agent to modulate the
effect/activity of an enzyme, block a receptor, stimulate a
receptor, modulate the expression level of one or more genes,
modulate cell proliferation, modulate cell division, modulate cell
morphology, or any combination thereof. In some instances, a
bioactivity can refer to the ability of a compound to produce a
toxic effect in a cell.
[0044] The bioactivity can be determined by assaying a cellular
response. Exemplary cellular responses include, but are not limited
to, lysis, apoptosis, growth inhibition, and growth promotion;
production, secretion, and surface exposure of a protein or other
molecule of interest by the cell; membrane surface molecule
activation including receptor activation; transmembrane ion
transports; transcriptional regulations; changes in viability of
the cell; changes in cell morphology; changes in presence or
expression of an internal component of the cell; changes in
presence or expression of a nucleic acid produced within the cell;
changes in the activity of an enzyme produced within the cell; and
changes in the presence or expression of a receptor. Methods for
assaying different cellular responses are well known to one of
skill in the art, e.g., western blot for determining changes in
presence or expression of an endogenous protein of the cell, or
microscopy for monitoring the cell morphology in response to the
active agent.
[0045] In reference to an antibody, the term "bioactivity"
includes, but is not limited to, epitope or antigen binding
affinity, the in vivo and/or in vitro stability of the antibody,
the immunogenic properties of the antibody, e.g., when administered
to a human subject, and/or the ability to neutralize or antagonize
the bioactivity of a target molecule in vivo or in vitro. The
aforementioned properties or characteristics can be observed or
measured using art-recognized techniques including, but not limited
to, scintillation proximity assays, ELISA, ORIGEN immunoassay
(IGEN), fluorescence quenching, fluorescence ELISA, competitive
ELISA, SPR analysis including, but not limited to, SPR analysis
using a BIAcore biosenser, in vitro and in vivo neutralization
assays (see, for example, International Publication No. WO
2006/062685), receptor binding, and immunohistochemistry with
tissue sections from different sources including human, primate, or
any other source as needed. In reference to an immunogen, the
"bioactivity" includes immunogenicity, the definition of which is
discussed in detail later. In reference to a virus, the
"bioactivity" includes infectivity, the definition of which is
discussed in detail later. In reference to a contrast agent, e.g.,
a dye, the "bioactivity" refers to the ability of a contrast agent
when administered to a subject to enhance the contrast of
structures or fluids within the subject's body. The bioactivity of
a contrast agent also includes, but is not limited to, its ability
to interact with a biological environment and/or influence the
response of another molecule under certain conditions.
[0046] By "original bioactivity" in reference to an active agent is
generally meant the bioactivity of an active agent as measured
immediately before or immediately after the active agent is
introduced into a silk fibroin matrix. That is, the original
bioactivity of an active agent can be measured, for example, within
about 20 minutes, before or after the active agent is introduced
into a silk fibroin matrix. In some instances, the original
bioactivity of an active agent can be measured, for example, about
10 seconds, about 15 seconds, about 20 seconds, about 25 seconds,
about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes,
about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes,
about 8 minutes, about 9 minutes, about 10 minutes, about 11
minutes, about 12 minutes, about 13 minutes, about 14 minutes,
about 15 minutes, about 16 minutes, about 17 minutes, about 18
minutes, about 19 minutes, or about 20 minutes, before or after the
active agent is introduced into a silk fibroin matrix. In one
embodiment, the silk fibroin matrix is a solid-state silk fibroin
matrix. In such embodiment, an active agent can lose some of its
bioactivity during handling before it is distributed into a
solid-state silk fibroin matrix. In another embodiment, the term
"original bioactivity," as used herein, can be used to describe the
bioactivity of an active agent before the active agent is
introduced into a silk-fibroin matrix. In some embodiments, the
term "original bioactivity" refers to the maximum bioactivity of an
active agent, e.g., bioactivity measured immediately after
activation of the active agent, e.g., by reconstitution or by
increasing the temperature. For example, if the active agent is
initially in powder, the original bioactivity of the active agent
can be measured immediately after reconstitution. In some
embodiments, the term "original bioactivity" refers to bioactivity
of an active agent when stored or transported in the absence of a
silk fibroin matrix under conditions specified by the manufacturer.
In some embodiments, the term "original bioactivity" refers to
bioactivity of an active agent when stored or transported in a
storage-stable composition as described herein under conditions
specified by the manufacturer. The definitions of the term
"original bioactivity" described herein are also applied to the
terms "original immunogenicity" and "original infectivity" as used
later herein.
[0047] In accordance with the methods described herein,
distributing, mixing, or embedding an active agent in a silk
fibroin matrix retains or stabilizes the bioactivity of the active
agent, e.g., at least about 30% of its original bioactivity,
regardless of the environmental or storage conditions (e.g.,
state-changing cycles, temperature, humidity, or light exposure).
The silk fibroin matrix can be in solution or in a solid state. In
various embodiments, when an active agent is distributed in a silk
fibroin matrix and such composition is subjected to a
state-changing cycle and/or is maintained for a period of time
under a specified condition, the active agent can retain at least
about 30% of its original bioactivity e.g., at least about 40%, at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, at least about 95% of the original
bioactivity or higher. In one embodiment, the active agent can
retain at least about 80% of its original bioactivity. Stated
another way, the stability of an active agent in a silk fibroin
matrix (i.e., the ability of an active agent to retain its
bioactivity (e.g., at least about 30% of its original bioactivity)
in a silk fibroin matrix) can be increased by at least about 30%,
at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, at least
about 95%, relative to the stability of an active agent in the
absence of a silk fibroin matrix. In one embodiment, the active
agent can retain at least about 80% of its original
bioactivity.
[0048] The compositions described herein can be maintained for any
period of time, e.g., hours, days, weeks, months or years. In some
embodiments, the compositions described herein can be maintained at
a temperature above 0.degree. C. for at least about 3 hours, at
least about 6 hours, at least about 9 hours, at least about 12
hours, at least about 24 hours or longer. In some embodiments, the
compositions described herein can be maintained for at least about
1 day, at least about 2 days, at least about 3 days, at least about
4 days, at least about 5 days, at least about 6 days, at least
about 7 days or longer. In some embodiments, the compositions
described herein can be maintained for at least about 1 week, at
least about 2 weeks, at least about 3 weeks, at least about 4 weeks
or longer. In some embodiments, the compositions described herein
can be maintained for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, at least about 12 months or
longer.
[0049] In methods and compositions described herein, the
compositions described herein can be maintained at any temperatures
or at a manufacturer's recommended temperature specified for an
active agent. In some embodiments, the compositions can be
maintained in liquid nitrogen or in dry ice. In some embodiments,
the compositions can be maintained, for example, between about
-80.degree. C. and about -20.degree. C., inclusive, or between
about -20.degree. C. and about 0.degree. C., inclusive. In some
embodiments, the compositions can be maintained at a temperature
above 0.degree. C. In those embodiments, the compositions can be
maintained at a temperature from about 0.degree. C. to about an
ambient temperature. As used herein, the term "ambient temperature"
is used to describe a surrounding temperature at which the
compositions described herein are maintained and it includes
temperatures between 0.degree. C. and 60.degree. C., between
0.degree. C. and 50.degree. C., or between 0.degree. C. and
40.degree. C. In some embodiments, the ambient temperature is the
fridge temperature (e.g., between 0.degree. C. and 15.degree. C.,
inclusive). In some embodiments, the ambient temperature is about
the body temperature of a subject (e.g., between 36.degree. C. and
38.degree. C., inclusive, for a human subject, or a higher or lower
body temperature range for other animals). In some embodiments, the
ambient temperature is the room temperature, e.g., between
20.degree. C. and 35.degree. C., and it can vary with geographical
conditions. For example, the room temperature in warm-climate
regions, e.g., Africa, can be generally warmer than that in
cool-climate regions, e.g., the United States or United Kingdom. In
some embodiments, the compositions can be maintained at a
temperature of at least about 37.degree. C. or greater than
37.degree. C. In some embodiments, the compositions can be
maintained at a temperature of at least about 40.degree. C. or
greater than 40.degree. C. In some embodiments, the compositions
can be maintained at a temperature of at least about 45.degree. C.
or greater than 45.degree. C.
[0050] Some embodiments described herein are beneficial for
development of implantable drug delivery devices in which an active
agent can retain at least 30% (including at least about 40%, at
least about 60%, at least about 80% or higher) of its original
bioactivity or higher for a period of time. In some embodiments, a
composition or an active agent in an implantable drug device can
retain at least about 30% of its original bioactivity or higher for
at least about 6 hours, at least about 12 hours, at least about 24
hours, at least about 36 hours, at least about 48 hours, at least 3
days, at least about 4 days, at least about 5 days, at least about
6 days, at least about 1 week, at least about 2 weeks, at least
about 3 weeks, at least about 4 weeks, at least about 2 months, at
least about 3 months, at least about 4 months, at least about 5
months, at least about 6 months, or at least after 1 year or
longer, after implantation.
[0051] In some embodiments, one or more active agents, e.g.,
immunogens such as vaccines, encapsulated in an injectable form of
silk fibroin matrix (e.g., but not limited to, hydrogel, gel-like
particles, and/or microspheres) can be administered to a subject
(e.g., by injection such as subcutaneous injection) as a depot of
the active agent (e.g., a vaccine depot) such that the active agent
(e.g., a vaccine) can be released, continuously or intermittently,
from the depot for an extended period of time, e.g., for a period
of hours, days, weeks, or months. In some embodiments, the active
agent (e.g., a vaccine) can be released at a rate at which at least
about 1% (including at least about 5%, at least about 10%, at least
about 20%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, or more) of the encapsulated
active agent is released over a period of at least 1 hour, at least
2 hours, at least 3 hours, at least about 4 hours, at least about 5
hours, at least about 6 hours, at least about 12 hours, at least
about 24 hours or longer. In some embodiments, the active agent
(e.g., a vaccine) can be released at a rate at which at least about
10% (including at least about 20%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%, or
more) of the encapsulated active agent is released over a period of
5 days, a period of 1 week, at least about 2 weeks, at least about
3 weeks, at least about 1 month, at least about 2 months, at least
about 3 months or longer.
[0052] In some embodiments, the active agent retains at least about
30% of its original bioactivity e.g., at least about 40%, at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, at least about 95% of the original
bioactivity or higher activity at about 4.degree. C., at about
25.degree. C., at about 37.degree. C., at about 45.degree. C., or
greater, for at least up to 6 months. In some embodiments, the
active agent retains at least about 8% of the original bioactivity
at temperatures of about 37.degree. C. or greater, for at least 6
months.
[0053] In some embodiments, the compositions described herein can
be maintained under exposure to light, e.g., light of different
wavelengths and/or from different sources. In some embodiments, the
compositions described herein can be maintained under exposure to
UV or infra-red irradiation. In some embodiments, the compositions
described herein can be maintained under visible lights.
[0054] In some embodiments, the composition described herein when
stored or transported can be subjected to at least one
state-changing cycle. The term "state-changing cycle" as used
herein refers to a change of a material state, including, but not
limited to, from a solid state to a fluid state, or from a fluid
state to a solid state. A fluid state can include, but is not
limited to, liquids, gases, slurries, flowable paste, plasmas, and
any combinations thereof. A solid state refers to a state that is
not flowable, and it can also encompass semi-solids, e.g., a gel.
The composition described herein can be maintained at a certain
state for any period of time, e.g., seconds, minutes, hours, weeks,
months, or years, before changing to another state. A
state-changing cycle can be resulted from at least one change in an
environmental condition described herein, e.g., a temperature
change, a change in ambient air pressure, light condition,
humidity, or any combinations thereof.
[0055] In one embodiment, the state-changing cycle refers to a
freeze-thaw cycle. In such embodiments, the composition described
herein when stored or transported can be subjected to at least one
freeze-thaw cycle, at least two freeze-thaw cycles, at least three
freeze-thaw cycles, at least four freeze-thaw cycles, at least five
freeze-thaw cycles, at least six freeze-thaw cycles, at least seven
freeze-thaw cycles, at least eight freeze-thaw cycles, at least
nine freeze-thaw cycles, at least ten free-thaw cycles or more. The
term "freeze-thaw cycles" is used herein to describe a series of
alternating freezing and thawing, and also encompasses a series of
alternating frozen (solid) and fluid state. For example, one
freeze-thaw cycle involves a change of state between a frozen
(solid) state and a fluid state. The time interval between freezing
and thawing, or frozen and fluid state, can be any period of time,
e.g., hours, days, weeks or months. For example, once an active
agent composition has been frozen or is in a frozen state, it can
be continually stored in the frozen state at sub-zero temperatures,
e.g., between about -20.degree. C. and -80.degree. C., until it
needs to be thawed for use again. Freezing of a composition can be
performed rapidly, e.g., in liquid nitrogen, or gradually, e.g., in
a freezing temperature, e.g., between about -20.degree. C. and
-80.degree. C. Thawing of a frozen composition can be performed at
any temperature above 0.degree. C. rapidly, e.g., at room
temperature, or gradually, e.g., on ice. Typically, an active agent
in non-silk fibroin matrix can lose its bioactivity over one or
more freeze-thaw cycles. As described herein, distributing an
active agent in a silk fiborin matrix can increase the stability of
the active agent and thus retain its bioactivity during one or more
freeze-thaw cycles.
[0056] In some embodiments, the compositions described herein can
be maintained at a relative humidity of at least about 5%, at least
about 10%, at least about 20%, at least about 30%, at least about
40%, at least about 50% or higher. The term "relative humidity" as
used herein is a measurement of the amount of water vapor in a
mixture of air and water vapor. It is generally defined as the
partial pressure of water vapor in the air-water mixture, given as
a percentage of the saturated vapor pressure under those
conditions.
[0057] In some embodiments, the compositions described herein can
be lyophilized to decrease residual moisture during storage. In
some embodiments, residual moisture is decreased by at least about
5%, at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90%, or at least
about 95%.
[0058] In some embodiments, the compositions described herein can
be maintained under or subjected to any air pressure. In some
embodiments, the compositions described herein can be maintained
under or subjected to about atmospheric pressure, or higher, e.g.,
about 1 atm, about 2 atms, about 3 atms, about 4 atms, about 5
atms, about 6 atms, about 7 atms, about 8 atms, about 9 atms or
about 10 atms. In some embodiments, the compositions described
herein can be maintained under or subjected to vacuum.
[0059] In one embodiment, the composition is maintained under two
or more conditions specified herein.
[0060] Without wishing to be bound by theory, silk can reduce the
degradation rate of an immunogen (e.g., vaccine) at an elevated
temperature (e.g., at room temperature or above, including at least
about 20.degree. C., at least about 30.degree. C., at least about
40.degree. C. or higher). Thus, an immunogen (e.g., vaccine)
distributed in a silk fibroin matrix can have a half-life longer at
an elevated temperature (e.g., at room temperature or above,
including at least about 20.degree. C., at least about 30.degree.
C., at least about 40.degree. C. or higher) by at least about
1.5-fold (e.g., at least about 2-fold, at least about 3-fold, at
least about 4-fold, at least about 5-fold, at least about 10-fold,
at least about 15-fold, at least about 20-fold, at least about
25-fold, at least about 30-fold, or more), as compared to an
immunogen without the silk matrix. As used herein, the term
"half-life" refers to the time at which an agent retains about 50%
of its original bioactivity (including original immunogenicity or
original infectivity). Accordingly, a method for extending the
half-life of an immunogen (e.g., vaccine), for example, at an
elevated temperature (e.g., at room temperature or above, including
at least about 20.degree. C., at least about 30.degree. C., at
least about 40.degree. C. or higher) is also provided herein. The
method comprises maintaining an immunogenic composition, wherein
the composition comprises a silk fibroin matrix and at least one
immunogen (e.g., vaccine) distributed therein, and wherein the
immunogen (e.g., vaccine) retains at least about 30% of its
original immunogenticity (e.g., infectivity) when the composition
is maintained for at least about 24 hours at a temperature of at
least about room temperature or higher. In some embodiments, the
immunogen (e.g., vaccine) can retain at least about 80% of its
original immunogenticity (e.g., infectivity). In some embodiments,
the composition can be maintained for at least up to about 6
months. In some embodiments, the composition can be maintained at a
temperature higher than 37.degree. C., or higher than 45.degree.
C., or higher.
Storage-Stable Compositions
[0061] Another aspects described herein are storage-stable
compositions, which comprise a silk fibroin matrix and an active
agent distributed, mixed or embedded therein, wherein the active
agent retains at least about 30% of its original bioactivity when
the composition is subjected to at least one state-changing cycle,
and/or is maintained for a period of time under one or more
conditions specified herein. In one embodiment, the state-changing
cycle is a freeze-thaw cycle. In one embodiment, the period of time
for maintaining the active agent is at least about 24 hours. In
some embodiments, the specified condition can be an environmental
condition under which an active agent is stored and/or transported.
Non-limiting examples of environmental conditions include
temperatures, air pressures, humidity, and light exposure. In some
embodiments, the compositions described herein can be immunogenic.
In such embodiments, the active agent is an immunogen. In some
embodiments, the active agent is a vaccine.
[0062] Any compositions described herein can be present in any
material state, e.g., a film, a fiber, a particle, a gel, a
microsphere, or a hydrogel. In various embodiments, the material
state of the compositions described herein can vary with the state
of the silk fibroin matrix, e.g., a film, a fiber, a particle, a
gel, a microsphere, or a hydrogel. In some embodiments, the silk
fibroin matrix is present in a solid state. In other embodiments,
the silk fibroin matrix can be a solution.
[0063] Any ratio of silk fibroin to active agent may be used. In
various embodiments, the ratio of a silk fibroin matrix to an
active agent is about 1:1000 to about 1000:1, about 1:500 to about
500:1, about 1:250 to about 250:1, about 1:125 to about 125:1,
about 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to
about 25:1, about 1:10 to about 10:1, about 1:5 to about 5:1, about
1:3 to about 3:1, or about 1:1. The ratio of the silk fibroin
matrix to the active agent can vary with a number of factors,
including the selection of an active agent, the storage condition
and duration, the concentration of the silk fibroin matrix and the
form of the silk matrix. One of skill in the art can determine
appropriate ratio of the silk fibroin matrix to the active agent,
e.g., by measuring the bioactivity of the active agent retained at
various ratios described herein over a pre-defined amount of time
under a defined condition, e.g., at a temperature of above
0.degree. C. Methods for measuring the bioactivity of various
active agents described herein, e.g., enzymes, vaccines, proteins,
antibodies and nucleic acids, are well known in the art. By way of
example, stability or bioactivity of a given active agent in silk
fibroin may be determined based on combinations of time and
temperature. For example, stabilization studies can be conducted
for 6 months. Activity assays can be conducted, for example, after
2 weeks, 4 weeks, then monthly. Samples can be prepared to provide
N=3 for each time point. The range of temperature storage
conditions to be assessed include 4.degree. C. (refrigeration),
25.degree. C. (room temperature), 37.degree. C. (body temperature),
45.degree. C. and/or 50.degree. C., inclusive. Additionally,
activity can be assayed after one, two, three or more freeze-thaw
cycles. These variables can be combined exhaustively to fully
characterize the optimum formulation for long-term stability of
active agent(s). In some embodiments, the results of the
silk-related active agent stability can be compared with e.g.,
lyophilized active agent preparations with the same storage
conditions, with the goal of improving the stability of the
manufacture-recommended storage conditions (e.g., 4.degree. C.) of
lyophilized active agent preparations.
[0064] When the silk fibroin matrix is in solid state, it can be
further processed. In some embodiments, the compositions comprising
a solid-state silk fibroin matrix can be further micronized. The
term "micronized" is used herein in reference to particles with an
average size of about 1000 .mu.m or less, and encompasses
nanoparticles and/or microparticles. As used herein, the term
"nanoparticles" is defined as particles with an average size
ranging from about 1 nm to about 1000 nm, from about 5 nm to about
900 nm, or from about 10 nm to about 800 nm. The term
"microparticles" refers to particles with an average size ranging
from about 1 .mu.m to 1000 .mu.m, from about 5 .mu.M to about 900
.mu.m, or from about 10 .mu.m to about 800 .mu.m. It should be
understood that "micronized" does not refer only to particles which
have been produced by the finely dividing such as mechanical
grinding, crushing or impinging jet, of materials which are in bulk
or other form, e.g., a solid-state silk fibroin film. In some
embodiments, micronized particles can also be formed by other
mechanical, chemical or physical methods known in the art, such as,
for example, formation in solution or in situ. A composition
described herein can be micronized, e.g., by pulverizing, crushing,
grinding, freeze-drying, or any combinations thereof.
Silk Fibroin
[0065] Silk fibroin is a particularly appealing biopolymer
candidate to be used for embodiments of various aspects described
herein, e.g., because of its all aqueous processing (Sofia et al.,
54 J. Biomed. Mater. Res. 139 (2001); Perry et al., 20 Adv. Mater.
3070-72 (2008)), relatively easy functionalization (Murphy et al.,
29 Biomat. 2829-38 (2008)), and biocompatibility (Santin et al., 46
J. Biomed. Mater. Res. 382-9 (1999)). For example, silk has been
approved by U.S. Food and Drug Administration as a tissue
engineering scaffold in human implants. See Altman et al., 24
Biomaterials: 401 (2003).
[0066] Silk can provide an immobilization matrix capable of
stabilizing bioactive molecules. Previous reports on entrapment of
enzymes, antibodies, and antibiotics entrapped in silk matrices
indicates stabilization and recovered activity even at elevated
temperatures and without specialized storage conditions or the
addition of additives (Pritchard et al., "Silk fibroin encapsulated
powder reservoirs for sustained release of adenosine" Journal of
Controlled Release (2010) 144:159-167; Lu et al., "Stabilization of
enzymes in silk films" Biomacromolecules (2009) 10:1032-1042).
However, these reports do not describe that silk fibroin can
stabilize vaccine (e.g., a live vaccine), which is a biological
preparation and is temperature sensitive.
[0067] As used herein, the term "silk fibroin" includes silkworm
fibroin and insect or spider silk protein. See e.g., Lucas et al.,
13 Adv. Protein Chem. 107 (1958). Any type of silk fibroin can be
used according to various aspects described herein. Silk fibroin
produced by silkworms, such as Bombyx mori, is the most common and
represents an earth-friendly, renewable resource. For instance,
silk fibroin used in a silk film may be attained by extracting
sericin from the cocoons of B. mori. Organic silkworm cocoons are
also commercially available. There are many different silks,
however, including spider silk (e.g., obtained from Nephila
clavipes), transgenic silks, genetically engineered silks, such as
silks from bacteria, yeast, mammalian cells, transgenic animals, or
transgenic plants (see, e.g., WO 97/08315; U.S. Pat. No.
5,245,012), and variants thereof, that can be used.
[0068] In various embodiments, the silk fibroin matrix can be
modified for different biomedical applications. For instance, to
maintain the stability of an active agent distributed in a silk
fibroin matrix when implanted in vivo for tissue engineering or
drug delivery purposes, the silk particles can be genetically
modified, which provides for further modification of the silk such
as the inclusion of a fusion polypeptide comprising a fibrous
protein domain and a mineralization domain, which can be used to
form an organic-inorganic composite. See WO 2006/076711.
Additionally, the silk matrix can be combined with one or more
biocompatible polymers such as polyethylene oxide, polyethylene
glycol, collagen, fibronectin, keratin, polyaspartic acid,
polylysin, alginate, chitosan, chitin, hyaluronic acid, and the
like. See, e.g., WO 04/062697; WO 05/012606. In some embodiments,
the silk fibroin can also be chemically modified, for example
through diazonium or carbodiimide coupling reactions, avidin-biodin
interaction, or gene modification and the like, to alter the
physical properties and functionalities of the silk protein. See,
e.g., WO 2011/011347, Functionalization of Silk Material by
Avidin-Biotin Interaction; WO 2010/057142, Surface Modification of
Silk Fibroin Matrices with PEG Useful as Anti-Adhesion Barriers
& Anti-Thrombotic Materials; U.S. Ser No. 12/192,588, Diazonium
Salt Modification of Silk Polymer. Additionally, the silk fibroin
matrix can be combined with a chemical, such as glycerol, that,
e.g., affects flexibility of the matrix. See, e.g., WO 2010/042798,
Modified Silk films Containing Glycerol.
Active Agents
[0069] As used herein, the term "active agent" refers to any
molecule, compound or composition, bioactivity of which is desired
to be stabilized when such molecule, compound, or composition is
subjected to at least one state-changing cycle, and/or is
maintained under certain conditions as described herein. For the
methods and compositions described herein, any active agent can be
maintained within a silk-fibroin matrix. Examples of active agents
include, but are not limited to, proteins, peptides, antigens,
immunogens, vaccines, antibodies or portions thereof (e.g.,
antibody-like molecules), enzymes, nucleic acids (e.g.,
oligonucleotides, polynucleotides, siRNA, shRNA), aptamers,
viruses, bacteria, small molecules, cells, photosynthetic and
energy-harvesting compounds, flavors, antibiotics, therapeutic
agents, diagnostic agents such as contrast agents or dye, viral
vectors, and anti-venom.
[0070] As used herein, the terms "proteins" and "peptides" are used
interchangeably herein to designate a series of amino acid residues
connected to the other by peptide bonds between the alpha-amino and
carboxy groups of adjacent residues. The terms "protein", and
"peptide", which are used interchangeably herein, refer to a
polymer of protein amino acids, including modified amino acids
(e.g., phosphorylated, glycated, etc.) and amino acid analogs,
regardless of its size or function. Although "protein" is often
used in reference to relatively large polypeptides, and "peptide"
is often used in reference to small polypeptides, usage of these
terms in the art overlaps and varies. The term "peptide" as used
herein refers to peptides, polypeptides, proteins and fragments of
proteins, unless otherwise noted. The terms "protein" and "peptide"
are used interchangeably herein when referring to a gene product
and fragments thereof. Thus, exemplary peptides or proteins include
gene products, naturally occurring proteins, homologs, orthologs,
paralogs, fragments and other equivalents, variants, fragments, and
analogs of the foregoing.
[0071] The term "nucleic acids" used herein refers to
polynucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA), polymers thereof in either
single- or double-stranded form. Unless specifically limited, the
term encompasses nucleic acids containing known analogs of natural
nucleotides, which have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g., degenerate codon substitutions)
and complementary sequences, as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer, et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka, et al., J. Biol. Chem. 260:2605-2608
(1985), and Rossolini, et al., Mol. Cell. Probes 8:91-98 (1994)).
The term "nucleic acid" should also be understood to include, as
equivalents, derivatives, variants and analogs of either RNA or DNA
made from nucleotide analogs, and, single (sense or antisense) and
double-stranded polynucleotides.
[0072] The term "short interfering RNA" (siRNA), also referred to
herein as "small interfering RNA" is defined as an agent which
functions to inhibit expression of a target gene, e.g., by RNAi. An
siRNA can be chemically synthesized, it can be produced by in vitro
transcription, or it can be produced within a host cell. siRNA
molecules can also be generated by cleavage of double stranded RNA,
where one strand is identical to the message to be inactivated. The
term "siRNA" refers to small inhibitory RNA duplexes that induce
the RNA interference (RNAi) pathway. These molecules can vary in
length (generally 18-30 base pairs) and contain varying degrees of
complementarity to their target mRNA in the antisense strand. Some,
but not all, siRNA have unpaired overhanging bases on the 5' or 3'
end of the sense 60 strand and/or the antisense strand. The term
"siRNA" includes duplexes of two separate strands, as well as
single strands that can form hairpin structures comprising a duplex
region.
[0073] The term "shRNA" as used herein refers to short hairpin RNA
which functions as RNAi and/or siRNA species but differs in that
shRNA species are double stranded hairpin-like structure for
increased stability. The term "RNAi" as used herein refers to
interfering RNA, or RNA interference molecules are nucleic acid
molecules or analogues thereof for example RNA-based molecules that
inhibit gene expression. RNAi refers to a means of selective
post-transcriptional gene silencing. RNAi can result in the
destruction of specific mRNA, or prevents the processing or
translation of RNA, such as mRNA.
[0074] The term "enzymes" as used here refers to a protein molecule
that catalyzes chemical reactions of other substances without it
being destroyed or substantially altered upon completion of the
reactions. The term can include naturally occurring enzymes and
bioengineered enzymes or mixtures thereof. Examples of enzyme
families include kinases, dehydrogenases, oxidoreductases, GTPases,
carboxyl transferases, acyl transferases, decarboxylases,
transaminases, racemases, methyl transferases, formyl transferases,
and .alpha.-ketodecarboxylases.
[0075] The term "vaccines" as used herein refers to any preparation
of killed microorganisms, live attenuated organisms, subunit
antigens, toxoid antigens, conjugate antigens or other type of
antigenic molecule that when introduced into a subjects body
produces immunity to a specific disease by causing the activation
of the immune system, antibody formation, and/or creating of a
T-cell and/or B-cell response. Generally vaccines against
microorganisms are directed toward at least part of a virus,
bacteria, parasite, mycoplasma, or other infectious agent. In one
embodiment, vaccine encapsulated in a silk fibroin matrix is a live
vaccine.
[0076] As used herein, the term "aptamers" means a single-stranded,
partially single-stranded, partially double-stranded or
double-stranded nucleotide sequence capable of specifically
recognizing a selected non-oligonucleotide molecule or group of
molecules. In some embodiments, the aptamer recognizes the
non-oligonucleotide molecule or group of molecules by a mechanism
other than Watson-Crick base pairing or triplex formation. Aptamers
can include, without limitation, defined sequence segments and
sequences comprising nucleotides, ribonucleotides,
deoxyribonucleotides, nucleotide analogs, modified nucleotides and
nucleotides comprising backbone modifications, branchpoints and
normucleotide residues, groups or bridges. Methods for selecting
aptamers for binding to a molecule are widely known in the art and
easily accessible to one of ordinary skill in the art.
[0077] As used herein, the term "antibody" or "antibodies" refers
to an intact immunoglobulin or to a monoclonal or polyclonal
antigen-binding fragment with the Fc (crystallizable fragment)
region or FcRn binding fragment of the Fc region. The term
"antibodies" also includes "antibody-like molecules", such as
fragments of the antibodies, e.g., antigen-binding fragments.
Antigen-binding fragments can be produced by recombinant DNA
techniques or by enzymatic or chemical cleavage of intact
antibodies. "Antigen-binding fragments" include, inter alia, Fab,
Fab', F(ab')2, Fv, dAb, and complementarity determining region
(CDR) fragments, single-chain antibodies (scFv), single domain
antibodies, chimeric antibodies, diabodies, and polypeptides that
contain at least a portion of an immunoglobulin that is sufficient
to confer specific antigen binding to the polypeptide. Linear
antibodies are also included for the purposes described herein. The
terms Fab, Fc, pFc', F(ab') 2 and Fv are employed with standard
immunological meanings (Klein, Immunology (John Wiley, New York,
N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations of
Modern Immunology (Wiley & Sons, Inc., New York); and Roitt, I.
(1991) Essential Immunology, 7th Ed., (Blackwell Scientific
Publications, Oxford)). Antibodies or antigen-binding fragments
specific for various antigens are available commercially from
vendors such as R&D Systems, BD Biosciences, e-Biosciences and
Miltenyi, or can be raised against these cell-surface markers by
methods known to those skilled in the art.
[0078] As used herein, the term "Complementarity Determining
Regions" (CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino
acid residues of an antibody variable domain the presence of which
are necessary for antigen binding. Each variable domain typically
has three CDR regions identified as CDR1, CDR2 and CDR3. Each
complementarity determining region may comprise amino acid residues
from a "complementarity determining region" as defined by Kabat
(i.e. about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the
light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102
(H3) in the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (i.e. about residues 26-32
(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain
and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain
variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917
(1987)). In some instances, a complementarity determining region
can include amino acids from both a CDR region defined according to
Kabat and a hypervariable loop.
[0079] The expression "linear antibodies" refers to the antibodies
described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995).
Briefly, these antibodies comprise a pair of tandem Fd segments
(VH-CH1-VH-CH1) which, together with complementary light chain
polypeptides, form a pair of antigen binding regions. Linear
antibodies can be bispecific or monospecific.
[0080] The expression "single-chain Fv" or "scFv" antibody
fragments, as used herein, is intended to mean antibody fragments
that comprise the VH and VL domains of antibody, wherein these
domains are present in a single polypeptide chain. Preferably, the
Fv polypeptide further comprises a polypeptide linker between the
VH and VL domains which enables the scFv to form the desired
structure for antigen binding. (Pl{umlaut over (.upsilon.)}ckthun,
The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).
[0081] The term "diabodies," as used herein, refers to small
antibody fragments with two antigen-binding sites, which fragments
comprise a heavy-chain variable domain (VH) Connected to a
light-chain variable domain (VL) in the same polypeptide chain
(VH-VL). By using a linker that is too short to allow pairing
between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create
two antigen-binding sites. (EP 404,097; WO 93/11161; Hollinger et
ah, Proc. Natl. Acad. Sd. USA, P0:6444-6448 (1993)).
[0082] As used herein, the term "small molecules" refers to natural
or synthetic molecules including, but not limited to, peptides,
peptidomimetics, amino acids, amino acid analogs, polynucleotides,
polynucleotide analogs, aptamers, nucleotides, nucleotide analogs,
organic or inorganic compounds (i.e., including heteroorganic and
organometallic compounds) having a molecular weight less than about
10,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 5,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 1,000
grams per mole, organic or inorganic compounds having a molecular
weight less than about 500 grams per mole, and salts, esters, and
other pharmaceutically acceptable forms of such compounds.
[0083] The term "bacteria" as used herein is intended to encompass
all variants of bacteria, for example, prokaryotic organisms and
cyanobacteria. Bacteria are small (typical linear dimensions of
around 1 m), non-compartmentalized, with circular DNA and ribosomes
of 70S.
[0084] The term "antibiotics" is used herein to describe a compound
or composition which decreases the viability of a microorganism, or
which inhibits the growth or reproduction of a microorganism. As
used in this disclosure, an antibiotic is further intended to
include an antimicrobial, bacteriostatic, or bactericidal agent.
Exemplary antibiotics include, but are not limited to, penicillins,
cephalosporins, penems, carbapenems, monobactams, aminoglycosides,
sulfonamides, macrolides, tetracyclins, lincosides, quinolones,
chloramphenicol, vancomycin, metronidazole, rifampin, isoniazid,
spectinomycin, trimethoprim, sulfamethoxazole, and the like.
[0085] The term "cells" used herein refers to any cell, prokaryotic
or eukaryotic, including plant, yeast, worm, insect and mammalian.
Mammalian cells include, without limitation; primate, human and a
cell from any animal of interest, including without limitation;
mouse, hamster, rabbit, dog, cat, domestic animals, such as equine,
bovine, murine, ovine, canine, feline, etc. The cells may be a wide
variety of tissue types without limitation such as; hematopoietic,
neural, mesenchymal, cutaneous, mucosal, stromal, muscle spleen,
reticuloendothelial, epithelial, endothelial, hepatic, kidney,
gastrointestinal, pulmonary, T-cells etc. Stem cells, embryonic
stem (ES) cells, ES-derived cells and stem cell progenitors are
also included, including without limitation, hematopoeitic, neural,
stromal, muscle, cardiovascular, hepatic, pulmonary,
gastrointestinal stem cells, etc. Yeast cells can also be used as
cells in some embodiments. In some embodiments, the cells can be ex
vivo or cultured cells, e.g. in vitro. For example, for ex vivo
cells, cells can be obtained from a subject, where the subject is
healthy and/or affected with a disease. Cells can be obtained, as a
non-limiting example, by biopsy or other surgical means know to
those skilled in the art.
[0086] The term "photosynthetic and energy-harvesting compounds"
refers to molecules that can obtain or absorb energy from light,
e.g., chlorophyll.
[0087] As used herein, the term "viral vector" typically includes
foreign DNA which is desired to be inserted in a host cell and
usually includes an expression cassette. The foreign DNA can
comprise an entire transcription unit, promoter gene-poly A or the
vector can be engineered to contain promoter/transcription
termination sequences such that only the gene of interest need be
inserted. These types of control sequences are known in the art and
include promoters for transcription initiation, optionally with an
operator along with ribosome binding site sequences. Viral vectors
include, but are not limited to, lentivirus vectors, retroviral
vectors, lentiviral vectors, herpes simplex viral vectors,
adenoviral vectors, adeno-associated viral (AAV) vectors, EPV, EBV
or variants or derivatives thereof. Various companies produce such
viral vectors commercially, including, but not limited to, Avigen,
Inc. (Alameda, Calif.; AAV vectors), Cell Genesys (Foster City,
Calif.; retroviral, adenoviral, AAV, and lentiviral vectors),
Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon
Hill, Pa.; adenoviral and AAV vectors), Genvec (France; adenoviral
vectors), IntroGene (Leiden, Netherlands; adenoviral vectors),
Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral
vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,
United Kingdom; lentiviral vectors), and Transgene (Strasbourg,
France; adenoviral, vaccinia, retroviral, and lentiviral
vectors).
[0088] As used herein, the term "antigens" refers to a molecule or
a portion of a molecule capable of being bound by a selective
binding agent, such as an antibody, and additionally capable of
being used in an animal to elicit the production of antibodies
capable of binding to an epitope of that antigen. An antigen may
have one or more epitopes. The term "antigen" can also refer to a
molecule capable of being bound by an antibody or a T cell receptor
(TCR) if presented by MHC molecules. The term "antigen", as used
herein, also encompasses T-cell epitopes. An antigen is
additionally capable of being recognized by the immune system
and/or being capable of inducing a humoral immune response and/or
cellular immune response leading to the activation of B- and/or
T-lymphocytes. This may, however, require that, at least in certain
cases, the antigen contains or is linked to a Th cell epitope and
is given in adjuvant. An antigen can have one or more epitopes (B-
and T-epitopes). The specific reaction referred to above is meant
to indicate that the antigen will preferably react, typically in a
highly selective manner, with its corresponding antibody or TCR and
not with the multitude of other antibodies or TCRs which may be
evoked by other antigens. Antigens as used herein may also be
mixtures of several individual antigens.
[0089] As used herein, the term "viruses" refers to an infectious
agent composed of a nucleic acid encapsidated in a protein. Such
infectious agents are incapable of autonomous replication (i.e.,
replication requires the use of the host cell's machinery). Viral
genomes can be single-stranded (ss) or double-stranded (ds), RNA or
DNA, and can or cannot use reverse transcriptase (RT).
Additionally, ssRNA viruses can be either sense (+) or antisense
(-). Exemplary viruses include, but are not limited to, dsDNA
viruses (e.g. Adenoviruses, Herpesviruses, Poxviruses), ssDNA
viruses (e.g. Parvoviruses), dsRNA viruses (e.g. Reoviruses),
(+)ssRNA viruses (e.g. Picornaviruses, Togaviruses), (-)ssRNA
viruses (e.g. Orthomyxoviruses, Rhabdoviruses), ssRNA-RT viruses,
i.e., (+)sense RNA with DNA intermediate in life-cycle (e.g.
Retroviruses), and dsDNA-RT viruses (e.g. Hepadnaviruses). In some
embodiments, viruses can also include wild-type (natural) viruses,
killed viruses, live attenuated viruses, modified viruses,
recombinant viruses or any combinations thereof. Other examples of
viruses include, but are not limited to, enveloped viruses,
respiratory syncytial viruses, non-enveloped viruses,
bacteriophages, recombinant viruses, and viral vectors. The term
"bacteriophages" as used herein refers to viruses that infect
bacteria.
[0090] The term "anti-venom," as used herein, refers to a
biological product used in the treatment of venomous bites or
stings. The anti-venom is created by milking venom from the desired
snake, spider or insect. The venom is then diluted and injected
into a horse, sheep, goat or cat. The subject animal will undergo
an immune response to the venom, producing antibodies against the
venom's active molecule which can then be harvested from the
animal's blood and used to treat envenomation.
[0091] The term "therapeutic agents" is art-recognized and refers
to any chemical moiety that is a biologically, physiologically, or
pharmacologically active substance that acts locally or
systemically in a subject. Examples of therapeutic agents, also
referred to as "drugs", are described in well-known literature
references such as the Merck Index, the Physicians Desk Reference,
and The Pharmacological Basis of Therapeutics, and they include,
without limitation, medicaments; vitamins; mineral supplements;
substances used for the treatment, prevention, diagnosis, cure or
mitigation of a disease or illness; substances which affect the
structure or function of the body; or pro-drugs, which become
biologically active or more active after they have been placed in a
physiological environment. Various forms of a therapeutic agent may
be used which are capable of being released from the subject
composition into adjacent tissues or fluids upon administration to
a subject. Examples include steroids and esters of steroids (e.g.,
estrogen, progesterone, testosterone, androsterone, cholesterol,
norethindrone, digoxigenin, cholic acid, deoxycholic acid, and
chenodeoxycholic acid), boron-containing compounds (e.g.,
carborane), chemotherapeutic nucleotides, drugs (e.g., antibiotics,
antivirals, antifungals), enediynes (e.g., calicheamicins,
esperamicins, dynemicin, neocarzinostatin chromophore, and
kedarcidin chromophore), heavy metal complexes (e.g., cisplatin),
hormone antagonists (e.g., tamoxifen), non-specific (non-antibody)
proteins (e.g., sugar oligomers), oligonucleotides (e.g., antisense
oligonucleotides that bind to a target nucleic acid sequence (e.g.,
mRNA sequence)), peptides, proteins, antibodies, photodynamic
agents (e.g., rhodamine 123), radionuclides (e.g., I-131, Re-186,
Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-67 and
Cu-64), toxins (e.g., ricin), and transcription-based
pharmaceuticals.
[0092] A "diagnostic agent" is any chemical moiety that can be used
for diagnosis. For example, diagnostic agents include imaging
agents containing radioisotopes such as indium or technetium;
contrast agents or dyes containing iodine, gadolinium or cyanine;
enzymes such as horse radish peroxidase, GFP, alkaline phosphatase,
or .beta.-galactosidase; fluorescent substances such as europium
derivatives; luminescent substances such as N-methylacrydium
derivatives or the like.
Immunogen and Vaccines
[0093] In certain embodiments, the active agents are immunogens. In
some embodiments, the immunogen is a vaccine. As shown herein, a
model vaccine MMR live attenuated vaccine entrapped and
subsequently recovered from a silk carrier maintained significant
biological activity compared to non-silk-entrapped vaccine. In one
embodiment, provided herein is a stabilized MMR vaccine that can be
stored at ambient temperature for several weeks while maintaining a
substantial proportion of original activity. The stabilization of
live attenuated vaccines provides an important breakthrough in
immunization programs, lowering costs for transportation, equipment
and training; reducing wastage; and thus expanding immunization
programs.
[0094] The term "immunogen" refers to any substance, e.g.,
vaccines, capable of eliciting an immune response in an organism.
An "immunogen" is capable of inducing an immunological response
against itself on administration to a subject. The term
"immunological" as used herein with respect to an immunological
response, refers to the development of a humoral (antibody
mediated) and/or a cellular (mediated by antigen-specific T cells
or their secretion products) response directed against an immunogen
in a recipient subject. Such a response can be an active response
induced by administration of an immunogen or immunogenic peptide to
a subject or a passive response induced by administration of
antibody or primed T-cells that are directed towards the immunogen.
A cellular immune response is elicited by the presentation of
polypeptide epitopes in association with Class I or Class II MHC
molecules to activate antigen-specific CD4+ T helper cells and/or
CD8+ cytotoxic T cells. Such a response can also involve activation
of monocytes, macrophages, NK cells, basophils, dendritic cells,
astrocytes, microglia cells, eosinophils or other components of
innate immunity.
[0095] The term "immunogenicity" refers to the ability of a
substance, such as an antigen or epitope, to provoke humoral and/or
cell-mediated immunological response in a subject. A skilled
artisan can readily measure immunogenicity of a substance. The
presence of a cell-mediated immunological response can be
determined by any art-recognized methods, e.g., proliferation
assays (CD4+ T cells), CTL (cytotoxic T lymphocyte) assays (see
Burke, supra; Tigges, supra), or immunohistochemistry with tissue
section of a subject to determine the presence of activated cells
such as monocytes and macrophages after the administration of an
immunogen. One of skill in the art can readily determine the
presence of humoral-mediated immunological response in a subject by
any well-established methods. For example, the level of antibodies
produced in a biological sample such as blood can be measured by
western blot, ELISA or other methods known for antibody
detection.
[0096] Immunogens useful in some embodiments of various aspects
described herein include killed pathogens, live attenuated
pathogens, protein subunits and conjugates thereof, inactivated
toxins, and synthetic peptides, carbohydrates and conjugates
thereof, and antigens. The term "pathogen" as used herein means any
disease-producing agent (especially a virus or bacterium or other
microorganism).
[0097] The term "killed pathogens" is used herein in reference to
pathogen that were previously virulent (i.e. able to cause disease)
but have been destroyed with chemicals or heat. Examples of
vaccines comprising killed pathogens include, without limitations,
the influenza vaccine, cholera vaccine, bubonic plague vaccine,
polio vaccine, hepatitis A vaccine, and rabies vaccine.
[0098] The term "live attenuated pathogens" as used herein refers
to pathogens that have not been inactivated, i.e. pathogens capable
of replicating on permissive cells and inducing a specific
immunological response, but do not induce diseases caused by the
corresponding wild-type pathogens in a subject. Live attenuated
pathogens can be produced by one of skill in the art, e.g., by
cultivating wild-type pathogens under conditions that disable their
virulent properties, or using closely-related but less virulent
organisms to produce such an immunological response. Exemplary live
attenuated pathogens include, but are not limited to, the viral
diseases yellow fever, measles, rubella, and mumps and the
bacterial disease typhoid. In some embodiments, the live
Mycobacterium tuberculosis vaccine is not made of a contagious
strain, but contains a virulently modified strain called "BCG" used
to elicit an immune response to the vaccine. The live attenuated
vaccine containing strain Yersinia pestis EV is used for plague
immunization.
[0099] In some embodiments, an immunogen used in the compositions
described herein can be inactivated toxins that cause diseases
rather than the pathogen. Such non-limiting compositions include
tetanus and diphtheria. In some embodiments, while an immunogen can
comprise an inactivated compound, e.g., an inactivated toxin, from
a pathogen, synthetic peptides, carbohydrates, or antigens can also
be used as an immunogen in the immunogenic compositions described
herein.
[0100] In certain embodiments, an immunogen used in the
compositions described herein can include a protein subunit, i.e.,
a fragment of a killed or live attenuated pathogen, or a conjugate
thereof. Such exemplary examples include, without limitations, the
subunit vaccine against Hepatitis B virus that is composed of only
the surface proteins of the virus (previously extracted from the
blood serum of chronically infected patients, but now produced by
recombination of the viral genes into yeast), the virus-like
particle (VLP) vaccine against human papillomavirus (HPV) that is
composed of the viral major capsid protein, and the hemagglutinin
and neuraminidase subunits of the influenza virus. In such
embodiments, certain pathogens have polysaccharide outer coats that
are poorly immunogenic. By linking these outer coats to proteins
(e.g. toxins), the immune system can recognize the polysaccharide
as if it were a protein antigen. An exemplary conjugated immunogen
is the one used in Haemophilus influenzae type B vaccine.
Accordingly, conjugated immunogens are also included in the aspects
described herein.
[0101] Additional examples of immunogens includes the ones that can
be derived from hepatitis B virus, Haemophilus influenzae Type B,
poliovirus, Neisseria meningitides C, influenza, Varicella, or
Mycobacteria tuberculosis bacille Calmette-Guerin, tetanus toxoid,
diphtheria toxoid, or Bordetella pertussis. The immunogen can also
be a combination immunogen, such as DTaP, DTwP, DTwP hepB, DTP hep
B Hib, or DTaP hep B Hib IPV.
[0102] In some embodiments, the immunogen is a bacterium, such as
Mycobacteria tuberculosis bacille Calmette-Guerin or Bordetella
pertussis. The bacterial immunogen can be killed or attenuated. The
immunogen can comprise a bacterial subunit. Example immunogenic
bacterial subunits include those derived from Neisseria
meningitides type C, Haemophilus influenzae type B, Streptococcus
pneumoniae, Group B streptococcus, or Bordetella pertussis. The
bacterial immunogen can be recombinant. The bacterial subunit can
be, or include, a polysaccharide. In still other embodiments, the
immunogen is a viral subunit, for example, derived from Hepatitis B
virus or Human Papillomavirus. The viral immunogen can also be
recombinant. The viral immunogen can also comprise killed
virus.
[0103] The immunogen stabilized as described herein can be a
vaccine product, for example, BIOTHRAX.RTM. (anthrax vaccine
adsorbed, Emergent Biosolutions, Rockville, Md.); TICE.RTM. BCG
Live (Bacillus Calmette-Guerin for intravesical use, Organon Tekina
Corp. LLC, Durham, N.C.); MYCOBAX.RTM. BCG Live (Sanofi Pasteur
Inc); DAPTACEL.RTM. (diphtheria and tetanus toxoids and acellular
pertussis [DTaP] vaccine adsorbed, Sanofi Pasteur Inc.);
INFANRIX.RTM. (DTaP vaccine adsorbed, GlaxoSmithKline);
TRIPEDIA.RTM. (DTaP vaccine, Sanofi Pasteur); TRIHIBIT.RTM.
(DTaP/Hib#, sanofi pasteur); KINRIX.RTM. (diphtheria and tetanus
toxoids, acellular pertussis adsorbed and inactivated poliovirus
vaccine, GlaxoSmithKline); PEDIARIX.RTM. (DTaP-HepB-IPV,
GlaxoSmithKline); PENTACEL.RTM. (diphtheria and tetanus toxoids and
acellular pertussis adsorbed, inactivated poliovirus and
Haemophilus b conjugate [tetanus toxoid conjugate] vaccine, sanofi
pasteur); Diphtheria and Tetanus Toxoids, adsorbed (for pediatric
use, Sanofi Pasteur); DECAVAC.RTM. (diphtheria and tetanus toxoids
adsorbed, for adult use, Sanofi Pasteur); ACTHIB.RTM. (Haemophilus
b tetanus toxoid conjugate vaccine, Sanofi Pasteur); PEDVAXHIB.RTM.
(Hib vaccine, Merck); Hiberix (Haemophilus b tetanus toxoid
conjugate vaccine, booster dose, GlaxoSmithKline); COMVAX.RTM.
(Hepatitis B-Hib vaccine, Merck); HAVRIX.RTM. (Hepatitis A vaccine,
pediatric, GlaxoSmithKline); VAQTA.RTM. (Hepatitis A vaccine,
pediatric, Merck); ENGERIX-B.RTM. (Hep B, pediatric, adolescent,
GlaxoSmithKline); RECOMBIVAX HB.RTM. (hepatitis B vaccine, Merck);
TWINRIX.RTM. (HepA/HepB vaccine, 18 years and up, GlaxoSmithKline);
CERVARIX.RTM. (human papillomavirus bivalent [types 16 and 18]
vaccine, recombinant, GlaxoSmithKline); GARDASIL.RTM. (human
papillomavirus bivalent [types 6, 11, 16 and 18] vaccine,
recombinant, Merck); AFLURIA.RTM. (Influenza vaccine, 18 years and
up, CSL); AGRIFLU.TM. (influenza virus vaccine for intramuscular
injection, Novartis Vaccines); FLUARIX.RTM. (Influenza vaccine, 18
years and up, GlaxoSmithKline); FLULAVAL.RTM. (Influenza vaccine,
18 years and up, GlaxoSmithKline); FLUVIRIN.RTM. (Influenza
vaccine, 4 years and up, Novartis Vaccine); FLUZONE.RTM. (Influenza
vaccine, 6 months and up, Sanofi Pasteur); FLUMIST.RTM. (Influenza
vaccine, 2 years and up, MedImmune); IPOL.RTM. (e-IPV polio
vaccine, sanofi Pasteur); JE-VAX.RTM. (Japanese encephalitis virus
vaccine inactivated, BIKEN, Japan); IXIARO.RTM. (Japanese
encephalitis virus vaccine inactivated, Novarits); MENACTRA.RTM.
(Meningococcal [Groups A, C, Y and W-135] and diphtheria vaccine,
Sanofi Pasteur); MENOMUNE.RTM.-A/C/Y/W-135 (Meningococcal
polysaccharide vaccine, sanofi pasteur); MMRII.RTM. (MMR vaccine,
Merck); MENVEO.RTM. (Meningococcal [Groups A, C, Y and W-135]
oligosaccharide diphtheria CRM.sub.197 conjugate vaccine, Novartis
Vaccines); PROQUAD.RTM. (MMR and varicella vaccine, Merck);
PNEUMOVAX 23.RTM. (pneumococcal polysaccharide vaccine, Merck);
PREVNAR.RTM. (pneumococcal vaccine, 7-valent, Wyeth/Lederle);
PREVNAR-13.RTM. (pneumococcal vaccine, 13-valent, Wyeth/Lederle);
POLIOVAX.TM. (poliovirus inactivated, sanofi pasteur); IMOVAX.RTM.
(Rabies vaccine, Sanofi Pasteur); RABAVERT.TM. (Rabies vaccine,
Chiron); ROTATEQ.RTM. (Rotavirus vaccine, live, oral pentavalent,
Merck); ROTARIX.RTM. (Rotavirus, live, oral vaccine,
GlaxoSmithKline); DECAVAC.TM. (tetanus and diphtheria toxoids
vaccine, sanofi pasteur); Td (generic) (tetanus and diphtheria
toxoids, adsorbed, Massachusetts Biol. Labs); TYPHIMVI.RTM.
(typhoid Vi polysaccharide vaccine, Sanofi Pasteur); ADACEL.RTM.
(tetanus toxoid, reduced diphtheria toxoid and acellular pertussis,
sanofi pasteur); BOOSTRIX.RTM. (tetanus toxoid, reduced diphtheria
toxoid and acellular pertussis, GlaxoSmithKline); VIVOTIF.RTM.
(typhoid vaccine live oral Ty21a, Berna Biotech); ACAM2000.TM.
(Smallpox (vaccinia) vaccine, live, Acambis, Inc.); DRYVAX.RTM.
(Smallpox (vaccinia) vaccine); VARIVAX.RTM. (varicella [live]
vaccine, Merck); YF-VAX.RTM. (Yellow fever vaccine, Sanofi
Pasteur); ZOSTAVAX.RTM. (Varicella zoster, Merck); or combinations
thereof. Any vaccine products listed in database of Center for
Disease Control and Prevention (CDC) can also be included in the
compositions described herein.
[0104] In some embodiments, animal vaccines such as canine and
feline vaccines can also be included in the methods and
compositions described herein. Examples of animal vaccines include,
but are not limited to, DURAMUNE.RTM. MAX 5 (5-way vaccine: Canine
Distemper, Infectious Canine Hepatitis, Adenovirus Type 2,
Parainfluenza, and Parvovirus, Fort Dodge); NEO PAR.RTM.
(parvovirus, Neo Tech); VANGUARD.RTM. PLUS 5 (Canine Distemper,
Adenovirus Type 1 and 2, Parainfluenza and Parvovirus; Pfizer);
BRONCHI-SHIELD.RTM. III (Canine Parainfluenza; Fort Dodge); and
ECLIPSE.RTM. 4 (feline rhinotracheitis, calici, and panleukopenia
viruses and Chlamydia psittaci, Schering-Plough/Intervet). Any
commercially available animal vaccines can be included in the
compositions described herein.
Live Attenuated Virus
[0105] Live attenuated immunogenic compositions, e.g., live
attenuated vaccines, can generally provoke more durable
immunological responses. Thus, they are sometimes the preferred
compositions for administration to a subject, e.g., a healthy
mammalian. In some embodiments, the immunogens used in the
compositions described herein is live, attenuated pathogens. In
particular embodiments, the immunogens are live attenuated viruses.
Accordingly, methods and immunogenic compositions comprising at
least one live attenuated virus (including at least two live
attenuated viruses, at least three live attenuated viruses, or
more) are also described herein. The immunogenic compositions
include a silk-fibroin matrix and at least one live attenuated
virus (including at least two live attenuated viruses, at least
three live attenuated viruses, or more) distributed therein,
wherein the live attenuated virus(es) retains at least about 30% of
its original infectivity when the composition is (a) subjected to
at least one state-changing cycle, and/or (b) maintained for a
period of time under a specified condition. In some embodiments,
the live attenuated virus(es) can retain at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90%, at least about
95% of original infectivity or higher.
[0106] As used herein, the term "infectivity" in reference to a
virus means the characteristic of a virus that embodies capability
of entering, surviving in, and multiplying or causing an
immunological response in a susceptible host. Any methods known to
a skilled artisan for determination of virus infectivity can be
used for the purposes described herein, e.g., the in vitro
infectivity assay described in Example 1 can be employed.
[0107] In particular embodiments, the live attenuated virus can be
an enveloped virus such as Paramyxoviridae, Togaviridae,
Orthomyxoviridae, Flaviviridae, Herpesviridae, Rhabdovirus, or
Retroviridae. These enveloped, live, attenuated virus can be
varicella, measles virus, mumps virus, German measles virus,
respiratory syncytial virus, yellow fever virus, or influenza
virus. By "enveloped virus" is meant a virus comprising a
lipid-containing or lipoprotein-containing membrane which surrounds
their protein capsids. These viral envelopes can be derived from
portions of the host cell membranes (phospholipids and proteins),
but include some viral glycoproteins. Functionally, viral envelopes
can be used to help viruses enter host cells. For example,
glycoproteins on the surface of the envelope serve to identify and
bind to receptor sites on the host's membrane. The viral envelope
then fuses with the host's membrane, allowing the capsid and viral
genome to enter and infect the host. However, as the viral envelope
is relatively sensitive to desiccation, heat and detergents, these
enveloped viruses can be sterilized more easily than non-enveloped
viruses, and thus have limited survival outside host environments.
Accordingly, methods and immunogenic compositions provided herein
are of particular importance to maintain the survival of live
attenuated enveloped virus outside host environments and thus their
infectivity once introduced into a host cell.
[0108] In other embodiments, the live, attenuated virus can be a
non-enveloped virus, i.e. a virus with no viral envelop as
described above. The non-enveloped virus can be rotavirus,
reovirus, hepatitis virus, rabies virus and/or poliovirus.
[0109] Further provided herein is a cell-free, stabilized virus
preparation comprising a silk fibroin matrix and infective virus
distributed, mixed or embedded therein, wherein the virus retains
at least about 30% of its original infectivity when the preparation
is (a) subjected to at least one state-changing cycle, and/or (b)
is maintained for a period of time under a condition specified
herein.
Additives and Pharmaceutically-Acceptable Carriers
[0110] Various embodiments of the compositions described herein can
further comprise an additive distributed, mixed or embedded in the
silk fibroin matrix. In some embodiments, the additive is a
stabilizing agent. The addition of "stabilizing agent" to the
compositions described herein can further increase the stability of
the active agent, i.e., the active agent can retain a higher
bioactivity, relative to the bioactivity in the absence of the
stabilizing agent. In some embodiments, the stabilizing agent is
selected from the group consisting of a saccharide, a sugar
alcohol, an ion, a surfactant, and any combinations thereof. In one
embodiment, the saccacharide, e.g., sucrose, is added into the
compositions described herein.
[0111] By way of example, additional stabilizing agents can be
added to the silk fibroin solution or matrix. Example stabilizers
previously shown to be effective on oral polio vaccine as well as
those discussed herein can be used. Stabilizing agents may include
cationic stabilizers (listed most to least stabilizing):
(CH.sub.3).sub.4N.sup.+>Me.sup.2+, K.sup.+>Na.sup.+,
NH.sup.4+>Li.sup.+; anionic stabilizers (most to least
stabilizing): CH.sub.3COO.sup.-, SO.sup.4-,
PO.sub.4.sup.2->Cl.sup.-, SCN.sup.-; and heavy water (D.sub.2O)
(Dorval et al, 1989). See, e.g., Mirchamsy et al., Stabilizing
effect of magnesium chloride and sucrose on Sabin live polio
vaccine, 41 Devel. Biol. Standardization 255 (1978); Rapp et al.,
Protection of measles virus by sulfate ions against thermal
inactivation, 90 J. Bact. 132 (1965). Other stabilizing agents
known in the art, e.g., for stabilizing other vaccines, can also be
included in the compositions described herein, for example, amino
acids, such as sodium glutamate, arginine, lysine, and cysteine;
monosaccharides, such as glucose, galactose, fructose, and mannose;
disaccharides, such as sucrose, maltose, and lactose; sugar
alcohols such as sorbitol and mannitol; polysaccharides, such as
oligosaccharide, starch, cellulose, and derivatives thereof; human
serum albumin and bovine serum albumin; gelatin, and gelatin
derivatives, such as hydrolyzed gelatin; and ascorbic acid as an
antioxidant. These materials are described in publications, e.g.,
"Toketsu-Kanso To Hogo Busshitsu (Lyophilization And Protective
Materials)" written by Nei, p. 1-176, published by Tokyo Daigaku
Shuppan Kai (Publishing Association of the University of Tokyo),
Japan in 1972; and "Shinku Gijutsu Koza (8): Sinku Kanso (Lecture
on Vacuum Technology (8): Vacuum Drying)" written by Ota et al., p
176-182, published by Nikkan Kogyo Shimbun Co., Ltd., Japan in
1964.
[0112] In some embodiments, the compositions or preparations
described herein can further comprise a pharmaceutically acceptable
carrier. Depending on the selected administration route, the
compositions or preparations can be in any form, e.g., a tablet, a
lozenge, a suspension, a free-flowing powder, an aerosol, and a
capsule. The term "pharmaceutically acceptable," as used herein,
refers to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0113] As used herein, the term "pharmaceutically acceptable
carrier" refers to a pharmaceutically-acceptable material,
composition or vehicle for administration of an active agent
described herein. Pharmaceutically acceptable carriers include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like which are compatible with the activity of the active agent and
are physiologically acceptable to the subject. Some examples of
materials which can serve as pharmaceutically-acceptable carriers
include: (i) sugars, such as lactose, glucose and sucrose; (ii)
starches, such as corn starch and potato starch; (iii) cellulose,
and its derivatives, such as sodium carboxymethyl cellulose,
methylcellulose, ethyl cellulose, microcrystalline cellulose and
cellulose acetate; (iv) powdered tragacanth; (v) malt; (vi)
gelatin; (vii) lubricating agents, such as magnesium stearate,
sodium lauryl sulfate and talc; (viii) excipients, such as cocoa
butter and suppository waxes; (ix) oils, such as peanut oil,
cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and
soybean oil; (x) glycols, such as propylene glycol; (xi) polyols,
such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG);
(xii) esters, such as ethyl oleate and ethyl laurate; (xiii) agar;
(xiv) buffering agents, such as magnesium hydroxide and aluminum
hydroxide; (xv) alginic acid; (xvi) pyrogen-free water; (xvii)
isotonic saline; (xviii) Ringer's solution; (xix) ethyl alcohol;
(xx) pH buffered solutions; (xxi) polyesters, polycarbonates and/or
polyanhydrides; (xxii) bulking agents, such as polypeptides and
amino acids (xxiii) serum component, such as serum albumin, HDL and
LDL; (xxiv) C2-C12 alcohols, such as ethanol; and (xxv) other
non-toxic compatible substances employed in pharmaceutical
formulations. Wetting agents, coloring agents, release agents,
coating agents, sweetening agents, flavoring agents, perfuming
agents, preservative and antioxidants can also be present in the
formulation. For compositions or preparations described herein to
be administered orally, pharmaceutically acceptable carriers
include, but are not limited to pharmaceutically acceptable
excipients such as inert diluents, disintegrating agents, binding
agents, lubricating agents, sweetening agents, flavoring agents,
coloring agents and preservatives. Suitable inert diluents include
sodium and calcium carbonate, sodium and calcium phosphate, and
lactose, while corn starch and alginic acid are suitable
disintegrating agents. Binding agents may include starch and
gelatin, while the lubricating agent, if present, will generally be
magnesium stearate, stearic acid or talc. If desired, the tablets
may be coated with a material such as glyceryl monostearate or
glyceryl distearate, to delay absorption in the gastrointestinal
tract.
[0114] Pharmaceutically acceptable carriers can vary in a
preparation described herein, depending on the administration route
and formulation. The compositions and preparations described herein
can be delivered via any administration mode known to a skilled
practitioner. For example, the compositions and preparations
described herein can be delivered in a systemic manner, via
administration routes such as, but not limited to, oral, and
parenteral including intravenous, intramuscular, intraperitoneal,
intradermal, and subcutaneous. In some embodiments, the
compositions and preparations described herein are in a form that
is suitable for injection. In other embodiments, the compositions
and preparations described herein are formulated for oral
administration.
[0115] When administering parenterally, a composition and
preparation described herein can be generally formulated in a unit
dosage injectable form (solution, suspension, emulsion). The
compositions and preparations suitable for injection include
sterile aqueous solutions or dispersions. The carrier can be a
solvent or dispersing medium containing, for example, water, cell
culture medium, buffers (e.g., phosphate buffered saline), polyol
(for example, glycerol, propylene glycol, liquid polyethylene
glycol, and the like), suitable mixtures thereof. In some
embodiments, the pharmaceutical carrier can be a buffered solution
(e.g. PBS).
[0116] An oral composition can be prepared in any orally acceptable
dosage form including, but not limited to, tablets, capsules,
emulsions and aqueous suspensions, dispersions and solutions.
Commonly used carriers for tablets include lactose and corn starch.
Lubricating agents, such as magnesium stearate, are also typically
added to tablets. For oral administration in a capsule form, useful
diluents include lactose and dried corn starch. When aqueous
suspensions or emulsions are administered orally, the active
ingredient can be suspended or dissolved in an oily phase combined
with emulsifying or suspending agents. If desired, certain
sweetening, flavoring, or coloring agents can be added. Liquid
preparations for oral administration can also be prepared in the
form of a dry powder to be reconstituted with a suitable solvent
prior to use.
[0117] The compositions can also contain auxiliary substances such
as wetting or emulsifying agents, pH buffering agents, gelling or
viscosity enhancing additives, preservatives, colors, and the like,
depending upon the route of administration and the preparation
desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL
SCIENCE", 17th edition, 1985, incorporated herein by reference, may
be consulted to prepare suitable preparations, without undue
experimentation. With respect to compositions described herein,
however, any vehicle, diluent, or additive used should have to be
biocompatible with the active agents described herein. Those
skilled in the art will recognize that the components of the
compositions should be selected to be biocompatible with respect to
the active agent. This will present no problem to those skilled in
chemical and pharmaceutical principles, or problems can be readily
avoided by reference to standard texts or by simple experiments
(not involving undue experimentation).
[0118] In some embodiments, the compositions and preparations
described herein can be formulated in an emulsion or a gel. Such
gel compositions and preparations can be implanted locally to a
diseased tissue region of a subject.
[0119] For in vivo administration, the compositions or preparations
described herein can be administered with a delivery device, e.g.,
a syringe. Accordingly, an additional aspect described herein
provides for delivery devices comprising at least one chamber with
an outlet, wherein the at least one chamber comprises a
pre-determined amount of any composition described herein and the
outlet provides an exit for the composition enclosed inside the
chamber. In some embodiments, a delivery device described herein
can further comprise an actuator to control release of the
composition through the outlet. Such delivery device can be any
device to facilitate the administration of any composition
described herein to a subject, e.g., a syringe, a dry powder
injector, a nasal spray, a nebulizer, or an implant such as a
microchip, e.g., for sustained-release or controlled release of any
composition described herein.
[0120] In some embodiments of the compositions described herein,
the silk fibroin matrix itself can be modified to control its
degradation and thus the release of active agents, e.g. such that
release occurs over a period of time ranging from hours to days, or
months. In some embodiments, the compositions described herein can
be combined with other types of delivery systems available and
known to those of ordinary skill in the art. They include, for
example, polymer-based systems such as polylactic and/or
polyglycolic acids, polyanhydrides, polycaprolactones,
copolyoxalates, polyesteramides, polyorthoesters,
polyhydroxybutyric acid, and/or combinations thereof. Microcapsules
of the foregoing polymers containing drugs are described in, for
example, U.S. Pat. No. 5,075,109. Other examples include nonpolymer
systems that are lipid-based including sterols such as cholesterol,
cholesterol esters, and fatty acids or neuka1 fats such as mono-,
di- and triglycerides; hydrogel release systems; liposome-based
systems; phospholipid based-systems; silastic systems; peptide
based systems; or partially fused implants. Specific examples
include, but are not limited to, erosional systems in which the
composition is contained in a form within a matrix (for example, as
described in U.S. Pat. Nos. 4,452,775, 4,675,189, 5,736,152,
4,667,014, 4,748,034 and -29 5,239,660), or diffusional systems in
which an active component controls the release rate (for example,
as described in U.S. Pat. Nos. 3,832,253, 3,854,480, 5,133,974 and
5,407,686). The formulation may be as, for example, microspheres,
hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric
systems. In some embodiments, the system may allow sustained or
controlled release of the composition to occur, for example,
through control of the diffusion or erosion/degradation rate of the
formulation containing the composition. In addition, a pump-based
hardware delivery system can be used to deliver one or more
embodiments of the compositions or preparations described herein.
Use of a long-term sustained release formulations or implants can
be particularly suitable for treatment of chronic conditions, such
as diabetes. Long-term release, as used herein, means that a
formulation or an implant is made and arranged to deliver
compositions or preparations described herein at a therapeutic
level for at least 30 days, or at least 60 days. In some
embodiments, the long-term release refers to a formulation or an
implant being configured to deliver an active agent at a
therapeutic level over several months.
Methods for Preparing a Storage-Stable Composition
[0121] Provided are methods for preparing storage-stable
compositions as described herein. In some embodiments, the
storage-stable compositions are immunogenic. The method includes
providing or obtaining a silk fibroin matrix comprising at least
one active agent, in which the at least one active agent retains at
least about 30% of its original bioactivity upon storage or
transported for a period of time under a specified condition. In
some embodiments, the method further comprises mixing, or adding at
least one active agent in a silk fibroin matrix. In some
embodiments, the method further comprises drying the silk fibroin
matrix comprising at least one active agent to form a solid-state
silk fibroin, in which the at least one active agent retains at
least about 30% of its original bioactivity upon storage or
transported for a period of time under a specified condition. In
these embodiments, the silk fibroin matrix can be a solution or a
gel-like solution. The silk fibroin matrix containing at least one
active agent can be dried in air or nitrogen, or by lyophilization.
In one embodiment, the silk fibroin matrix (e.g., silk solution)
containing at least one active agent can be subjected to
lyophilization to form lyophilized solid-state silk fibroin loaded
with the active agent, in which the at least one active agent
retains at least about 60%, at least about 70%, or at least about
80% of its original bioactivity (e,g, viral potency, see e.g.
Example 3) upon storage or transport for a period of time (e.g. for
at least 6 months, or for up to 6 months) under a specified
condition (e.g. storage or transport at 37.degree. C., at
45.degree. C., or at greater than at 45.degree. C.).
[0122] In some embodiments, the method can further comprise
lyophilization of the solid-state or dried silk fibroin comprising
at least one active agent, e.g., to further decrease residual
moisture of the composition, in which the at least one active agent
retains at least about 60%, at least about 70%, or at least about
80% of its original bioactivity (e,g, viral potency, see e.g.
Example 3) upon storage or transport for a period of time (e.g. for
at least 6 months, or for up to 6 months) under a specified
condition (e.g. storage or transport at 37.degree. C., at
45.degree. C., or at greater than at 45.degree. C.).
[0123] In one embodiment, the method of producing a solid-state
storage-stable composition includes (a) providing or obtaining a
silk fibroin matrix comprising at least one active agent; and (b)
drying the silk fibroin matrix comprising the at least one active
agent to form a solid-state silk fibroin, in which the at least one
active agent retains at least about 30% of its original bioactivity
upon storage or transported for a period of time under a specified
condition. In some embodiments, the method further comprises step
(c) of lyophilizing the solid state silk-fibroin of step (b), e.g.
to retain at least about 60%, at least about 70%, or at least about
80% original bioactivity of the active agent at temperatures above
0.degree. C., e.g. greater than 30.degree. C., greater than
37.degree. C., greater than 40.degree. C. In some embodiments, the
solid state silk-fibroin of step (b) is subjected to a post
treatment, e.g. treatment with methanol, ethanol, shear stress,
electric filed, pressure etc., prior to the lyophilization of step
(c). In some embodiments the active agent is an immunogenic
composition. In one embodiment, the immunogenic composition
comprises a monovalent vaccine. In another embodiment, the
immunogenic composition comprises a multivalent or polyvalent
vaccine, e.g., a divalent vaccine or a trivalent vaccine.
[0124] As used herein, the term "a monovalent vaccine" refers to a
vaccine that is designed to immunize against a single antigen or
single microorganism.
[0125] As used herein, the term "a multivalent or polyvalent
vaccine" refers to a vaccine that is designed to immunize against
two or more different strains of a microorganism, or against two or
more different microorganisms. For example, a divalent vaccine is
generally a vaccine that is designed to immunize against two
different strains of a microorganism or against two different
microorganisms. A trivalent vaccine is generally a vaccine that is
designed to immunize against three different strains of a
microorganism or against three different microorganisms. An
exemplary trivalent vaccine is a vaccine that is designed to
immunize against measles, mumps, and rubella.
[0126] Without wishing to be bound by theory, silk can prevent the
virus proteins from undergoing heat-induced aggregation and/or
raise the glass-transition temperature of the vaccine (or melting
point of a viral protein), thus maintaining infectivity at elevated
temperatures. Accordingly, in some embodiments, the method of
preparing an immunogenic composition described herein can be
employed for decreasing the likelihood of or preventing viral
protein aggregation at a temperature at which a virus would
otherwise aggregate in the absence of silk matrix. For example, in
some embodiments, the method of preparing an immunogenic
composition described herein can be used to decrease the likelihood
of viral protein aggregation by at least about 10%, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, at least about 95% or more, as compared to an immunogen
without the silk fibroin matrix. In some embodiments, viral protein
aggregation in an immunogenic composition described herein can be
reduced by at least about 1.5 fold, at least about 2-fold, at least
about 3-fold, at least about 4-fold, at least about 5-fold, or
more, as compared to an immunogen without the silk fibroin matrix.
The viral protein aggregation can be determined, e.g., by measuring
the effective diameter of viral particles using dynamic light
scattering as shown in Example 3.
[0127] Stated another way, in some embodiments, the methods of
preparing an immunogenic composition described herein can be
employed for increasing the viral protein aggregation temperature
by at least about 10.degree. C., at least about 20.degree. C., at
least about 30.degree. C., at least about 40.degree. C., at least
about 50.degree. C., at least about 60.degree. C., at least about
70.degree. C., at least about 80.degree. C., at least about
90.degree. C., at least about 100.degree. C., or higher, as
compared to an immunogen without the silk fibroin matrix. The viral
protein aggregation temperature can be determined, e.g., by
measuring the effective diameter of viral particles over a range of
temperatures using dynamic light scattering as shown in Example 3.
The temperature at which the effective diameter of viral particles
begins to increase can be the viral protein aggregation
temperature.
[0128] In some embodiments, the methods of preparing an immunogenic
composition described herein can be employed for increasing the
glass-transition temperature and/or melting point of a vaccine by
at least about 10.degree. C., at least about 20.degree. C., at
least about 30.degree. C., at least about 40.degree. C., at least
about 50.degree. C., at least about 60.degree. C., at least about
70.degree. C., at least about 80.degree. C., at least about
90.degree. C., at least about 100.degree. C., at least about
125.degree. C., at least about 150.degree. C. or higher, as
compared to an immunogen without the silk fibroin matrix. The
glass-transition temperature and/or melting point of a vaccine can
be determined, e.g., by differential scanning calorimetry as shown
in Example 3.
[0129] The aqueous silk fibroin solution used for making a
solid-state silk fibroin can be prepared using techniques known in
the art. The concentration of silk fibroin in solutions used to
embed or carry active agent can be suited to the particular active
agent. Any concentration of silk fibroin solution may be used. In
one embodiment, e.g. for vaccine stabilization, the concentrations
of silk may be at least about 2%, at least about 4%, at least about
5%, at least about 6%, at least about 7%, at least about 8%, at
least about 9%, at least about 10%, at least about 12%, at least
about 14%, at least about 15%, at least about 16%, at least about
18%, or at least about 20% (w/v), inclusive. Suitable processes for
preparing silk fibroin solution are disclosed, for example, in U.S.
patent application Ser. No. 11/247,358; WO/2005/012606; and
WO/2008/127401. The silk aqueous solution can then be processed
into silk matrix such as silk films, conformal coatings or layers,
or 3-dimensional scaffolds, or electrospun fibers for further
processing into the silk reflectors. A micro-filtration step may be
used herein. For example, the prepared silk fibroin solution may be
processed further by centrifugation and syringe based
micro-filtration before further processing into silk matrix.
[0130] Additional polymers, e.g., biocompatible and biodegradable
polymers, can also be blended in the silk fibroin. For example,
additional biopolymers, such as chitosan, exhibit desirable
mechanical properties, can be processed in water, blended with silk
fibroin, and form generally films. Other biopolymers, such as
chitosan, collagen, gelatin, agarose, chitin,
polyhydroxyalkanoates, pullan, starch (amylose amylopectin),
cellulose, alginate, fibronectin, keratin, hyaluronic acid, pectin,
polyaspartic acid, polylysin, pectin, dextrans, and related
biopolymers, or a combination thereof, may be utilized in specific
applications, and synthetic biodegradable polymers such as
polyethylene oxide, polyethylene glycol, polylactic acid,
polyglycolic acid, polycaprolactone, polyorthoester,
polycaprolactone, polyfumarate, polyanhydrides, and related
copolymers may also be selectively used.
[0131] A silk fibroin matrix can be in solution or in a solid
state. The solid-state silk fibroin matrix can be in any material
format, such as silk fibers, electrospun fibers, films, mats, 3-D
scaffolds, dried gels, spheres (including microsphere and/or
nanospheres), particles or composites of one or more different
formats of silk materials, as described herein. In other
embodiments, the solid-state silk fibroin is a particle.
[0132] In one embodiment, the solid-state silk fibroin is a silk
film. For example, a silk fibroin film can be prepared by
depositing an aqueous silk fibroin-containing solution (e.g., silk
concentration of about 3% (w/v) to about 30% (w/v), or about 5%
(w/v) to about 15% (w/v)) on a support substrate and allowing the
silk fibroin solution to dry into a film. In this regard, the
substrate coated with silk fibroin-based solution may be exposed in
air for a period of time, such as 12 hours. Depositing the silk
fibroin solution can be performed by, e.g., using a spin coating
method, where the silk fibroin solution is spin coated onto the
substrate to allow the fabrication of thin membranes of non-uniform
in height; or simply by pouring silk fibroin solution over the top
of the substrate. The properties of the silk fibroin film, such as
thickness and content of other components, may be altered based on
the concentration and/or the volume of the silk fibroin solution
applied to the substrate, and the techniques used for processing
the silk fibroin solution into silk film. For instance, the
thickness of the silk film may be controlled by changing the
concentration of the silk fibroin in the solution, or by using
desired volumes of silk fibroin solution, resulting silk fibroin
film with a thickness ranging from approximately 2 nm to 1 mm
thick. In one embodiment, one can spin coat the silk fibroin onto a
substrate to create films having thickness from about 2 nm to about
100 .mu.m using various concentrations of silk fibroin and spinning
speeds.
[0133] In some embodiments, instead of drying the silk fibroin
solution containing one or more active agents (e.g., immunogens) in
gas such air or nitrogen, the active-agent containing silk fibroin
solution can be subjected to lyophilization to form lyophilized
silk fibroin matrix, e.g., lyophilized silk fibroin film.
Subjecting the silk fibroin solution containing one or more active
agents (e.g., immunogens such as vaccines) to lyophilization for
drying not only improve the initial recovery of the active agent
(e.g., immunogens such as vaccines) during the fabrication process,
but it also surprisingly provides greater stabilization of the
active agent (e.g., immunoagens such as vaccines) at elevated
temperatures (e.g., at room temperature or above, or 37.degree. C.
or above, or at 45.degree. C. or above) for an extended period of
time, e.g., for at least about 1 weeks, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months or longer.
[0134] In some embodiments, the solid-state silk fibroin can be a
composite of one or more layers of silk fibroin. Each layer of silk
fibroin can possess the same or different composition or
properties. For instance, each layer of silk fibroin can possess
the same or different concentration of silk fibroin, and/or each
layer can possess the same or different mechanical and/or
degradation properties. In one embodiment, the solid-state silk
fibroin can be a multi-layered silk fibroin, e.g., which can be
tuned to reflect specific wavelengths.
[0135] In some embodiments, the solid-state silk fibroin can be a
silk hydrogel. Methods for making a silk hydrogel are known in the
art. For example, a silk hydrogel can be produced by applying a
shear stress to a silk fibroin solution (comprising one or more
active agents such as immunogens, and silk fibroin at a
concentration of about 0.5% (w/v) to about 20% (w/v), or about 1%
(w/v) to about 15% (w/v), or about 2% (w/v) to about 10% (w/v)). In
such embodiments, the weight ratio of the active agent(s) (e.g.,
immunogen(s)) to silk solution can range from about 1:10 to about
10:1. In one embodiment, the weight ratio of the active agent(s)
(e.g., immunogen(s)) to silk solution can be around 1:1. See, e.g.,
International App. No.: WO 2011/005381, the content of which is
incorporated herein by reference for methods of producing
vortex-induced silk fibroin gelation for encapsulation and
delivery. Without limitations, other methods for making a silk
hydrogel with one or more active agents such as immunogens
distributed herein can also be used, such as by sonication (e.g.,
U.S. Pat. App. No. U.S. 2010/0178304 and International App. No.: WO
2008/150861), or by pH adjustment (e.g., U.S. App. No.: US
2011/0171239). The contents of those patent applications are
incorporated herein by reference.
[0136] In some embodiments, the solid-state silk fibroin can
include a silk microsphere. Various methods of producing silk
microspheres or nanospheres are known in the art. In some
embodiments, the silk microparticles or nanoparticles can be
produced by a polyvinyl alcohol (PVA) phase separation method as
described in, e.g., International App. No. WO 2011/041395, the
content of which is incorporated herein by reference. In such
embodiments, the silk concentration used in the PVA phase
separation method can range from about 0.5% (w/v) to about 20%
(w/v), or about 1% (w/v) to about 15% (w/v), or about 3% (w/v) to
about 10% (w/v). In one embodiment, the silk concentration used in
the PVA phase separation method can be about 5% (w/v). In some
embodiments, the weight ratio of active agent(s) (e.g.,
immunogen(s)) to silk solution can be about 1:300 to about 1:2000,
or about 1:500 to about 1:1500. In one embodiment, the weight ratio
of active agent(s) (e.g., immunogen(s)) to silk solution can be
about 1:1000. Other methods for producing silk microspheres or
nanospheres, e.g., described in U.S. App. No. U.S. 2010/0028451 and
International App. No.: WO 2008/118133 (using lipid as a template
for making silk microspheres or nanospheres), and in Wenk et al. J
Control Release 2008; 132: 26-34 (using spraying method to produce
silk microspheres or nanospheres) can be used for the purpose of
making silk microparticles or nanoparticles encapsulating an active
agent such as an immunogen described herein.
[0137] In some embodiments, the silk microspheres or nanospheres
can be further embedded in a biopolymer, e.g., to prolong the
release of an active agent such as an immunogen over a period of
time. In some embodiments, the biopolymer can be a silk hydrogel to
encapsulate the active agent (e.g., immunogen)-loaded silk
microspheres or nanospheres. See, e.g., International App. No.: WO
2010/141133 for methods of producing silk fibroin scaffolds for
antibiotic delivery.
[0138] In some embodiments, the solid-state silk fibroin
compositions (storage-stable compositions described herein) can be
subjected to post-treatment, e.g., to modify the degradation rate
of the silk fibroin. Additional treatment can include, but are not
limited to, organic solvent treatment, mechanical treatment, or
electromagnetic treatment. By way of example, the degradation rate
of the silk fibroin can be controlled, e.g., by modifying the
amount of beta-sheet crystal, and/or crystal orientation.
Accordingly, the amount of beta-sheet crystal, and/or crystal
orientation in a silk fibroin can be controlled by contacting the
silk fibroin with alcohol, e.g., methanol or ethanol, as
established in the art. In some embodiments, the silk fibroin can
be subjected to a mechanical force, e.g., stretching or shear
stress to vary the amount beta-sheet crystal, and/or alignment of
the crystal orientation. In some embodiments, the silk fibroin can
be subjected to an electric filed or pressure. In some embodiments,
the silk fibroin can be contacted with salt.
[0139] Without wishing to be bound by theory, the release rate of
an active agent from a silk fibroin matrix can be controlled by the
content of beta-sheet crystalline structures, silk concentration
and/or porosity of the silk fibroin matrix. Methods for forming
pores in a silk matrix are known in the art, e.g., porogen-leaching
method, freeze-drying method, and/or gas-forming method. Such
methods are described, e.g., in U.S. Pat. App. Nos.: US
2010/0279112, US 2010/0279112, and U.S. Pat. No. 7,842,780, the
contents of which are incorporated herein by reference.
[0140] In some embodiments, the methods of preparing storage-stable
compositions described herein can further comprise reducing the
dried solid-state silk fibroin by a mechanical means to obtain
micronized particles as defined herein. Exemplary mechanical means
to obtain micronized particles include micronizing, pulverizing,
crushing, grinding, freeze-drying or any combination thereof.
[0141] In accordance with the conventional practice, the
compositions described herein are desirably processed under aseptic
conditions using components which preliminarily have been rendered
bacterially sterile. Sterility on storage can be maintained by
incorporation of an antigen-compatible germicidal substance such as
thimerosal.
Kits and Devices
[0142] Packages and kits comprising at least one storage-stable
composition or preparation are also described herein. The packages
can be prepared in various types of containers, which can be
selected from the group consisting of a vial, an ampule, a capsule,
a tube, a delivery device, a bottle, and a packet. In some
embodiments, the delivery device is a syringe. In some embodiments,
the syringe can be needleless. The storage-stable composition
contained in a package can be in a form of a hydrogel, gel-like
particles, powder, microspheres, nanospheres, or any combinations
thereof. In some embodiments, the storage-stable composition
contained in a package can be lyophilized. In some embodiments, the
storage-stable composition can be loaded in a syringe for
injection.
[0143] Kits provided herein comprise a package described herein,
and a pharmaceutically acceptable solution, e.g., PBS. In some
embodiments, the kits can further comprise at least one delivery
device for administering a composition or a preparation described
herein to a subject. In other embodiments, the kits can further
comprise a disinfectant. In certain embodiments, such packages, and
kits described herein can be used for vaccination purposes.
[0144] As used herein, a "subject" means a human or animal. Usually
the animal is a vertebrate such as a primate, rodent, domestic
animal or game animal. Primates include chimpanzees, cynomologous
monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents
include mice, rats, woodchucks, ferrets, rabbits and hamsters.
Domestic and game animals include cows, horses, pigs, deer, bison,
buffalo, feline species, e.g., domestic cat, canine species, e.g.,
dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and
fish, e.g., trout, catfish and salmon. In certain embodiments of
the aspects described herein, the subject is a mammal, e.g., a
primate, e.g., a human. A subject can be male or female.
Preferably, the subject is a mammal. The mammal can be a human,
non-human primate, mouse, rat, dog, cat, horse, or cow, but are not
limited to these examples. In addition, the methods and
compositions described herein can be used to treat domesticated
animals and/or pets.
[0145] Delivery devices pre-loaded with at least one composition or
preparation described herein are also within the scope of various
aspects described herein. Embodiments of a delivery device
comprises at least one chamber with an outlet, wherein the at least
one chamber comprises a pre-determined amount of the composition
described herein, and the outlet provides an exit for the
composition.
[0146] The term "chamber" as used herein refers to any structure
configured to store and/or convey a composition described herein.
The chamber can be of any shape or any size, depending on users'
applications, needs, and/or preferences. An exemplary chamber
includes, but is not limited to, a barrel, a tube, a cassette, and
a depression, e.g., a microwell.
[0147] In some embodiments, the delivery device described herein
can further comprise an actuator to control release of the
composition through the outlet, thereby administering the
composition therein to a subject. As used herein, the term "an
actuator" is a mechanical device that can convert any kind of
energy to move the composition through the outlet of the device. By
way of example, an actuator can convert electrical energy to move
or control release of the composition through the outlet. In some
embodiments, an actuator can convert pressure to remove or control
release composition through the outlet. For example, a plunger of a
syringe converts force or pressure to release a composition from
the barrel (chamber), thereby injecting the composition to a
subject.
[0148] Examples of delivery devices described herein include, but
are not limited to, a syringe, a dry powder injector, a nasal
spray, a nebulizer, and an implant. In some embodiments, an implant
can be a microchip, e.g., the ones described in U.S. Pat. Nos.
5,797,898; 6,669,683; 7,052,488; and 7,582,080. In some
embodiments, the delivery devices can be used for vaccination. In
such embodiments, vaccine delivery devices/systems can include, but
are not limited to, the ones described in U.S. Patent Application
Nos.: US 2004/0133160; US 2004/0096455; US 2005/0112135; US
2005/0123565; US 2009/0043280; and US 2009/0143724, as well as U.S.
Pat. Nos. 5,346,481; and 5,900,238.
[0149] The term "pre-determined amount" is generally used in
reference to an amount of a composition desired and/or determined
by a user, e.g., depending on applications or treatment. In some
embodiments, the term "pre-determined amount" refers to an amount
of a composition effective to treat or prevent a disease or a
disorder, e.g., increasing immunity to the disease; reducing,
inhibiting or delaying at least one symptom of the disease; or
producing an improvement in the disease, for example, beneficial or
desired clinical results. For the purposes of various aspects
described herein, beneficial or desired clinical results include,
but are not limited to, alleviation of one or more symptoms,
diminishment of extent of disease, stabilized (e.g., not worsening)
state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission
(whether partial or total), whether detectable or undetectable. In
some embodiments, treating can refer to prolonging survival as
compared to expected survival if not receiving treatment. Thus, one
of skill in the art realizes that a treatment may improve the
disease condition, but may not be a complete cure for the disease.
In reference to immunogenic or vaccine compositions, the term
"pre-determined amount" can mean an amount of the composition
effective to provide or increase immunity to a particular disease.
A blood test or any methods known to a skilled artisan can be used
to check immunity. Accordingly, in some embodiments, the delivery
device comprises an effective dose of immunogenic or vaccine
composition.
[0150] Embodiments of the various aspects described herein can be
illustrated by the following numbered paragraphs. [0151] 1. A
method comprising a step of: maintaining a composition, wherein the
composition comprises a silk fibroin matrix and at least one active
agent distributed therein, and wherein the active agent retains at
least about 30% of its original bioactivity when the composition is
(a) subjected to at least one freeze-thaw cycle, or (b) maintained
for at least about 24 hours at a temperature above 0.degree. C., or
(c) both (a) and (b). [0152] 2. The method of paragraph 1, wherein
the active agent retains at least about 50% of its original
bioactivity. [0153] 3. The method of paragraph 1 or 2, wherein the
active agent retains at least about 80% of its original
bioactivity. [0154] 4. The method of any of paragraphs 1-3, wherein
the composition is maintained for at least about 1 month. [0155] 5.
The method of any of paragraphs 1-4, wherein the composition is
maintained for at least about 6 months. [0156] 6. The method of any
of paragraphs 1-5, wherein the composition is a film, a fiber, a
particle, a gel, or a hydrogel. [0157] 7. The method of any of
paragraphs 1-6, wherein the composition is lyophilized. [0158] 8.
The method of any of paragraphs 1-7, wherein the composition is
micronized. [0159] 9. The method of paragraph 8, wherein the
micronized composition are nanoparticles or microparticles. [0160]
10. The method of paragraph 9, wherein the nanoparticles or
microparticles have a size of about 10 nm to about 1000 .mu.m.
[0161] 11. The method of any of paragraphs 1-10, wherein the
composition further comprises an additive. [0162] 12. The method of
paragraph 11, wherein the additive is selected from a stabilizing
agent, a pharmaceutically acceptable carrier or any combinations
thereof. [0163] 13. The method of any of paragraphs 1-12, wherein
the composition is maintained at a temperature of about 0.degree.
C. to above room temperature. [0164] 14. The method of paragraph
13, wherein the composition is maintained at a temperature of about
room temperature to about 37.degree. C. [0165] 15. The method of
any of paragraphs 1-14, wherein the composition is maintained at a
temperature greater than 37.degree. C. [0166] 16. The method of any
of paragraphs 1-15, wherein the composition is maintained under
exposure to light. [0167] 17. The method of any of paragraphs 1-16,
wherein the composition is maintained at a relative humidity of at
least about 10%. [0168] 18. The method of any of paragraphs 1-17,
wherein the active agent is selected from the group consisting of
proteins, peptides, antigens, immunogens, vaccines, antibodies or
portions thereof, antibody-like molecules, enzymes, nucleic acids,
siRNA, shRNA, aptamers, viruses, bacteria, small molecules, cells,
photosynthetic and energy-harvesting compounds, flavors,
antibiotics, therapeutic agents, diagnostic agents, viral vectors,
and anti-venom. [0169] 19. The method of any of paragraphs 1-18,
wherein the active agent is an immunogen. [0170] 20. The method of
paragraph 19, wherein the immunogen is selected from the group
consisting of killed pathogens, live attenuated pathogens, protein
subunits and conjugate thereof, inactivated toxins, and synthetic
peptides, carbohydrates and antigens. [0171] 21. The method of
paragraph 19 or 20, wherein the immunogen is derived from hepatitis
B virus, Haemophilus influenzae Type B, poliovirus, Neisseria
meningitides C, influenza, Varicella, or Mycobacteria tuberculosis
bacille Calmette-Guerin, tetanus toxoid, diphtheria toxoid, and
Bordetella pertussis. [0172] 22. The method of paragraph 19 or 20,
wherein the immunogen is a combination immunogen selected from the
group consisting of DTaP, DTwP, DTwP hepB, DTP hep B Hib, DTaP hep
B Hib IPV, and any combinations thereof. [0173] 23. The method of
paragraph 19 or 20, wherein the immunogen is live, attenuated
virus. [0174] 24. The method of paragraph 23, wherein the live,
attenuated virus is an enveloped virus. [0175] 25. The method of
paragraph 24, wherein the enveloped virus is selected from the
group consisting of Paramyxoviridae, Togaviridae, Orthomyxoviridae,
Flaviviridae, Herpesviridae, Rhabdovirus, Retroviridae, and any
combinations thereof. [0176] 26. The method of any of paragraphs
23-25, wherein the virus is varicella. [0177] 27. The method of any
of paragraphs 23-25, wherein the virus is influenza. [0178] 28. The
method of paragraph 23, wherein the live, attenuated virus causes
measles, mumps, or rubella. [0179] 29. The method of paragraph 19
or 20, wherein the immunogen is a live, attenuated, non-enveloped
virus. [0180] 30. The method of paragraph 29, wherein the
non-enveloped virus is rotavirus, reovirus, hepatitis virus, rabies
virus or poliovirus. [0181] 31. The method of paragraph 19, wherein
the immunogen is a bacterium. [0182] 32. The method of paragraph
31, wherein the bacterium is Mycobacteria tuberculosis bacille
Calmette-Guerin or Bordetella pertussis. [0183] 33. The method of
paragraph 19, wherein the immunogen is a bacterial subunit. [0184]
34. The method of paragraph 33, wherein the bacterial subunit is
derived from Neisseria meningitides type C, Haemophilus influenzae
type B, Streptococcus pneumoniae, or Group B streptococcus. [0185]
35. The method of paragraph 33, wherein the bacterial subunit is a
polysaccharide. [0186] 36. The method of paragraph 19, wherein the
immunogen is a viral subunit. [0187] 37. The method of paragraph
36, wherein the viral subunit is derived from Hepatitis B virus or
Human Papillomavirus. [0188] 38. The method of paragraph 19,
wherein the immunogen is recombinant. [0189] 39. The method of
paragraph 19, wherein the immunogen is a vaccine product selected
from the group consisting of Anthrax vaccine (BioThrax); BCG
(Bacillus Calmette-Guerin) (Tice, Mycobax); DTaP (Daptacel); DTaP
(Infanrix); DTaP (Tripedia); DTaP/Hib (TriHIBit); DTaP-IPV
(Kinrix); DTaP-HepB-IPV (Pediarix); DtaP-IPV/Hib (Pentacel); DT
(diphtheria vaccine plus tetanus vaccine) (Sanofi); Hib vaccine
(ACTHib); DT (Massachusetts); Hib (PedvaxHib); Hib/Hep B (Comvax);
Hep A (Havrix), Hepatitis A vaccine; Hep A (Vaqta), Hepatitis A
vaccine; Hep B (Engerix-B), Hepatitis B vaccine; Hep B
(Recombivax), Hepatitis B vaccine; HepA/HepB vaccine (Twinrix);
Human Papillomavirus (HPV) (Gardasil); Influenza vaccine (Afluria);
Influenza vaccine (Fluarix); Influenza vaccine (Flulaval);
Influenza vaccine (Fluvirin); Influenza vaccine (Fluzone);
Influenza vaccine (FluMist); IPV (Ipol), Polio vaccine; Japanese
encephalitis vaccine (JE-Vax); Japanese encephalitis vaccine
(Ixiaro); Meningococcal vaccine (Menactra); MMR vaccine (MMR-II);
MMRV vaccine (ProQuad); Pneumococcal vaccine (Pneumovax);
Pneumococcal vaccine (Prevnar); Poliovirus inactivated (Poliovax),
Polio vaccine; Rabies vaccine (Imovax); Rabies vaccine (RabAvert);
Rotavirus vaccine (RotaTeq); Rotavirus vaccine (Rotarix); Td
vaccine (Decavac); Td vaccine (Massachusetts); Tdap vaccine
(Adacel); Tdap vaccine (Boostrix); Typhoid (inactivated--Typhim
Vi), Typhus vaccine; Typhoid (oral--Ty21a), Typhus vaccine;
Vaccinia (ACAM2000); Varicella vaccine (Varivax); Yellow fever
vaccine (YF-Vax); Zoster vaccine (Zostavax); and any combinations
thereof. [0190] 40. The method of any of paragraphs 1-39, wherein
the ratio of the silk fibroin matrix to the active agent is about
1:1000 to about 1000:1. [0191] 41. A storage-stable composition
comprising a silk fibroin matrix and an active agent distributed
therein, wherein the active agent retains at least about 30% of its
original bioactivity when the composition is (a) subjected to at
least one freeze-thaw cycle, or (b) maintained for at least about
24 hours at a temperature above 0.degree. C., or (c) both (a) and
(b). [0192] 42. The composition of paragraph 41, wherein the active
agent retains at least about 50% of its original bioactivity.
[0193] 43. The composition of paragraph 41 or 42, wherein the
active agent retains at least about 80% of its original
bioactivity. [0194] 44. The composition of any of paragraphs 41-43,
wherein the composition is maintained for at least about 1 month.
[0195] 45. The composition of any of paragraphs 41-44, wherein the
composition is maintained for at least about 6 months. [0196] 46.
The composition of any of paragraphs 41-45, wherein the composition
is a film, a fiber, a particle, a gel, or a hydrogel. [0197] 47.
The composition of any of paragraphs 41-46, wherein the composition
is lyophilized. [0198] 48. The composition of any of paragraphs
41-47, wherein the composition is micronized. [0199] 49. The
composition of paragraph 48, wherein the micronized composition are
nanoparticles or microparticles. [0200] 50. The composition of
paragraph 49, wherein the nanoparticles or microparticles have a
size of about 10 nm to about 1000 .mu.m. [0201] 51. The composition
of any of paragraphs 41-50, further comprising an additive
distributed through the silk fibroin matrix. [0202] 52. The
composition of paragraph 51, wherein the additive is selected from
a stabilizing agent, a pharmaceutically acceptable carrier, or any
combinations thereof. [0203] 53. The composition of any of
paragraphs 41-52, wherein the composition is maintained at a
temperature of about 0.degree. C. to above room temperature. [0204]
54. The composition of any of paragraphs 41-53, wherein the
composition is maintained at a temperature of about room
temperature to about 37.degree. C. [0205] 55. The composition of
any of paragraphs 41-54, wherein the composition is maintained at a
temperature greater than 37.degree. C. [0206] 56. The composition
of any of paragraphs 41-55, wherein the composition is maintained
under exposure to light. [0207] 57. The composition of any of
paragraphs 41-56, wherein the composition is maintained at a
relative humidity of at least about 10%. [0208] 58. The composition
of any of paragraph 41-57, wherein the active agent is selected
from the group consisting of proteins, peptides, antigens,
immunogens, vaccines, antibodies or portions thereof, antibody-like
molecules, enzymes, nucleic acids, siRNA, shRNA, aptamers, viruses,
bacteria, small molecules, cells, photosynthetic and
energy-harvesting compounds, flavors, antibiotics, therapeutic
agents, diagnostic agents, viral vectors, anti-venom, and any
combinations thereof. [0209] 59. The composition of any of
paragraphs 41-58, wherein the active agent is an immunogen. [0210]
60. The composition of paragraph 59, wherein the immunogen is
selected from the group consisting of killed pathogens, live
attenuated pathogens, protein subunits and conjugate thereof,
inactivated toxins, and synthetic peptides, carbohydrates and
antigens. [0211] 61. The composition of paragraph 59 or 60, wherein
the immunogen is derived from hepatitis B virus, Haemophilus
influenzae Type B, poliovirus, Neisseria meningitides C, influenza,
Varicella, or Mycobacteria tuberculosis bacille Calmette-Guerin,
tetanus toxoid, diphtheria toxoid, and Bordetella pertussis. [0212]
62. The composition of paragraph 59 or 60, wherein the immunogen is
a combination immunogen selected from the group consisting of DTaP,
DTwP, DTwP hepB, DTP hep B Hib, DTaP hep B Hib IPV, and any
combinations thereof. [0213] 63. The composition of paragraph 59 or
60, wherein the immunogen is live, attenuated virus. [0214] 64. The
composition of paragraph 63, wherein the live, attenuated virus is
an enveloped virus. [0215] 65. The composition of paragraph 64,
wherein the enveloped virus is selected from the group consisting
of Paramyxoviridae, Togaviridae, Orthomyxoviridae, Flaviviridae,
Herpesviridae, Rhabdovirus, Retroviridae, and any combinations
thereof. [0216] 66. The composition of any of paragraphs 63-65,
wherein the virus is varicella. [0217] 67. The composition of any
of paragraphs 63-65, wherein the virus is influenza. [0218] 68. The
composition of paragraph 63, wherein the live, attenuated virus
causes measles, mumps, or rubella. [0219] 69. The composition of
paragraph 59 or 60, wherein the immunogen is a live, attenuated,
non-enveloped virus [0220] 70. The composition of paragraph 69,
wherein the non-enveloped virus is rotavirus, reovirus, hepatitis
virus, rabies virus or poliovirus. [0221] 71. The composition of
paragraph 59, wherein the immunogen is a bacterium. [0222] 72. The
composition of paragraph 71, wherein the bacterium is Mycobacteria
tuberculosis bacille Calmette-Guerin or Bordetella pertussis.
[0223] 73. The composition of paragraph 59, wherein the immunogen
is a bacterial subunit. [0224] 74. The composition of paragraph 73,
wherein the bacterial subunit is derived from Neisseria
meningitides type C, Haemophilus influenzae type B, Streptococcus
pneumoniae, or Group B streptococcus. [0225] 75. The composition of
paragraph 73, wherein the bacterial subunit is a polysaccharide.
[0226] 76. The composition of paragraph 59, wherein the immunogen
is a viral subunit. [0227] 77. The composition of paragraph 76,
wherein the viral subunit is derived from Hepatitis B virus or
Human Papillomavirus. [0228] 78. The composition of paragraph 59,
wherein the immunogen is recombinant. [0229] 79. The composition of
paragraph 59, wherein the immunogen is a vaccine product selected
from the group consisting of Anthrax vaccine (BioThrax); BCG
(Bacillus Calmette-Guerin) (Tice, Mycobax); DTaP (Daptacel); DTaP
(Infanrix); DTaP (Tripedia); DTaP/Hib (TriHIBit); DTaP-IPV
(Kinrix); DTaP-HepB-IPV (Pediarix); DtaP-IPV/Hib (Pentacel); DT
(diphtheria vaccine plus tetanus vaccine) (Sanofi); Hib vaccine
(ACTHib); DT (Massachusetts); Hib (PedvaxHib); Hib/Hep B (Comvax);
Hep A (Havrix), Hepatitis A vaccine; Hep A (Vaqta), Hepatitis A
vaccine; Hep B (Engerix-B), Hepatitis B vaccine; Hep B
(Recombivax), Hepatitis B vaccine; HepA/HepB vaccine (Twinrix);
Human Papillomavirus (HPV) (Gardasil); Influenza vaccine (Afluria);
Influenza vaccine (Fluarix); Influenza vaccine (Flulaval);
Influenza vaccine (Fluvirin); Influenza vaccine (Fluzone);
Influenza vaccine (FluMist); IPV (Ipol), Polio vaccine; Japanese
encephalitis vaccine (JE-Vax); Japanese encephalitis vaccine
(Ixiaro); Meningococcal vaccine (Menactra); MMR vaccine (MMR-II);
MMRV vaccine (ProQuad); Pneumococcal vaccine (Pneumovax);
Pneumococcal vaccine (Prevnar); Poliovirus inactivated (Poliovax),
Polio vaccine; Rabies vaccine (Imovax); Rabies vaccine (RabAvert);
Rotavirus vaccine (RotaTeq); Rotavirus vaccine (Rotarix); Td
vaccine (Decavac); Td vaccine (Massachusetts); Tdap vaccine
(Adacel); Tdap vaccine (Boostrix); Typhoid (inactivated--Typhim
Vi), Typhus vaccine; Typhoid (oral--Ty21a), Typhus vaccine;
Vaccinia (ACAM2000); Varicella vaccine (Varivax); Yellow fever
vaccine (YF-Vax); Zoster vaccine (Zostavax); and any combinations
thereof. [0230] 80. The composition of any of paragraphs 41-79,
wherein the ratio of the silk fibroin matrix to the active agent is
about 1:1000 to about 1000:1. [0231] 81. A method for preparing a
storage-stable composition of any of paragraphs 41 to 80, the
method comprising the steps of:
[0232] a. providing a silk fibroin solution comprising at least one
active agent; and [0233] b. drying the silk fibroin solution of
step (a) to form a solid-state silk fibroin, thereby obtaining a
composition in which the at least one active agent retains at least
about 30% of its original bioactivity upon storage. [0234] 82. The
method of paragraph 81, wherein the drying is lyophilization.
[0235] 83. The method of paragraph 81, wherein the drying is
air-dry. [0236] 84. The method of any of paragraphs 81-83, further
comprising lyophilizing the solid-state silk fibroin from step (b).
[0237] 85. The method of any of paragraphs 81-84, further
comprising post-treatment of the composition. [0238] 86. The method
of paragraph 85, wherein the post-treatment alters the
crystallinity of the composition. [0239] 87. The method of
paragraph 85 or 86, wherein the post-treatment is contacting the
composition with methanol or ethanol. [0240] 88. The method of any
of paragraphs 85-87, wherein the post-treatment is subjecting the
composition to shear stress. [0241] 89. The method of any of
paragraphs 85-88, wherein the post-treatment is subjecting the
composition to an electric field. [0242] 90. The method of any of
paragraphs 85-89, wherein the post-treatment is subjecting the
composition to pressure. [0243] 91. The method of any of paragraphs
85-90, wherein the post-treatment is contacting the composition
with salt. [0244] 92. The method of any of paragraphs 81-91,
further comprising reducing the solid-state silk fibroin of step
(b) by a mechanical means to obtain micronized particles. [0245]
93. The method of paragraph 92, wherein the mechanical means is
selected from micronizing, pulverizing, crushing, grinding,
freeze-drying or any combinations thereof. [0246] 94. The method of
paragraph 92 or 93, wherein the micronized particles have a size of
about 10 nm to about 1000 .mu.m. [0247] 95. The method of any of
paragraphs 81-94, wherein the at least one active agent retains at
least about 80% of its original bioactivity upon storage. [0248]
96. The method of any of paragraphs 81-95, wherein the storage is
over a period of at least about 6 months. [0249] 97. The method of
any of paragraphs 81-96, wherein the storage is at a temperature of
about room temperature to about 37.degree. C. [0250] 98. The method
of any of paragraphs 81-97, wherein the storage is at a temperature
greater than 37.degree. C. [0251] 99. A method comprising a step of
maintaining an immunogenic composition, wherein the composition
comprises a silk fibroin matrix and at least one immunogen
distributed therein, and wherein the immunogen retains at least
about 30% of its original immunogenicity when the composition is
(a) subjected to at least one freeze-thaw cycle, or (b) maintained
for at least about 24 hours at a temperature above 0.degree. C., or
(c) both (a) and (b). [0252] 100. The method of paragraph 99,
wherein the immunogen retains at least about 50% of its original
immunogenicity. [0253] 101. The method of paragraph 99 or 100,
wherein the immunogen retains at least about 80% of its original
immunogenicity. [0254] 102. The method of any of paragraphs 99-101,
wherein the composition is maintained for at least about 1 month.
[0255] 103. The method of any of paragraphs 99-102, wherein the
composition is maintained for at least about 6 months. [0256] 104.
The method of any of paragraphs 99-103, wherein the composition is
a film, a fiber, a particle, a gel, or a hydrogel. [0257] 105. The
method of any of paragraphs 99-104, wherein the composition is
lyophilized. [0258] 106. The method of any of paragraphs 99-105,
wherein the composition is micronized. [0259] 107. The method of
paragraph 106, wherein the micronized composition are nanoparticles
or microparticles. [0260] 108. The method of paragraph 107, wherein
the nanoparticles or microparticles have a size of about 10 nm to
about 1000 .mu.m. [0261] 109. The method of any of paragraphs
99-108, wherein the composition further comprises an additive
distributed through the silk fibroin matrix. [0262] 110. The method
of paragraph 109, wherein the additive is selected from the group
consisting of a stabilizing agent, a pharmaceutically acceptable
carrier, and any combinations thereof. [0263] 111. The method of
paragraph 110, wherein the stabilizing agent is selected from the
group consisting of a saccharide, a sugar alcohol, an ion, a
surfactant, and any combinations thereof. [0264] 112. The method of
paragraph 111, wherein the saccharide is sucrose. [0265] 113. The
method of any of paragraphs 99-112, wherein the composition is
maintained at a temperature of about 0.degree. C. to above room
temperature. [0266] 114. The method of any of paragraphs 99-113,
wherein the composition is maintained at a temperature of about
room temperature to about 37.degree. C. [0267] 115. The method of
any of paragraphs 99-114, wherein the composition is maintained at
a temperature greater than 37.degree. C. [0268] 116. The method of
any of paragraphs 99-115, wherein the composition is maintained
under exposure to light. [0269] 117. The method of any of
paragraphs 99-116, wherein the composition is maintained at a
relative humidity of at least about 10%. [0270] 118. The method of
any of paragraphs 99-117, wherein the immunogen is selected from
the group consisting of killed pathogens, live attenuated
pathogens, protein subunits and conjugate thereof, inactivated
toxins, synthetic peptides, carbohydrates, antigens, and any
combinations thereof. [0271] 119. The method of any of paragraphs
99-118, wherein the immunogen is derived from hepatitis B virus,
Haemophilus influenzae Type B, poliovirus, Neisseria meningitides
C, influenza, Varicella, or Mycobacteria tuberculosis bacille
Calmette-Guerin, tetanus toxoid, diphtheria toxoid, and Bordetella
pertussis. [0272] 120. The method of any of paragraphs 99-118,
wherein the immunogen is a combination immunogen selected from the
group consisting of DTaP, DTwP, DTwP hepB, DTP hep B Hib, DTaP hep
B Hib IPV, and any combinations thereof. [0273] 121. The method of
any of paragraphs 99-118, wherein the immunogen is live, attenuated
virus. [0274] 122. The method of paragraph 121, wherein the live,
attenuated virus is an enveloped virus. [0275] 123. The method of
paragraph 122, wherein the enveloped virus is selected from the
group consisting of Paramyxoviridae, Togaviridae, Orthomyxoviridae,
Flaviviridae, Herpesviridae, Rhabdovirus, Retroviridae, and any
combinations thereof. [0276] 124. The method of paragraph 121-123,
wherein the virus is varicella. [0277] 125. The method of paragraph
121-123, wherein the virus is influenza. [0278] 126. The method of
paragraph 121, wherein the live, attenuated virus causes measles,
mumps, or rubella. [0279] 127. The method of any of paragraphs
99-118, wherein the immunogen is a live, attenuated, non-enveloped
virus. [0280] 128. The method of paragraph 127, wherein the
non-enveloped virus is rotavirus, reovirus, hepatitis virus, rabies
virus or poliovirus. [0281] 129. The method of any of paragraphs
99-118, wherein the immunogen is a bacterium. [0282] 130. The
method of paragraph 129, wherein the bacterium is Mycobacteria
tuberculosis bacille Calmette-Guerin or Bordetella pertussis.
[0283] 131. The method of any of paragraphs 99-118, wherein the
immunogen is a bacterial subunit. [0284] 132. The method of
paragraph 131, wherein the bacterial subunit is derived from
Neisseria meningitides type C, Haemophilus influenzae type B,
Streptococcus pneumoniae, or Group B streptococcus. [0285] 133. The
method of paragraph 131, wherein the bacterial subunit is a
polysaccharide. [0286] 134. The method of any of paragraphs 99-118,
wherein the immunogen is a viral subunit. [0287] 135. The method of
paragraph 134, wherein the viral subunit is derived from Hepatitis
B virus or Human Papillomavirus. [0288] 136. The method of any of
paragraphs 99-118, wherein the immunogen is recombinant. [0289]
137. The method of any of paragraphs 99-118, wherein the immunogen
is a vaccine product selected from the group consisting of Anthrax
vaccine (BioThrax); BCG (Bacillus Calmette-Guerin) (Tice, Mycobax);
DTaP (Daptacel); DTaP (Infanrix); DTaP (Tripedia); DTaP/Hib
(TriHIBit); DTaP-IPV (Kinrix); DTaP-HepB-IPV (Pediarix);
DtaP-IPV/Hib (Pentacel); DT (diphtheria vaccine plus tetanus
vaccine) (Sanofi); Hib vaccine (ACTHib); DT (Massachusetts); Hib
(PedvaxHib); Hib/Hep B (Comvax); Hep A (Havrix), Hepatitis A
vaccine; Hep A (Vaqta), Hepatitis A vaccine; Hep B (Engerix-B),
Hepatitis B vaccine; Hep B (Recombivax), Hepatitis B vaccine;
HepA/HepB vaccine (Twinrix); Human Papillomavirus (HPV) (Gardasil);
Influenza vaccine (Afluria); Influenza vaccine (Fluarix); Influenza
vaccine (Flulaval); Influenza vaccine (Fluvirin); Influenza vaccine
(Fluzone); Influenza vaccine (FluMist); IPV (Ipol), Polio vaccine;
Japanese encephalitis vaccine (JE-Vax); Japanese encephalitis
vaccine (Ixiaro); Meningococcal vaccine (Menactra); MMR vaccine
(MMR-II); MMRV vaccine (ProQuad); Pneumococcal vaccine (Pneumovax);
Pneumococcal vaccine (Prevnar); Poliovirus inactivated (Poliovax),
Polio vaccine; Rabies vaccine (Imovax); Rabies vaccine (RabAvert);
Rotavirus vaccine (RotaTeq); Rotavirus vaccine (Rotarix); Td
vaccine (Decavac); Td vaccine (Massachusetts); Tdap vaccine
(Adacel); Tdap vaccine (Boostrix); Typhoid (inactivated--Typhim
Vi), Typhus vaccine; Typhoid (oral--Ty21a), Typhus vaccine;
Vaccinia (ACAM2000); Varicella vaccine (Varivax); Yellow fever
vaccine (YF-Vax); Zoster vaccine (Zostavax); and any combinations
thereof. [0290] 138. The method of any of paragraphs 99-137,
wherein the ratio of the silk fibroin matrix to the immunogen is
about 1:1000 to about 1000:1. [0291] 139. A storage-stable
immunogenic composition comprising a silk fibroin matrix and an
immunogen distributed therein, wherein the immunogen retains at
least about 30% of its original immunogenicity when the composition
is (a) subjected to at least one freeze-thaw cycle, or (b)
maintained for at least about 24 hours at a temperature above
0.degree. C., or (c) both (a) and (b). [0292] 140. The composition
of paragraph 139, wherein the immunogen retains at least about 50%
of its original immunogenicity. [0293] 141. The composition of
paragraph 139 or 140, wherein the immunogen retains at least about
80% of its original immunogenicity. [0294] 142. The composition of
any of paragraphs 139-141, wherein the composition is maintained
for at least about 1 month. [0295] 143. The composition of any of
paragraphs 139-142, wherein the composition is maintained for at
least about 6 months. [0296] 144. The composition of any of
paragraphs 139-143, wherein the composition is a film, a fiber, a
particle, a gel or a hydrogel. [0297] 145. The composition of any
of paragraphs 139-144, wherein the composition is lyophilized.
[0298] 146. The composition of any of paragraphs 139-145, wherein
the composition is micronized. [0299] 147. The composition of
paragraph 146, wherein the micronized composition are nanoparticles
or microparticles. [0300] 148. The composition of paragraph 147,
wherein the nanoparticles or microparticles have a size of about 10
nm to about 1000 .mu.m. [0301] 149. The composition of any of
paragraphs 139-148, further comprising an additive distributed
through the silk fibroin matrix. [0302] 150. The composition of
paragraph 149, wherein the additive is selected from the group
consisting of a stabilizing agent, a pharmaceutically acceptable
carrier, and any combinations thereof. [0303] 151. The composition
of paragraph 150, wherein the stabilizing agent is selected from
the group consisting of a saccharide, a sugar alcohol, an ion, a
surfactant, and any combinations thereof. [0304] 152. The
composition of paragraph 151, wherein the saccharide is sucrose.
[0305] 153. The composition of any of paragraphs 139-152, wherein
the composition is maintained at a temperature of about
0.quadrature.C to above room temperature. [0306] 154. The
composition of any of paragraphs 139-153, wherein the composition
is maintained at a temperature of about room temperature to about
37.degree. C. [0307] 155. The composition of any of paragraphs
139-154, wherein the composition is maintained at a temperature
greater than 37.degree. C. [0308] 156. The composition of any of
paragraphs 139-155, wherein the composition is maintained under
exposure to light. [0309] 157. The composition of any of paragraphs
139-156, wherein the composition is maintained at a relative
humidity of at least about 10%. [0310] 158. The composition of any
of paragraphs 139-157, wherein the immunogen is selected from the
group consisting of killed pathogens, live attenuated pathogens,
protein subunits and conjugate thereof, inactivated toxins,
synthetic peptides, carbohydrates, antigens and any combinations
thereof. [0311] 159. The composition of any of paragraphs 139-158,
wherein the immunogen is derived from hepatitis B virus,
Haemophilus influenzae Type B, poliovirus, Neisseria meningitides
C, influenza, Varicella, or Mycobacteria tuberculosis bacille
Calmette-Guerin, tetanus toxoid, diphtheria toxoid, and Bordetella
pertussis. [0312] 160. The composition of any of paragraphs
139-158, wherein the immunogen is a combination immunogen selected
from the group consisting of DTaP, DTwP, DTwP hepB, DTP hep B Hib,
DTaP hep B Hib IPV, and any combinations thereof. [0313] 161. The
composition of any of paragraphs 139-158, wherein the immunogen is
live, attenuated virus. [0314] 162. The composition of paragraph
161, wherein the live, attenuated virus is an enveloped virus.
[0315] 163. The composition of paragraph 162, wherein the enveloped
virus is selected from the group consisting of Paramyxoviridae,
Togaviridae, Orthomyxoviridae, Flaviviridae, Herpesviridae,
Rhabdovirus, Retroviridae, and any combinations thereof. [0316]
164. The composition of paragraph 161-163, wherein the virus is
varicella. [0317] 165. The composition of paragraph 161-163,
wherein the virus is influenza. [0318] 166. The composition of
paragraph 161, wherein the live, attenuated virus causes measles,
mumps, or rubella. [0319] 167. The composition of any of paragraphs
139-158, wherein the immunogen is a live, attenuated, non-enveloped
virus. [0320] 168. The composition of paragraph 167, wherein the
non-enveloped virus is rotavirus, reovirus, hepatitis virus, rabies
virus or poliovirus. [0321] 169. The composition of any of
paragraphs 139-158, wherein the immunogen is a bacterium. [0322]
170. The composition of paragraph 169, wherein the bacterium is
Mycobacteria tuberculosis bacille Calmette-Guerin or Bordetella
pertussis. [0323] 171. The composition of any of paragraphs
139-158, wherein the immunogen is a bacterial subunit. [0324] 172.
The composition of paragraph 171, wherein the bacterial subunit is
derived from Neisseria meningitides type C, Haemophilus influenzae
type B, Streptococcus pneumoniae, or Group B streptococcus. [0325]
173. The composition of paragraph 171, wherein the bacterial
subunit is a polysaccharide.
[0326] 174. The composition of any of paragraphs 139-158, wherein
the immunogen is a viral subunit. [0327] 175. The composition of
paragraph 174, wherein the viral subunit is derived from Hepatitis
B virus or Human Papillomavirus. [0328] 176. The composition of any
of paragraphs 139-158, wherein the immunogen is recombinant. [0329]
177. The composition of any of paragraphs 139-158, wherein the
immunogen is a vaccine product selected from the group consisting
of Anthrax vaccine (BioThrax); BCG (Bacillus Calmette-Guerin)
(Tice, Mycobax); DTaP (Daptacel); DTaP (Infanrix); DTaP (Tripedia);
DTaP/Hib (TriHIBit); DTaP-IPV (Kinrix); DTaP-HepB-IPV (Pediarix);
DtaP-IPV/Hib (Pentacel); DT (diphtheria vaccine plus tetanus
vaccine) (Sanofi); Hib vaccine (ACTHib); DT (Massachusetts); Hib
(PedvaxHib); Hib/Hep B (Comvax); Hep A (Havrix), Hepatitis A
vaccine; Hep A (Vaqta), Hepatitis A vaccine; Hep B (Engerix-B),
Hepatitis B vaccine; Hep B (Recombivax), Hepatitis B vaccine;
HepA/HepB vaccine (Twinrix); Human Papillomavirus (HPV) (Gardasil);
Influenza vaccine (Afluria); Influenza vaccine (Fluarix); Influenza
vaccine (Flulaval); Influenza vaccine (Fluvirin); Influenza vaccine
(Fluzone); Influenza vaccine (FluMist); IPV (Ipol), Polio vaccine;
Japanese encephalitis vaccine (JE-Vax); Japanese encephalitis
vaccine (Ixiaro); Meningococcal vaccine (Menactra); MMR vaccine
(MMR-II); MMRV vaccine (ProQuad); Pneumococcal vaccine (Pneumovax);
Pneumococcal vaccine (Prevnar); Poliovirus inactivated (Poliovax),
Polio vaccine; Rabies vaccine (Imovax); Rabies vaccine (RabAvert);
Rotavirus vaccine (RotaTeq); Rotavirus vaccine (Rotarix); Td
vaccine (Decavac); Td vaccine (Massachusetts); Tdap vaccine
(Adacel); Tdap vaccine (Boostrix); Typhoid (inactivated--Typhim
Vi), Typhus vaccine; Typhoid (oral--Ty21a), Typhus vaccine;
Vaccinia (ACAM2000); Varicella vaccine (Varivax); Yellow fever
vaccine (YF-Vax); Zoster vaccine (Zostavax); and any combinations
thereof. [0330] 178. The composition of any of paragraphs 139-177,
wherein the ratio of the silk fibroin matrix to the immunogen is
about 1:1000 to about 1000:1. [0331] 179. A method for preparing a
storage-stable immunogenic composition of any of paragraphs 139 to
178, the method comprising the steps of: [0332] a. providing a silk
fibroin solution comprising at least one immunogen; and [0333] b.
drying the silk fibroin solution of step (a) to form a solid-state
silk fibroin, thereby obtaining an immunogenic composition in which
the at least one immunogen retains at least about 30% of its
original immunogenicity upon storage. [0334] 180. The method of
paragraph 179, wherein the drying is lyophilization. [0335] 181.
The method of paragraph 179, wherein the drying is air-dry. [0336]
182. The method of any of paragraphs 179-181, further comprising
lyophilizing the solid-state silk fibroin from step (b). [0337]
183. The method of any of paragraphs 179-182, further comprising
post-treatment of the composition. [0338] 184. The method of
paragraph 183, wherein the post-treatment alters the crystallinity
of the composition. [0339] 185. The method of paragraph 183 or 184,
wherein the post-treatment is contacting the composition with
methanol or ethanol. [0340] 186. The method of any of paragraphs
183-185, wherein the post-treatment is subjecting the composition
to shear stress. [0341] 187. The method of any of paragraphs
183-186, wherein the post-treatment is subjecting the composition
to an electric field. [0342] 188. The method of any of paragraphs
183-187, wherein the post-treatment is subjecting the composition
to pressure. [0343] 189. The method of any of paragraphs 183-188,
wherein the post-treatment is contacting the composition with salt.
[0344] 190. The method of any of paragraphs 179-189, further
comprising reducing the solid-state silk fibroin of step (b) by a
mechanical means to obtain micronized particles. [0345] 191. The
method of paragraph 190, wherein the mechanical means is selected
from micronizing, pulverizing, crushing, grinding, freeze-drying or
any combination thereof. [0346] 192. The method of paragraph 190 or
191, wherein the micronized particles have a size of about 10 nm to
about 1000 .mu.m. [0347] 193. The method of any of paragraphs
179-192, wherein the at least one immunogen retains at least about
80% of its original immunogenicity upon storage. [0348] 194. The
method of any of paragraphs 179-193, wherein the storage is over a
period of at least 6 months. [0349] 195. The method of any of
paragraphs 179-194, wherein the storage is at a temperature of
about room temperature to about 37.degree. C. [0350] 196. The
method of any of paragraphs 179-195, wherein the storage is at a
temperature greater than 37.degree. C. [0351] 197. An immunogenic
composition comprising a silk fibroin matrix and at least one live,
attenuated virus distributed therein; wherein the live, attenuated
virus retains at least about 30% of its original infectivity when
the composition is (a) subjected to at least one freeze-thaw cycle,
or (b) maintained for at least 24 hours at a temperature above
0.degree. C. [0352] 198. The composition of paragraph 197, wherein
the virus retains at least about 50% of its original infective
stability. [0353] 199. The composition of paragraph 197 or 198,
wherein the virus retains at least about 80% of its original
infective stability. [0354] 200. The composition of any of
paragraphs 197-199, wherein the composition is maintained for at
least about 6 months. [0355] 201. The composition of any of
paragraphs 197-200, wherein the composition is maintained at a
temperature of about room temperature to about 37.degree. C. [0356]
202. The composition of any of paragraphs 197-201, wherein the
composition is maintained at a temperature greater than 37.degree.
C. [0357] 203. The composition of any of paragraphs 197-202,
wherein the composition is lyophilized. [0358] 204. The composition
of any of paragraphs 197-203, wherein the live, attenuated virus is
an enveloped virus. [0359] 205. The composition of paragraph 204,
wherein the enveloped virus is selected from the group consisting
of Paramyxoviridae, Togaviridae, Orthomyxoviridae, Flaviviridae,
Retroviridae, Herpesviridae, Rhabdovirus, and any combinations
thereof. [0360] 206. The composition of paragraph 204 or 205,
wherein the enveloped virus is varicella, measles virus, mumps
virus, German measles virus, respiratory syncytial virus, yellow
fever virus, or influenza virus. [0361] 207. The composition of any
of paragraphs 197-203, wherein the live, attenuated virus is a
non-enveloped virus. [0362] 208. The composition of paragraph 207,
wherein said non-enveloped virus is rotavirus. [0363] 209. The
composition of any of paragraphs 197-208, further comprising an
additive. [0364] 210. The composition of paragraph 209, wherein the
additive is selected from the group consisting of a stabilizing
agent, a pharmaceutically acceptable carrier, and any combinations
thereof. [0365] 211. The composition of paragraph 210, wherein the
stabilizing agent is selected from the group consisting of a
saccharide, a sugar alcohol, an ion, a surfactant, and any
combinations thereof. [0366] 212. The composition of paragraph 211,
wherein said saccharide is sucrose. [0367] 213. A cell-free,
stabilized virus preparation comprising a silk fibroin matrix and
infective virus distributed therein, wherein the virus retains at
least about 30% of its original infectivity when the preparation is
(a) subjected to at least one freeze-thaw cycle, or (b) maintained
for at least about 24 hours at a temperature above 0.degree. C., or
(c) both (a) and (b). [0368] 214. The preparation of paragraph 213,
wherein the virus and silk fibroin matrix are lyophilized. [0369]
215. The preparation of paragraph 213 or 214, wherein the virus
retains at least about 80% of its original infectivity. [0370] 216.
The preparation of any of paragraphs 213-215, wherein the
preparation is maintained for at least about 6 months. [0371] 217.
The preparation of any of paragraphs 213-216, wherein the
preparation is maintained at a temperature of about room
temperature to about 37.degree. C. [0372] 218. The preparation of
any of paragraphs 213-217, wherein the preparation is maintained at
a temperature greater than 37.degree. C. [0373] 219. The
preparation of any of paragraphs 213-218, wherein the virus is an
enveloped virus. [0374] 220. The preparation of any of paragraphs
213-218, wherein the virus is respiratory syncytial virus. [0375]
221. The preparation of any of paragraphs 213-218, wherein the
virus is a non-enveloped virus. [0376] 222. The preparation of any
of paragraphs 213-218, wherein the virus is a bacteriophage. [0377]
223. The preparation of any of paragraphs 213-218, wherein the
virus is a recombinant virus. [0378] 224. The preparation of any of
paragraphs 213-218, wherein the virus is a viral vector. [0379]
225. The preparation of paragraph 224, wherein the viral vector is
selected from the group consisting of a retroviral vector, a
lentiviral vector, an adenoviral vector, an adeno-associated viral
vector, and any combinations thereof. [0380] 226. A preparation
comprising at least one composition of any of paragraphs 41-80,
139-178, or 197-212. [0381] 227. The preparation of paragraph 226,
wherein the preparation is selected from a group consisting of a
tablet, a lozenge, a suspension, a free-flowing powder, an aerosol,
a capsule, and any combinations thereof. [0382] 228. The
preparation of any of paragraphs 226-227, further comprising a
pharmaceutically acceptable carrier. [0383] 229. A package
comprising at least one composition of any of paragraphs 41-80,
139-178, or 197-212, or a preparation of any of paragraphs 213-225
or 226-228. [0384] 230. The package of paragraph 229, wherein the
container is selected from a group consisting of a vial, an ampule,
a capsule, a tube, a syringe, a bottle, a packet, and any
combinations thereof. [0385] 231. The package of paragraph 230,
wherein the syringe is needleless. [0386] 232. A kit comprising the
package of any of paragraphs 229-231, and a pharmaceutically
acceptable solution. [0387] 233. The kit of paragraph 232, further
comprising at least one syringe. [0388] 234. The kit of paragraph
232 or 233, further comprising a disinfectant. [0389] 235. A
delivery device comprising: at least one chamber with an outlet,
wherein the at least one chamber comprises a pre-determined amount
of the composition of any of paragraphs 41-80, 139-178 or 197-212,
and the outlet provides an exit for the composition. [0390] 236.
The device of paragraph 235, wherein the delivery device is
selected from the group consisting of a syringe, a dry powder
injector, a nasal spray, a nebulizer, an implant, and any
combinations thereof. [0391] 237. The device of paragraph 235,
wherein the implant is a microchip. [0392] 238. The device of any
of paragraphs 235-237, further comprising an actuator to control
release of the composition through the outlet.
[0393] To the extent not already indicated, it will be understood
by those of ordinary skill in the art that any one of the various
embodiments herein described and illustrated may be further
modified to incorporate features shown in any of the other
embodiments disclosed herein.
[0394] The following examples illustrate some embodiments and
aspects of the invention. It will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be performed without altering the
spirit or scope of the invention, and such modifications and
variations are encompassed within the scope of the invention as
defined in the claims which follow. The following examples do not
in any way limit the invention.
EXAMPLES
Example 1
Stability of MMR Vaccine in Silk Fibroin
[0395] A commercial batch of trivalent vaccine, MMR.RTM. II
(measles, mumps and rubella virus vaccine live) (Merck & Co.,
INC., USA) was used. This lyophilized live virus vaccine contains
the Enders' attenuated Edmonston measles, the Jeryl Lynn mumps, and
the Wistar RA 27/3 rubella viruses. Prior to use, the vaccine is
reconstituted in the provided diluent and each 0.5 mL dose contains
no less than 1,000 TCID.sub.50 (tissue culture infectious doses) of
measles virus; 12,500 TCID.sub.50 of mumps virus; and 1,000
TCID.sub.50 of rubella virus. Manufacture conditions state that the
vaccine must be used within 8 hours of reconstitution and stored at
4.degree. C. or otherwise be discarded. Each dose of the vaccine is
calculated to contain sorbitol (14.5 mg), sodium phosphate, sucrose
(1.9 mg), sodium chloride, hydrolyzed gelatin (14.5 mg),
recombinant human albumin (.ltoreq.0.3 mg), fetal bovine serum
(<1 ppm), other buffer and media ingredients and approximately
25 mcg of neomycin. The product contains no preservative. Before
reconstitution, the lyophilized vaccine is a light yellow compact
crystalline plug.
[0396] A mixture of sterilized 9% (w/v) silk solution and
reconstituted MMR vaccine was prepared at a concentration of 1:1 by
weight ratio of MMR to silk solution. Films were then cast onto a
Teflon-coated surface. The films were allowed to dry in a sterile
hood for 24 hr at room temperature (RT). A sample of reconstituted
vaccine in solution for 24 hr was also prepared for comparison.
[0397] A standard curve was generated by reconstituting the
vaccine, considered as the 1 log.sub.10 dilution, and the vaccine
solution was then serially diluted in 0.5 log.sub.10 steps from 1.5
log.sub.10 to 3.5 log.sub.10. The silk films were redissolved in an
aliquot of water that diluted the final concentration of vaccine in
the solution to 1.5 log.sub.10. Vero cells (African green monkey
kidney cells; ATCC, Manassas, Va.) were grown to confluence,
trypsinized, counted, and adjusted to 50,000 cells/mL and plated in
a 24-well plate. Next, 50 .mu.L of the vaccine dilution,
redissolved silk film, and 24 hr-stored reconstituted vaccine were
added to a well of Vero cells, in triplicate. The virus was allowed
to replicate in the cell for 3 days, then the RNA from the infected
cells isolated, converted to cDNA and quantitated using qPCR. A
log-linear relationship exists between the amount of target RNA and
the PCR cycle where the fluorescence raises above the background
(threshold cycle, Ct). The more viable the viruses that are present
in the sample, the lower the Ct value.
[0398] The results of the qPCR infectivity assay show a linear
relationship between the increase of vaccine dilution and the
greater the Ct value (FIG. 1). The assay produced expected results
as the low vaccine dilution contained a greater quantity of viable
viruses, therefore producing a lower Ct value. Using this result as
a standard curve, the results for the silk-encapsulated vaccine
sample and the reconstituted vaccine sample can be quantified by
extrapolating the final log.sub.10 dilution from their Ct
values.
[0399] The log.sub.10 dilution values were then converted into
potency values of log.sub.10 TCID.sub.50/dose. The log.sub.10
TCID.sub.50/dose values of the MMR-silk films were then related to
the log.sub.10 TCID.sub.50/dose of the original dilution at which
they were casted (1.5 log.sub.10 dilution), producing the residual
potency. The reconstituted control vaccine stored at RT for 24
hours produced no Ct values, indicating that it had lost nearly, if
not all, its potency. The initial potencies recovered from the
MMR-silk films for measles, mumps and rubella were 75.89%, 58.04%
and 62.48%, respectively (Table 1).
TABLE-US-00001 TABLE 1 Initial potency recovery of 1:1 (w/w)
MMR-silk films Initial Potency Recovery (log10 TCID50/dose) (%)
Measles 75.89 Mumps 58.04 Rubella 62.48
[0400] This is a substantial improvement over the reconstituted
control vaccine stored at room temperature for the same period of
time, for which residual potency was below the lower limit of
detection. The initial loss of potency in the silk-embedded vaccine
was likely caused by reconstituting the lyophilized vaccine during
the preparation of the films rather than the effect of interactions
of the vaccine with the silk. To show that the loss of activity
most likely occurred while the MMR-silk was still in solution
before completely drying into films, aliquots of lyophilized
vaccine were reconstituted in water for 24, 18, 12, and 6 hr at
room temperature prior to cell inoculation (FIG. 2).
[0401] As seen in FIG. 2, the vaccine potency decreases
significantly the longer it remains in solution. Vaccine samples in
solution for 24 hr prior to cell inoculation exhibited no residual
potency for any of the viral components. Once the films were dried
and stored, the silk exhibited a stabilizing effect on the vaccine.
As seen in FIG. 3, after 3 months of storage at room temperature,
the silk films retained 96%, 92% and 80% potency for the measles,
mumps and rubella virus, respectively. The results indicate that
MMR vaccines stored in only unprocessed silk films are capable of
extending the potency of the commercially prepared lyophilized
vaccine at RT.
Example 2
Addition of Additives
[0402] Whether the addition of stabilizing additives further
increases the thermostability of the virus was examined. Oral polio
virus vaccine (OPV), also a live attenuated vaccine, has been
widely studied and was commercially prepared with the addition of
MgCl.sub.2 stabilizer. Studies on the effects of stabilizing
additives OPV have been conducted and previously tested stabilizers
include sucrose, magnesium sulfate and magnesium chloride
(Mirchamsy et al., 1978; Rapp et al., 1965). MMR-silk films were
then prepared from solutions of MMR, silk, and stabilizer. The
initial recovery of MMR-films stabilized with 1M MgCl.sub.2, 1M
MgSO.sub.4 and 70% sucrose is shown in FIG. 4. Although MgCl.sub.2
is an excellent stabilizer for OPV, it had negatively stabilizing
effects on measles, mumps, and rubella. MgSO.sub.4 provided the
best stabilization of the measles component of the vaccine but
little effect on mumps and had negative effects on rubella.
Sucrose, however, acted as a stabilizer for all three components of
the MMR vaccine. Furthermore, the initial recovery of the viral
components stabilized in sucrose shows an improvement over the
initial recovery of MMR-silk only films (Table 1). Also,
reconstitution of the vaccine in 70% sucrose solution in RT (FIG.
5) showed higher retained viral potency over 24 hr than the vaccine
reconstituted in water at RT (FIG. 2). Compared to MMR vaccine
reconstituted in water, vaccine reconstituted in 70% sucrose also
provided better stability at 4.degree. C. and 37.degree. C. (FIG.
6). Using stabilizing additives such as sucrose may minimize the
initial loss of vaccine activity during the film preparation. The
sucrose can stabilize the vaccine during the film drying process
while the MMR and silk are still in solution.
Example 3
Long Term Stabilization of Measles, Mumps and Rubells Vaccine
[0403] We show that vaccine-entrapped in silk films and lyophilized
silk films are significantly extended in terms of half-life at
storage temperatures as high as 45.degree. C. In the presence of
silk the degradation rate of viral proteins is reduced. Biophysical
characterization has demonstrated that the silk provides structural
stability to the vaccine by reducing residual moisture during
storage and increasing the melting point of the viral proteins.
Thus, we present a vaccine stabilizing system capable of extending
vaccine potency without the need for refrigeration. The silk can be
molded into a variety of delivery systems including films,
hydrogels, microspheres and microneedles, capable of being tailored
for specific stabilization and delivery needs. This material system
has may be used ex vivo or in vivo as delivery vehicles due to the
biocompatibility and FDA-approved history of silk use in biomedical
devices.
[0404] Residual Vaccine Activity after Immobilization in Silk
Films
[0405] The silk films were prepared with a weight ratio of vaccine
to silk of 1:1. Initial recovered vaccine potency was determined
directly after the films were prepared to determine how much
vaccine infectivity was lost during the film fabrication process.
In order to incorporate the vaccine into the silk solution, the
lyophilized vaccine must be reconstituted. The lyophilized vaccine
was reconstituted immediately prior to cell inoculation. Once the
freeze-dried vaccine has been reconstituted, potency decreases
rapidly (Galazka, 1998; WHO 2006) and according the manufacturer's
specifications, the MMR vaccine must be used within 8 hours of
reconstitution or otherwise discarded. Once completely dried, the
films, containing the same amount of lyophilized vaccine, were
dissolved in sterile water and the solution was added to the Vero
cells for potency testing. The potency results of the silk films
were compared to the potency of the lyophilized vaccine to
establish an initial recovered potency. The initial residual
potency observed from the MMR-silk films are summarized in Table 2
with 84.7%, 73.9%, and 87.0% residual potency of the measles,
mumps, and rubella components, respectively.
TABLE-US-00002 TABLE 2 Comparison of the Initial recovered viral
potency of vaccine- entrapped silk films and lyophilized silk films
Initial Recovered Vaccine-silk form Viral Component Potency (%)
MMR-silk film Measles 84.7 .+-. 6.40 Mumps 73.9 .+-. 2.24 Rubella
87.0 .+-. 2.23 Lyophilized Measles 94.7 .+-. 0.34 MMR-silk film
Mumps 89.6 .+-. 1.30 Rubella 98.4 .+-. 0.35
[0406] Enough vaccine entrapped silk films were fabricated with
this method of air-drying and stored for a 6 month study to assess
long-term stability at four different temperatures (4.degree. C.,
25.degree. C., 37.degree. C. and 45.degree. C.). However, we
hypothesized that the majority of the loss of viral potency
observed with the films occurred while the MMR-silk was in solution
for the preparation process. To verify this hypothesis, lyophilized
vaccine was reconstituted in the diluents provided by the
manufacturer and the solutions were stored at 4.degree. C.,
25.degree. C. and 37.degree. C. (FIG. 8A-8C). Potency measurements
were taken after 6 hours, 12 hours, 18 hours and 24 hours after
reconstitution. As predicted, the vaccine rapidly lost potency
within hours in solution and storage in higher temperatures
resulted in a more rapid decline in potency. Compared to the drying
time of the films, at 12 hours in solution at 4.degree. C. 53.4%,
73.4%, 76.3% residual potency remains for the measles, mumps and
rubella components, respectively. At 12 hours at 25.degree. C.,
only 57.8%, 53.1%, 46.6% residual potency of the measles, mumps,
rubella remained, respectively. A more dramatic decline was
observed for vaccine reconstituted for 12 hours at 37.degree. C. as
only 36.5%, 46.6%, and 23.9% of the measles, mumps and rubella
potency was recovered, respectively. Based on these results, we
postulated that reducing the time that the initial vaccine was in
solution would improve recovery. Therefore, lyophilized MMR-silk
films were prepared to shorten the MMR-silk solution stage. The
lyophilization process significantly improved the long-term thermal
stability of the vaccine in the silk films. As shown in Table 2,
compared to the initial potency recovered from the air-dried silk
films, the lyophilized films improved the recovery of measles,
mumps, and rubella to 94.7%, 89.6%, 98.4%, respectively
[0407] Thermostability of Vaccine-Encapsulated Silk Films
[0408] Vaccine stability was quantitatively expressed as the
residual potency observed from the films after storage. Residual
potency was measured and compared with the initial residual potency
(Table 2) to demonstrate vaccine stability. Measuring the residual
potency of all the viral components of the vaccine stored in silk
films over six months, with the exception of storage at 4.degree.
C., the general trend showed that the silk films enhanced
stabilization of the measles, mumps, and rubella viral particles by
displaying higher residual potencies when compared to the
manufacturer's lyophilized vaccine stored at the same temperature.
FIGS. 9A-9D show the comparison of residual potency of the measles
component of the vaccine for silk films and lyophilized MMR vaccine
powder stored for six months at 4.degree. C., 25.degree. C.,
37.degree. C. and 45.degree. C. With the exception of storage at
4.degree. C., the silk films showed greater residual potency of the
vaccine. Even at 4.degree. C., the residual potency of the silk
films was similar that of the MMR powder. While the residual
potency of the powder did not fluctuate, the silk films displayed
greater variance in measured potency at this temperature. For the
first 3 months the silk films outperformed the powder with greater
recovered residual potency. In the last 3 months the silk films
displayed a slight decrease in residual potency while that of the
powder remained relatively constant. At the end of the six month
study, the residual potency of the measles component of the MMR
vaccine stored in silk films was 87.2% compared to 92.2% for the
powder. The silk films, however, show improved measles residual
potency when stored at 25.degree. C., 37.degree. C. and 45.degree.
C. Stored at 25.degree. C., the silk films showed greater recovered
potency at each time point and at the end of the six months
displayed 83.9% recovered potency compared to 74.5% for the
powder.
[0409] The stabilization provided by the silk was even more evident
for films and powder stored at elevated temperatures, 37.degree. C.
and 45.degree. C. At 37.degree. C., the silk films showed a
dramatic improvement in stability of the measles infectivity
through the course of six months, resulting in 56.5% potency
recovered compared to 9.9% from the powder. In the case of films
and powder stored at 45.degree. C., the measles component of the
vaccine lost all potency after 20 weeks in storage while the silk
films retained 53.5% activity after 24 weeks. Similar trends were
shown for both the mumps (FIG. 10A-10D) and rubella components
(FIG. 11A-11D). Again, similar to the results for the measles
component, the silk films displayed more stabilization of the mumps
component at 37.degree. C. and 45.degree. C. At both temperatures
the silk films displayed a slight drop in residual activity during
the first month in storage, but beyond the first month the decrease
in residual potency was much slower than the decrease in vaccine
activity of the stored powder. At the conclusion of the study, the
silk films stored at 37.degree. C. retained 61.3% of the mumps
infectivity while the powder retained 13.0%. After 6 months of
storage at 45.degree. C., the silk films recovered 59.6% of the
mumps potency while the powder lost all potency after 20 weeks. The
rubella component displayed similar trends in potency. For silk
films stored at 4.degree. C., 25.degree. C., 37.degree. C. and
45.degree. C., 88.4%, 78.4%, 60.3%, and 58.3% residual potency was
recovered, respectively. With the exception of powders stored at
4.degree. C., these results showed that the silk provided enhanced
stabilization and maintenance of potency over the powder. The
residual potency of the powder at 4.degree. C., 25.degree. C.,
37.degree. C. and 45.degree. C. was 89.4%, 56.3%, 15.3% and 0%,
respectively.
[0410] While the lyophilized silk films were initially prepared to
improve the initial recovery of vaccine lost during the film
fabrication process, they provided even greater stabilization of
the vaccines at elevated temperature. The residual potency of the
measles, mumps, and rubella components stored in the lyophilized
silk films are shown in FIGS. 12A-12D, 13A-13D, and 14A-14D,
respectively. Regardless of the storage temperature, the
lyophilized films provided comparable levels of stabilization to
all components of the vaccine. While the lyophilized silk films
showed improved stabilization and residual potency recovered over
all the temperatures, the level of stabilization was more dramatic
at the higher temperatures. After 6 months of storage, the
lyophilized silk films retained 85.2% and 85.1% of the residual
measles potency at 37.degree. C. and 45.degree. C., respectively.
Similarly, 86.2%, and 86.0% of the mumps potency remained after 6
months when stored at 37.degree. C. and 45.degree. C.,
respectively. The rubella component appeared to be most stabilized
by the lyophilized silk films as 88.2% of the viral potency
remained after 6 months storage at 37.degree. C., while storage at
45.degree. C. still resulted in retention of 87.5% of the viral
potency.
[0411] Evaluation of Thermostability by Kinetics of Degradation
[0412] The degradation rate, k.sub.obs, for each temperature was
calculated by linear regression of the log.sub.10 drop of virus
titer against the exposure time in weeks. The slopes of the
resulting curves represent the degradation rates. The virus
half-lives (t.sub.1/2) at each temperature and the corresponding
95% confidence interval were then calculated from the k.sub.obs and
their standard errors. The virus half-life is the predicted
half-life or time required for the average potency to decrease to
50% of the initial value. The estimated degradation rates and
corresponding half-lives for each viral component and vaccine
encapsulation method are summarized in Table 3 and the half-lives
are plotted against temperature in FIG. 16A-16C. The degradation
rates of the three systems over the range of temperatures tested
also fitted well on the Arrhenius plot (FIG. 14A-15C). As seen from
the plot, the values for the slope of the powder vaccine form were
consistently higher than both the silk systems, indicating the
rates of degradation of the powder increased faster with an
increase in storage temperature.
TABLE-US-00003 TABLE 3 Summary of degradation rates and predicted
half-lives of the vaccine powder, and silk film- and lyophilized
silk film-encapsulated vaccines Predicted Half-life, Temperature
Degradation rate, k.sub.obs t.sub.1/2 (weeks) (.degree. C.) Vaccine
System (log.sub.10 TCID.sub.50/weeks) (Lower, Upper Limit) Measles
4 Powder 0.009 .+-. 0.0005 178.6 weeks (169.2, 197.4) Films 0.0136
.+-. 0.0025 96.6 weeks (81.6, 126.6) Lyophilized Films 0.009 .+-.
0.0001 135.3 weeks (133.8, 138.3) 25 Powder 0.0331 .+-. 0.0037 47.1
weeks (42.4, 56.6) Films 0.0123 .+-. 0.0002 100.8 weeks (99.1,
104.0) Lyophilized Films 0.0098 .+-. 0.0013 123.8 weeks (109.8,
151.8) 37 Powder 0.1287 .+-. 0.0004 9.4 weeks (9.4, 9.5) Films
0.0458 .+-. 0.0034 21.9 weeks (20.4, 25.0) Lyophilized Films 0.0131
.+-. 0.0008 93.8 weeks (88.4, 104.6) 45 Powder 0.139 .+-. 0.0261
5.1 weeks (4.3, 6.7) Films 0.0401 .+-. 0.0010 19.8 weeks (19.4,
20.8) Lyophilized Films 0.0128 .+-. 0.0019 94.9 weeks (82.6, 119.4)
Mumps 4 Powder 0.0173 .+-. 0.0005 102.3 weeks (99.4, 108.0) Films
0.0302 .+-. 0.005 51.3 weeks (44.0, 65.9) Lyophilized Films 0.0091
.+-. 0.0018 152.1 weeks (127.0, 202.3) 25 Powder 0.0677 .+-. 0.0001
26.5 weeks (26.5, 26.6) Films 0.0408 .+-. 0.0056 34.3 weeks (30.2,
42.6) Lyophilized Films 0.0106 .+-. 0.0005 129.3 weeks (123.5,
140.4) 37 Powder 0.1435 .+-. 0.0052 10.7 weeks (10.3, 11.5) Films
0.0439 .+-. 0.001 25.7 weeks (25.1, 26.8) Lyophilized Films 0.0129
.+-. 0.0025 106.0 weeks (88.8, 140.4) 45 Powder 0.1967 .+-. 0.0357
8.2 weeks (6.9, 10.7) Films 0.0384 .+-. 0.0021 28.6 weeks (27.1,
31.6) Lyophilized Films 0.0121 .+-. 0.0027 111.9 weeks (91.5,
152.7) Rubella 4 Powder 0.0119 .+-. 0.0022 124.5 weeks (105.1,
163.3) Films 0.0101 .+-. 0.0012 123.6 weeks (110.5, 149.9)
Lyophilized Films 0.0036 .+-. 0.0001 383.7 weeks (373.3, 404.4) 25
Powder 0.0521 .+-. 0.0021 29.5 weeks (28.3, 31.8) Films 0.022 .+-.
0.0024 56.8 weeks (51.2, 68.0) Lyophilized Films 0.0121 .+-. 0.0012
117.3 weeks (106.7, 138.5) 37 Powder 0.1192 .+-. 0.0006 10.9 weeks
(10.8, 11.0) Films 0.0387 .+-. 0.0004 28.9 weeks (28.6, 29.5)
Lyophilized Films 0.0144 .+-. 0.0037 99.3 weeks (79.0, 139.8) 45
Powder 0.1681 .+-. 0.0298 8.2 weeks (6.9, 10.6) Films 0.0387 .+-.
0.0003 27.3 weeks (27.1, 27.7) Lyophilized Films 0.0144 .+-. 0.0025
97.1 weeks (82.8, 125.9)
[0413] With the exception of samples stored at 4.degree. C., the
silk- and lyophilized silk-encapsulated vaccine systems exhibited
lower degradation rates over the powder vaccine at 25.degree. C.,
37.degree. C. and 45.degree. C. for the measles, mumps and rubella
components. The decreased degradation rates correspond to an
increase in predicted half-lives. From the Table, the trends appear
similar for all the viral components and show that at elevated
temperatures, the silk films increased the half-lives of the
vaccine over the powder formulation with the lyophilized silk films
showing dramatic improvements of vaccine half-lives over both the
powder and silk films. Furthermore, as the storage temperature was
increased, the degradation rates and predicted half-lives
significantly increased and decreased, respectively, for both the
powder and silk film formulations. The lyophilized silk film
samples, however, maintained slow degradation rates across all the
tested storage temperatures. Evaluating the change in rate of
degradation of the measles component from 4.degree. C. to
45.degree. C., the powder vaccine exhibited a 1.444% increase in
degradation rate from 0.0136.+-.0.0025 to 0.0401.+-.0.0010
log.sub.10 TCID.sub.50/weeks, while the MMR-silk film and
lyophilized MMR-silk film had a 195% increase from
0.0.139.+-.0.0261 to 0.009.+-.0.0005 log.sub.10 TCID.sub.50/weeks
and 42% increase from 0.0128.+-.0.0019 to 0.009.+-.0.0001
log.sub.10 TCID.sub.50/weeks in degradation rate over the
temperature range, respectively. The mumps and rubella components
exhibit similar trends. With the exception of storage at 4.degree.
C., in the powder vaccine data shows a predicted measles half-life
of 178.6 weeks, greater than the 96.6 weeks for the silk films and
the 135.3 weeks of the lyophilized silk films, the silk films and
lyophilized silk films displayed a much more dramatic increase in
half-life of the virus at elevated temperatures. The difference was
especially evident in the predicted half-life of the virus at
37.degree. C. and 45.degree. C. At 35.degree. C., the 21.9 weeks
half-life of the silk films was a significant improvement of the
9.4 weeks provided by the powder but the lyophilized silk films
dramatically increased the half-life to 93.8 weeks. Storage at
45.degree. C. provided similar results as the powder, silk films
and lyophilized silk films showed half-lives of 5.1, 19.8, and 94.9
weeks, respectively.
[0414] The temperature dependence of the vaccine degradation rates
was further evaluated using regression analysis. From plot of drop
of virus titer against time (not shown) for the powder, silk film
and lyophilized silk film systems, the data can be reasonably
approximated by a straight line, exhibiting pseudo zero-order
behavior. The plot of virus titer drop against time corresponds to
and exhibits similar trends to the residual potency curves. As seen
from the plot, a linear response of change in viral concentration
suggests that the degradation mechanism follows zero-order
kinetics. Though upon closer inspection of the data, the linearity
of the drop of virus titer against time decreases at long time
intervals. These data suggest that the degradation rate may deviate
from zero-order behavior and decrease over longer periods of time.
The kinetic model of the degradation of the vaccine over time is
therefore considered a pseudo zero-order model. Pseudo reaction
orders are encountered in drug stability studies as only the change
in concentration of the active pharmaceutical ingredient is usually
monitored over time, while other reactants and buffer components
are in large excess though not analyzed (Zhou et al., 2009). Hence,
a first-order reaction can appear to be a zero-order reaction, or
pseudo zero-order. In zero-order reactions, the rate of the
reaction does not depend on the concentration of the reactant and
is constant. The zero-order rate equation is as follows
- C t = k obs ##EQU00001##
and the data obtained at each temperature was fitted using the
following equation:
C t C 0 = - k obs t ##EQU00002##
where C.sub.t is the measured potency in log.sub.10 TCID.sub.50 at
time t, C.sub.0 is the measured potency at time zero, k.sub.obs is
the degradation rate determined by linear regression and t is the
time of storage in weeks. The predicted half-life, t.sub.1/2, is
the time required for the average potency to decrease to 50% of the
initial value. The half-life for each temperature was estimated by
the equation
t 1 / 2 = C 0 2 k obs ##EQU00003##
Thermal degradation rates of the three vaccine systems should
follow the Arrhenius law expression with respect to absolute
temperature as shown in the following equation:
k(T)=Ae.sup.-E/RT
where k is the thermal degradation rate, A is the preexponential
factor, E is the activation energy, R is the gas constant and T is
the absolute temperature. A plot of degradation rates against the
reciprocal of absolutely temperature can show the
temperature-dependent degradation trends over large temperature
ranges. The Arrhenius plots show that the powder vaccine has the
steepest slope, indicating a greater dependence of degradation on
temperature than both of the silk systems. By comparison, the more
shallow slopes of the silk systems demonstrate that the silk
greatly reduces the degradation rate of the vaccine at elevated
temperatures and is capable of maintaining vaccine potency at high
ambient temperatures. As seen in the comparison of half-lives of
the three vaccine systems, the improved half-lives of the silk
systems become more evident and dramatic as the storage temperature
increases.
[0415] Effect of Processing Conditions on Residual Moisture of
Vaccine Systems
[0416] The MMR vaccine is supplied in freeze-dried form and the
presence of stabilizers in the vaccine to reduce the moisture
content have greatly improved their thermal stability (Galazka et
al., 1998). The shelf life of lyophilized vaccines is dependent on
both adherence to the cold chain and maintenance of low residual
moisture content. As the storage temperature increases, so does the
amount of water present in the air. The MMR-silk films were
fabricated and air-dried at ambient conditions therefore had a
higher initial residual moisture than both the lyophilized MMR
powder and the lyophilized MMR-silk films. The residual moisture of
the MMR-silk films calculated immediately after determination of a
dried state was determined to be 4.42%.+-.0.65. The residual
moisture of the MMR powder and lyophilized MMR-silk films were
calculated to be 2.39%.+-.0.23 and 1.89%.+-.0.13, respectively.
With the exception of MMR-silk films, storage of the lyophilized
vaccine systems after six months at 4.degree. C. produced very
little change in residual moisture. In fact, the residual moisture
of the lyophilized films stored for six months at all the
temperature points exhibited very little aberration from the
initially measured value, displaying only a net increase of 6.9% in
residual moisture from the initial value to the highest recorded
value at 45.degree. C. Due to the processing conditions, the
MMR-silk films had higher residual moisture, but the net increase
to the highest residual moisture value recorded at six months of
storage at 45.degree. C. was only 30.1% compared to the 61.5%
increase shown in the MMR powder system. The residual moisture
content of the vaccine systems corresponded to the residual
potency. At the elevated temperatures, the MMR powder exhibited
greater losses in potency than the silk films, correlating well
with the greater increase in residual moisture. Similarly, the
minimal increase of residual moisture of the lyophilized MMR-silk
films resulted in a greater retention of viral potency.
TABLE-US-00004 TABLE 4 Comparison of the residual moistures of the
MMR powder, MMR-silk films and lyophilized MMR- silk films at
specific storage conditions. Vaccine System Storage Condition
Residual Moisture (%) MMR Powder At t = 0 (Initial Condition) 2.39
.+-. 0.23 After 6 months in 4.degree. C. 2.37 .+-. 0.15 After 6
months in 25.degree. C. 2.73 .+-. 0.54 After 6 months in 37.degree.
C. 3.52 .+-. 0.35 After 6 months in 45.degree. C. 3.86 .+-. 0.28
Lyophilized At t = 0 (Initial Condition) 1.89 .+-. 0.13 MMR-silk
films After 6 months in 4.degree. C. 1.81 .+-. 0.20 After 6 months
in 25.degree. C. 1.97 .+-. 0.43 After 6 months in 37.degree. C.
1.83 .+-. 0.35 After 6 months in 45.degree. C. 2.02 .+-. 0.52
MMR-silk films At t = 0 (Initial Condition) 4.42 .+-. 0.65 After 6
months in 4.degree. C. 5.26 .+-. 0.51 After 6 months in 25.degree.
C. 5.47 .+-. 0.53 After 6 months in 37.degree. C. 5.53 .+-. 0.70
After 6 months in 45.degree. C. 5.75 .+-. 0.91
[0417] Structural Characterization of Vaccine-Silk Systems
[0418] To investigate how the silk provided enhanced
thermostability to the vaccines, physical measurements using
calorimetry and light scattering were studied. DSC (FIG. 17) was
used to demonstrate that the silk encapsulant provided
stabilization to the vaccine by an increase in the glass transition
(Tg) of the vaccine. Solid-state DSC of lyophilized silk films
showed a Tg at 178.degree. C. The DSC thermogram of the MMR powder
showed a Tg at 68.9.degree. C. while the lyophilized MMR-silk films
showed a Tg at 89.2.degree. C. as well as two peaks at
116.6.degree. C. and 164.8.degree. C. that could indicate a Tm,
melting temperature, and Td, degradation temperature. It is unclear
whether the shift in Tg was due to structural stabilization by the
silk or the interaction between silk and the various excipients
present in the vaccine sample. The vaccine was then purified to
remove the added excipients. The resulting solution contained
purified viral particles suspended in sterile, nuclease-free water.
As the viral sample is a liquid solution, nano-DSC was run on these
samples. The nano-DSC thermogram (FIG. 18) of the purified viral
particles in water showed a Tm at 16.8.degree. C., indicating the
viral proteins were undergoing a conformational change. The
solution of purified viral particles in silk showed an elevated Tm
at 68.3.degree. C. While the range of the nano-DSC does not extend
far enough to show the Tg of the silk, a thermogram of silk
solution is still shown to illustrate that neither of the Tg values
can be attributed to a change in the silk structure. To investigate
whether the Tm corresponded to viral particle denaturation due to
aggregation, the size of the viral particles was examined by DLS
(FIG. 19). The average mean effective diameter of a naked viral
particle is around 250 nm. This is consistent with reported values
of measles, mumps and rubella (Russell et al., 1967; Hall and
Martin, 1973). The results indicated that the purified virus
solution showed an increase in mean effective diameter around
16.degree. C., indicating the presence of protein aggregation. The
DLS of the viral particles in silk solution, on the other hand, did
not show an increase in the mean effective diameter until around
70.degree. C., showing protein aggregation at a more elevated
temperature. The results of the DLS correspond well with the
results of the nano-DSC as the aggregation detected by light
scattering occur within the temperature range of the protein
unfolding measured by DSC.
[0419] Vaccine Release from Silk Films, Lyophilized Silk Film, Silk
Hydrogels and Silk Microspheres.
[0420] In order to demonstrate that silk provides not only
stabilization of the vaccine but also control over vaccine release
kinetics, release studies were conducted with a variety of silk
formulations. Each silk formulation (film, lyophilized film,
hydrogel and microsphere) contained the same initial amount of
vaccine. Cumulative release profiles of the vaccine-encapsulated
silk film and lyophilized silk films are shown in FIG. 20A-20B. In
vitro release studies for these two systems were conducted with
single films placed between two blocks of gelatin hydrogels,
allowing for the release of vaccine as the silk films dissolved
into the hydrogels. Vaccine-entrapped silk films prepared from 8%
(w/v) silk solution exhibited a slight burst release, typical of
diffusional release. At the last collection of 6 hours, nearly all
the encapsulated vaccine was released as 92.43% of the vaccine was
recovered and active. The silk films prepared with 4% (w/v)
exhibited a similar release profile, only the burst release was
faster as the mass released neared complete release more rapidly.
The lyophilized silk films showed a more pronounced burst effect as
the 8% silk films released 44% and the 4% films released 56% of the
loaded MMR within 10 minutes of placement in the hydrogel. The
lyophilized silk films released nearly all the MMR loaded within 90
minutes. The silk hydrogels and microsphere preparations exhibited
a much slower release by comparison, on the order of days (FIG.
21A-21B). The 4% and 8% silk hydrogels extended the release of
vaccine to 8 days. Furthermore, the 16% silk hydrogels were able to
extend the release further as only 73.53% of the vaccine was
released by day 8. Vaccine release from silk microspheres showed
greater potential as an extended and sustained release system.
Similarly, increasing the silk concentration of the microspheres
slowed the release rate; the 16% silk microspheres released 65.35%
of the loaded vaccine by day 8. The relative linearity of the
release from the 16% silk microspheres is also of interest as the
regression coefficient (R.sup.2) for the curve was 0.988,
indicating nearly zero-order release.
[0421] Biophysical characterization of the measles, mumps, and
rubella vaccine through use of calorimetry and light-scattering
allowed for a description of the physical stabilization of the
virus as a function of elevated temperature during vaccine storage.
Due to the temperature-sensitivity of the vaccines, the process of
distribution and storing vaccines is fragile as these issues often
come with considerable logistical cost (Zweig, 2006). It is known
that the immobilization of bioactive molecules such as enzymes
leads to an increase in stability while improving handling.
Immobilization is important as is maintaining constant
environmental conditions in order to protect bioactive molecules
against changes in pH, temperature or ionic strength (Kumakura,
1995). The chemistry, structure and assembly of silk generates a
unique nano-scale environment and makes this protein polymer an
attractive candidate for the stabilization of bioactive molecules
over extended periods of time (Jin and Kaplan, 2003). Without
chemical processing, silk can be used to entrap bioactive molecules
in the amiphilic, self-assembly domains from aqueous solution.
[0422] MMR.RTM. II, a live attenuated measles, mumps, and rubella
vaccine, is provided as a lyophilized preparation to be
reconstituted at time of use. Prior to reconstitution, the vaccine
must be stored at 2 to 8.degree. C. and will be stable for 24
months, and once reconstituted must be used within 8 hours. The WHO
requirement for heat stability of measles, mumps and rubella
vaccines employs two indices of stability: 1) the vaccine should
retain at least 1,000 live virus particles in each human dose after
incubation at 37.degree. C. for seven days; and 2) the virus titre
should not have decreased by more than 1 log.sub.10 during storage
(WHO, 1982; WHO 1994). Live viruses vaccines, unlike other vaccine
forms, depend on their immunogenicity to establish an appropriate
immune response, which requires the retention of a sufficient
numbers of live viral particles. Storage of these thermally labile
viral particles requires the addition of stabilizers. Each dose of
MMR.RTM. II is stabilized in 14.5 mg sorbitol, sodium phosphate,
1.9 mg sucrose, sodium chloride, 14.5 mg hydrolyzed gelatin,
.ltoreq.0.3 mg recombinant human albumin, <1 ppm fetal bovine
serum, other buffer and media ingredients and approximately 25
.mu.g of neomycin.
[0423] Excessive exposure to temperatures above recommended storage
conditions can damage vaccines in a number of ways, most notably
alteration of the tertiary structure of the viral proteins,
reducing viral infectivity and thereby decreasing vaccine potency
(Chen and Kristensen, 2009). Changes in protein structure can lead
to aggregation and altered cellular uptake, affecting vaccine
activity (Brandau et al., 2003; Manning et al., 1989; Middaugh,
1996). Measles and mumps belong to the Paramyxovirus family,
characterized as an enveloped virus containing the nucleocapsid
that encloses the single-stranded negative-sense viral RNA
surrounded by fusion (F) and haemagglutinin (H) glycoproteins
expressed on the surface of the virus (FIG. 22A) (Kingston et al.,
2008; Woelk et al., 2002) Similarly, rubella belongs to the
Togavirus family and is an enveloped virus with two virus-specific
glycoproteins E1 and E2 that surround a icosahedral nucleocapsid
enclosing single-stranded positive-sense viral RNA (Dorsett et al.,
1985; Nakhasi et al., 1991).
[0424] The method of infection of paramyxoviruses involve the
attachment of the virus to the CD46 and CD150 receptors of host
cells by interaction between both the haemagglutinin (H)
glycoprotein and fusion (F) glycoprotein (FIG. 22C) (Wild et al.,
1991; Malvoisin and Wild, 1993; Moss and Griffin, 2006). Fusion of
the virus and cell allow for viral entry and release of viral
nucleic material into the cell. The main cause of viral
inactivation is disruption of viral surface proteins and stresses
such as elevated temperatures can induce conformational changes in
the viral proteins (Rexroad et al., 2006; Ausar et al., 2006).
These conformational changes may affect the stability of the virus
by inducing viral particle aggregation that prevents cell binding
and uptake, thus leading to virus inactivation (FIG. 22D) (Ohtake
et al., 2010). Developing a vaccine formulation that is resistant
to heat damage and improving thermostability of the vaccine would
have major benefits including extending the shelf-life, decreasing
the loss of vaccines and reducing dependency on cold-chain
requirements.
[0425] The improvement in vaccine stability provided by silk
protein was more pronounced at the elevated temperatures of
37.degree. C. and 45.degree. C., while the stability was comparable
or slightly better than the manufactured powder form stored at
4.degree. C. and 25.degree. C. The lyophilized vaccine is
stabilized with a variety of excipients that according to the
manufacturer should remain stable and active for at least two
years. Vaccine spoilage, however, occurs when the cold chain is
broken and the vaccine is stored above refrigeration temperature.
In such cases, silk would provide sufficient stability to the
vaccine to maintain stability over a range of environmental
conditions. Lyophilization of the MMR-silk films improved initial
vaccine potency recovered as the longer the viral particles
remained in the hydrated state, the more likely they are to be
exposed to degradative reactions such as hydrolysis and deamidation
that may contribute to instability (Li, 1994). The continued
enhanced stabilization provided by the lyophilized silk films over
six months in storage could also be due in part to the conditions
of storage for the lyophilized films. While the regular silk films
were stored in Eppendorf tubes, the lyophilized films were stored
in vacuum-sealed vials with low residual moisture content.
[0426] Evaluation of the experimental potency data by kinetic
models of unfolding and degradation serves to illustrate the
projected accelerated stability of the different vaccine systems
under select temperatures while also relating the potency data to
the temperature-induced conformational changes to be explained by
the biophysical characterizations. Under accelerated stability
study conditions, the intrinsic stability of protein
pharmaceuticals is often decreased dramatically. At elevated
temperatures, protein unfolding rates increase and most chemical
interactions involved in destabilization of the protein are
accelerated as compared to at lower temperatures (Creighton, 1990).
Accelerating the degradation process via storage at elevated
temperatures allows for the assessment of silk as a suitable
candidate stabilizer to inhibit the inactivation process.
Examination of the potency data suggested that the degradation
observed by the three vaccine systems could be fitted to a
zero-order kinetic system. Observation of a concentration-time
profile of zero-, first-, and second-order reactions show that
zero-order kinetics always predict the greatest degree of
composition as a function of time amongst the three. Therefore,
when predicting the order of a reaction, assumption of a zero-order
kinetic model will predict the maximum amount of degradation that
could occur at some future time (Zhou et al, 2009). Therefore, in
assuming the degradation of the MMR virus is pseudo zero-order,
using zero-order kinetics to model the degradation profile, we
predicted the most conservative estimates for the half-life of the
vaccine-encapsulated silk film and lyophilized silk film
systems.
[0427] The general trend observed for all three virus components
was that both types of the silk films decreased the degradation
rates of the viruses and the lyophilized silk systems exhibited the
lowest degradation rates, and thus largest half-lives, amongst the
three systems, while the powder form of the vaccine degraded the
fastest at all the temperature points tested. Deviation from this
trend was observed in the 4.degree. C. samples as the powder
vaccines show a lower degradation rate with a high predicted
half-life over the non-lyophilized silk films. A possible
explanation for the results is that the powder vaccine has the
advantage of lyophilized preparation over the regular silk films.
During the film preparation, additional moisture was introduced to
the encapsulated vaccine that increases the intermolecular mobility
and chances for degradation within the silk films compared to the
manufacturer-provided vaccine powder. Undoubtedly, the silk films
provide increased stability at elevated temperatures even with the
handicap of additional moisture. However, the lyophilized vaccine
powder has been formulated to remain stable at the optimum
temperature range of 2 to 8.degree. C. This advantage of
lyophilization could also explain the success of the lyophilized
silk films at stabilizing the vaccine as they provide the stability
offered by both the silk and lyophilization.
[0428] In addition to the kinetic studies performed using the
potency data, biophysical characterizations using calorimetry and
light scattering were conducted to corroborate the results. Due to
the high glass transition temperature of silk fibroin, 178.degree.
C., silk protein is thermodynamically stable once self-assembled
into the .beta.-sheet conformation. These features provide an
environment to stabilize the vaccine. The vaccine-silk solution
consisted of vaccine molecules in native form and the silk fibroin
mainly present in random coils. Once the films are cast, there is
some conversion of the fibroin into .beta.-sheets, which contain
hydrophobic regions, while the random coils contain the more
hydrophilic regions. The vaccines seem to be stabilized in this
environment, possibly due to interactions with the fibroin chains
or the constraints on chain mobility (FIG. 21B). The sold-state DSC
(FIG. 17) showed a Tg of the MMR powder at 68.9.degree. C. This
value, however, can be misleading as it may not reflect the Tg and
corresponding structural change of the viral particles themselves
but rather an averaged value from the contributions of the various
protein excipients and stabilizers already present in the MMR
powder vaccine formulation. MMR-silk film showed a Tg at
89.2.degree. C., indicating a shift in the Tg of the vaccine due to
the presence of the silk. The thermogram of the lyophilized
MMR-silk film, however, also showed the presence of two exothermic
peaks at 116.6.degree. C. and 164.8.degree. C. The peak at
116.6.degree. C. was mostly likely a Tm, indicative of protein
unfolding, while the second peak was degradation, Td. It can not be
determined whether the unfolding and degradation contributed to the
viral proteins or the various excipients present in the vaccine
formulation. It is, therefore, difficult to conclusively say that
the increase in Tg of the vaccine in the presence of silk was
directly contributing to the structural stability of the viral
particles.
[0429] In order to clarify the situation, the vaccine was purified
to remove excipients. Since the result of the purification is a
liquid preparation, the viral particles are in a less stable
environment. Therefore, the viral particle solution was stored at
-80.degree. C. until used. The Tm by nano-DSC appeared when a
protein unfolds, exposing the hydrophobic and hydrophilic regions
to the aqueous buffer solution. Adjacent hydrophobic protein
molecules will aggregate to shield these regions from the
surrounding aqueous solution. Because the unfolded state of
proteins have more surface area than the native state, the degree
of preferential exclusion offered by the stabilizing agent of silk
from the less structured state would raise the chemical potential
of this stabilized form even above that of the native state
(Brandau et al, 2003). This increased stability can be reflected in
the increase of the midpoint of the unfolding transition of the
proteins, Tm. The elevated Tm of the vaccine-encapsulated silk
solution is due to structural stabilization provided by the silk to
prevent viral protein denaturation and aggregation. The Tm value at
16.8.degree. C. is likely due to the unfolding of the viral surface
glycoproteins (F and N of measles and mumps and E1 and E2 of
rubella) due to the elevated heat applied to the viral particles.
This denaturation most likely leads to the aggregation of the viral
particles (FIG. 22D), preventing them from binding and fusing with
the animal cells (FIG. 22C), leading to loss of infectivity of the
viral particles and vaccine as a whole. This associated aggregation
is shown on the DSC thermogram as the sharp change in heat flow
immediately following the Tm. It appears the aggregation, an
exothermic process, is a direct result of the endothermic unfolding
of the protein. The broadness of the Tm peaks is most likely a
result of the combined contribution of the unfolding of several
proteins present in the sample. Furthermore, the protein
aggregation may also contribute to the broad peaks by overlapping
with the denaturational endotherm (Packer et al., 2002). The
infectivity of MMR is dependent on the conformational stability of
the viral proteins (Kissman et al, 2008). The increase in Tm by the
presence of silk molecules indicates that the silk provided
structural stability to the viral proteins that shielded them from
thermal denaturation. The interaction between the viral particles
and the silk hydrophobic regions, as well as limited chain
mobility, might prevent viral protein aggregation (FIG. 22B),
thereby preserving viral and vaccine infectivity.
[0430] Unfolding of a protein is usually associated with an
increase in its hydrodynamic size as the partial or fully unfolded
proteins states are unstable and form aggregates (Roberts, 2007).
The results of the light scattering indicated that the naked viral
particles aggregated at a much lower temperature than a solution of
viral particles in silk. The onset of particle aggregation as
verified by DLS occurs around the same temperature of protein
unfolding as shown by DSC, indicating the protein unfolding
directly resulted in aggregation. The DLS results corroborate the
results of the DSC that the silk provides structural stability to
the viral particles, particularly to the viral surface
glycoproteins, preventing intermolecular collisions and thus
minimizing aggregation at elevated temperatures.
[0431] Humidity can also have a significant effect on vaccine
products as the excess water introduced to the system can lead to
increase in mobility and corresponding reactivity of the viral
proteins (Waterman and Adami, 2005). The residual moisture analysis
of the films reveal that over the course of the stability testing
the increase in residual moisture, especially in the high
temperature ranges, is most likely due to the lack of
moisture-controlled environment provided by the Eppendorf tubes.
While the MMR powder and lyophilized MMR-silk films were stored in
low humidity conditions as they are extremely hygroscopic, provided
by the lyophilization vials and stopper, and sealed in a
nitrogen-rich environment, the MMR-silk films were stored in
Eppendorf tubes that would allow greater chances for absorption of
the atmospheric humidity into the containers than the vacuum-sealed
vials. The increased water activity in the Eppendorf tubes is also
due to the amount of initial water associated with the silk films,
already at a higher level than both the powder and lyophilized
films, and the initial packaging relative humidity. The increase in
residual moisture exhibited in the silk films at elevated
temperatures could also be explained by possible water desorption
from the silk, that though allowed to air dry still contain trace
amounts of intermolecularly bound water molecules within the silk
matrices (Hu et al., 2007). Moreover, for the silk films stored at
room temperature, the variability of the atmospheric humidity
dictated by the weather patterns would affect the relative humidity
inside the Eppendorf tubes.
[0432] Though the effect of residual moisture on the vaccine was
taken into consideration, the silk films were stored in Eppendorf
tubes simply to show that with minimal processing conditions and
with no special storage consideration, vaccine-entrapped silk films
are able to display enhance stability at elevated temperatures over
commercially available lyophilized vaccines tested at the same
temperatures. The mobility increase due to moisture and temperature
would explain how the lyophilized silk films exhibited such
dramatic increases in stability of the regular silk films. The
lyophilization process and low moisture storage of the lyophilized
films significantly limited the mobility of the viral proteins at
high temperatures, causing the viral proteins to be resistant to
thermally-induced unfolding and the associated aggregation. Even
though the absolute residual moisture of the silk films is higher
than that of the MMR powder, the percent increase of the residual
moisture in the powder over the tested temperature range is greater
than that observed in the silk films. The increase in temperature
appears to have had a greater impact in the temperature-induced
moisture in the powder than the silk films. It would appear the
silk provided an inhibition of molecular mobility during storage to
prevent protein unfolding and subsequent aggregation whereas the
increased water activity in powder resulting from
temperature-induced moisture, without the conformational stability
provided by the silk, increased viral protein aggregation.
[0433] Release studies were carried on a variety of
vaccine-entrapped silk delivery vehicles to show that silk was able
to stabilize the vaccine and control release kinetics.
Vaccine-loaded silk films and lyophilized silk films were untreated
and water-soluble. They have the potential to be molded into
delivery formats such as microneedles, a safe and pain-free
alternative to transdermal drug delivery over hypodermic needles
(Tsioris et al., 2011). Conceivably, a patch could be applied to
the skin and the vaccine-loaded silk microneedles would puncture
the skin, the silk needles would dissolve and release the vaccine
subcutaneously. The release profile of the silk films show promise
toward this goal. The release studies for the silk films and
lyophilized silk films were conducted in a gelatin hydrogel due to
a consistency analogous to tissue (Wightmas et al., 2007). The silk
films cast from 4% and 8% silk displayed similar release profiles
(FIG. 20A-20B), showing an initial burst of release followed by a
decreased rate of release. The 4% silk film exhibited a more rapid
release due to the lower concentration of silk protein, allowing
the vaccine to diffuse faster from the matrix while also allowing
the film to dissolve more rapidly. As the silk concentration
increased to 8%, the release rate slowed due to the increased
.beta.-sheet content of the film, forming a more rigid matrix and
slowing the diffusion of vaccine into the hydrogel. The release
profiles of the lyophilized silk films showed a more pronounced
initial burst effect. Rather than the vaccine quickly diffusing
from the silk upon initial contact with the hydrogel, the initial
burst from the lyophilized films was most likely due to the rapid
dissolution of the lyophilized films. The subsequent release was
likely due to the diffusion of the vaccine from the undissolved
film. For the same reason as the silk films, the 4% lyophilized
silk films exhibited a faster release profile than the 8%. While
the time scale of silk film release was on the order hours, 96.85%
of the encapsulated MMR was released by 90 minutes.
[0434] Vaccine-silk delivery vehicles were also fabricated in
insoluble formats of silk hydrogels and microspheres. These forms
could be incorporated into injectable vaccine delivery that forms a
subcutaneous vaccine depot able to slowly release the vaccine over
an extended period of time. The hydrogels and microspheres released
the vaccine over a period of days. The slower drug release can be
explained by diffusion of the vaccine, limited by the increased
.beta.-sheet content of the hydrogels and microspheres. Also, as
the concentration of the hydrogels and microspheres was increased,
the linearity of release of improved, corresponding to a decrease
in release rate. Increasing the silk concentration of the hydrogels
resulted in a decrease in vaccine release rate. Increasing the silk
concentration of the MMR-silk microspheres, however, slowed the
release rate and the release rates became more linear, approaching
zero-order. The 8% microspheres had a regression coefficient
(R.sup.2) of 0.95 and the 16% silk had a value of 0.988. The
increased linearity of vaccine release from microspheres could be
due to the small volumes that generate a smaller diffusion gradient
for the vaccine particles to cross into the bulk phase.
[0435] These results indicate that these silk entrapped vaccine
systems can be fabricated to provide thermal stability for vaccines
at elevated temperatures, while also functioning as a controlled
and sustained vaccine delivery system. Entrapment of MMR vaccine in
lyophilized silk films showed enhanced stability at elevated
temperatures outside the recommended cold-chain for a period of
time well beyond that of the manufacturer's vaccines. The choice of
silk as a encapsulating polymer also supports the safety of the
implantable or injectable vaccine-silk system since silk is a
biocompatible, biodegradable and FDA-approved biomaterial (Altman
et al, 2003; Horan et al, 2005).
[0436] Vaccine-encapsulated silk films and lyophilized silk films
provide a highly effective carrier for the long-term
thermostabilization of the measles, mumps and rubella vaccine. Both
silk film systems were able to increase the half-lives of all three
viral components of the vaccine compared to the manufacturer
supplied vaccines at 25.degree. C., 37.degree. C. and 45.degree. C.
The silk reduces the temperature-induced viral protein unfolding
and subsequent aggregation by reducing the residual moisture of the
samples during storage at elevated temperatures and also providing
structural stability to the vaccine to elevate the temperature at
which the viral proteins denature. Furthermore, the silk carriers
can be fabricated into different delivery vehicles capable of
tailoring the release kinetics of the vaccine. This silk carrier
system provides a novel vaccine delivery system easily fabricated
without special processing considerations and capable of
maintaining the potency of the vaccine without the need for strict
adherence to the cold chain.
Example 3
Methods
[0437] Trivalent Vaccine.
[0438] For potency estimation, we used a commercial source of
trivalent measles, mumps, rubella vaccine MMR.RTM. II (Merck &
Co., Inc., USA), a sterile lyophilized live virus vaccine
containing the Enders' attenuated Edmonston measles, the Jeryl Lynn
mumps and Wistar RA 27/3 rubella. Prior to use, the vaccine was
reconstituted in diluent and each 0.5 mL dose contained no less
than 1,000 TCID.sub.50 (tissue culture infectious dose) of measles
virus; 12,500 TCID.sub.50 of mumps virus; and 1,000 TCID.sub.50 of
rubella virus. Manufacturer conditions state that the vaccine must
be used within 8 hours of reconstitution and stored at 4.degree. C.
or otherwise be discarded. Each 0.5 mL dose contains sorbitol (14.5
mg), sodium phosphate, sucrose (1.9 mg), sodium chloride,
hydrolyzed gelatin (14.5 mg), recombinant human albumin (<0.3
mg), fetal bovine serum (<1 ppm), other buffer and media
ingredients and approximately 25 .mu.g of neomycin.
[0439] Silk Fibroin Purification.
[0440] Silk fibroin aqueous solutions were prepared as previously
described (Wang et al., 2008). Cocoons of B. mori silkworm silk
were boiled for 30 minutes in an aqueous solution of 0.02 M
Na.sub.2CO.sub.3 and then thoroughly rinsed with dI water to
extract the sericin. After drying, the silk was dissolved in a 9.3
M LiBr solution at 60.degree. C. for 4-6 hours and then dialyzed in
distilled water using Slide-a-Lyzer dialysis cassettes (MWCO 3,500,
Pierce) for 48 hours. The solution was centrifuged to remove silk
aggregates and other insoluble residues. The final concentration of
silk fibroin was approximately 9% (w/v). The solutions were then
autoclaved for sterility.
[0441] Virus Purification.
[0442] Lyophilized vaccine powder was reconstituted in sterile
water and loaded into 0.5 kDa dialysis tubing (Sigma Aldrich) and
dialyzed against a 0.15M NaCl solution to remove the excipients
from the vaccine solution. The recovered vaccine solution was then
run through a PD-10 desalting column (GE Healthcare) to remove
excess salt. The spin protocol was followed per manufacturer
specifications. The recovered purified viral particle solution was
collected and stored in an Eppendorf tube at -80.degree. C. until
use.
[0443] Vaccine Entrapment in Silk Films.
[0444] The process for vaccine-encapsulated silk film fabrication
is illustrated in FIG. 7. A mixture of sterilized 9% (w/v) silk
solution and lyophilized MMR vaccine were prepared at a
concentration of 1:1 by weight ratio of MMR to silk solution. Films
were then cast onto a Teflon-coated surface. The films were allowed
to dry in a sterile hood for 12 hours at room temperature,
protected from light. Individual films were placed in Eppendorf
tubes, under ambient conditions, and stored at 4.degree. C.,
25.degree. C., 37.degree. C. and 45.degree. C. for stability
studies.
[0445] Lyophilization of Vaccine-Entrapped Silk Films.
[0446] MMR-silk solutions (1:1 weight ratio) were aliquoted into
96-well plates and freeze dried using a VirTis 25L Genesis SQ Super
XL-70 Freeze Dryer. The samples were frozen at -45.degree. C. for
480 minutes. The primary drying occurred at -20.degree. C. for
2,400 minutes and secondary drying at 35.degree. C. for 620
minutes. The samples were held at -45.degree. C. until they were
removed from the lyophilizer. The films were then removed from the
well plates and transferred to 5 cc glass serum vials. Five mm
lyophilization stoppers were applied to the vials under nitrogen
and vacuum conditions in a MBRAUN LABmaster glovebox (Garching,
Germany) and a 5 mm crimper was used to tighten the 5 mm alum seal
on the vials. The vials were stored at 4.degree. C., 25.degree. C.,
37.degree. C., and 45.degree. C. for stability studies. The vials,
stoppers, seals and crimper were supplied by VWR (Bridgeport,
N.J.).
[0447] Vaccine Entrapment in Silk Hydrogels.
[0448] The silk solution was adjusted to concentrations of 2-5 wt %
and then autoclaved for sterility. Then 1 mL of the silk solution
was transferred into an autoclaved 2 mL Fisher glass vial, and the
solution was mixed in the glass vial for 7 minutes at 3,200 rpm
using a Fisher vortexer. The turbid solution was collected and
transferred into 2 mL Eppendorf tubes, the vaccine was gently mixed
into the solution at a 1:1 wt. ratio solution. The vaccine-silk
solution was incubated in the Eppendorf tube at room temperature
until gelation. The gels were then refrigerated for longer term
storage.
[0449] Vaccine Entrapment in Silk Microspheres.
[0450] The production of vaccine-loaded microspheres from silk/PVA
blends used the procedure described by Wang et al. (2010). Vaccine
powder was added to a 5 wt % silk solution to reach a weight ratio
of 1:1000. This solution was gently blended with a 5% (w/v) PVA
(polyvinyl alcohol, mol wt 30,000-70,000, Sigma Aldrich) stock
solution. Using a consistent weight ratio of 1:4, a 5% (w/v)
silk-PVA solution was prepared by mixing 1 mL of 5 wt % silk
solution with 4 mL of 5 wt % PVA solution. The silk solution was
autoclaved for sterility. After mixing, the solution was stirred
for 2 hours at room temperature. Then the 5% (w/v) solution was
transferred to a 35 mm Petri dish. The solution was allowed to dry
overnight in a fume hood. The dried films were dissolved in 30 mL
of ultrapure water with 10 minutes of gentle shaking at room
temperature and then centrifuge at 16,000 rpm for 20 minutes at
4.degree. C. The supernatant was discarded and the pellet
resuspended in 30 mL ultrapure water and centrifuged again. The
final pellet was suspended in 2 mL of ultrapure water.
[0451] Quantitative Real-Time RT-PCR Infectivity Assay.
[0452] A standard curve was generated by serially diluting a
solution of the only the vaccine reconstituted in sterile water.
The reconstituted vaccine, considered as the 1 log.sub.10 dilution,
and was serially diluted in 0.5 log.sub.10 steps from 1.5
log.sub.10 to 3.5 log.sub.10. The MMR-silk films (containing 1
log.sub.10 dilution of vaccine) were redissolved in an aliquot of
water and the solution was added directly to the cultured cells.
Vero cells (African green monkey kidney cells) (ATCC, Manassas,
Va.) were cultured in M199 medium with 25 mM hepes and L-glutamine
(Sigma-Aldrich, St. Louis, Mo.), 1% penicillin/streptomycin
(Invitrogen Life Technologies, Carlsbad, USA) and 5% fetal calf
serum (Invitrogen). The cells were trypsinized, counted and
adjusted to 50,000 cells/mL and plated in 24-well plates. Next, 50
.mu.L of the vaccine dilution and redissolved silk film were added
to a well of Vero cells in triplicate. The virus was allowed to
replicate in the cells for 3 days, then the RNA from the infected
cells was isolated, converted to cDNA and quantitated using qPCR. A
log-linear relationship exists between the amount of target RNA and
the PCR cycle where the fluorescence raises above the background
(threshold cycle, Ct). The more viable the viruses that are present
in the sample, the quicker and fewer cycles it takes for the
fluorescence of the PCR product to be above background and
therefore, the lower the Ct value. To account for variable cell
growth, each time an assay is run, a standard curve was generated
by serially diluting a vaccine solution containing the same amount
of vaccine loaded in the MMR-silk films. Viral infectivity was
measured right after the initial film preparation to establish
baseline activity (time 0). Measurements at subsequent time points
were compared to the time 0 value to establish residual potency.
The initial recovered potency from silk films, viral activity
immediately after casting film, was determined by comparison of
viral infectivity measured from the MMR-silk films to the
infectivity of a solution of vaccine alone, containing the same
concentration of vaccine as loaded in the silk films. Residual
potency in the MMR-silk films was calculated by the viral
infectivity measured at the certain time point compared to the
initial recovered potency and the residual potency of the vaccine
powder in storage was measure by comparison of viral activity
compared to the infectivity of the vaccine solution used to
determine the initial recovered potency from silk films. As
controls, viral activities were also measure for the silk films
with no vaccines loaded.
[0453] RNA was isolated from the Vero cells using TRIzol reagent
(Invitrogen) and chloroform. The RNA was purified using the Qiagen
RNEasy kit (Qiagen, Valencia, Calif.). Reverse transcription was
performed in the purified RNA to synthesize cDNA using the High
Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster
City, Calif.). Real time RT-PCR was performed on a Strategene
Mx3000P QPCR System (Strategene, La Jolla, Calif.). PCR reaction
were carried out in 50 .mu.L mixture volumes containing TaqMan
Universal PCR Mater Mix (1.times.) (Applied Biosystems, Foster
City, Calif.), 0.9 .mu.M of each PCR primer and 0.25 .mu.M of the
probe with 5 .mu.L of the cDNA sample. For measles detection, a 114
bp fragment (nt 584-697) was amplified with forward primer
(5'-CCCTGAGGGATTCAACATGATTCT-3'), reverse primer (5'-ATCCACCTTCTTAG
CTCCGAATC-3') and probe (5' FAM-TCTTGCTCGCAAAGGCGGTTACGG-BHQ1 3')
(Hubschen et al., 2008). For rubella detection, forward primer
(5'-TGATACCCAGACCTGTGTTCAC-3'), reverse primer (5'-GGTCGATGAGG
ACGTGTAGG-3') and probe (5' JOE-GATCACCCAGCACTCCACGCAA-BHQ1 3')
were used to amplify a 129 bp region (nt 195-323) (Hubschen et al.,
2008). For detection of a 134 bp region of the mumps virus, forward
primer (5'-CATAGGAGATATGTGGGG-3'), reverse primer
(5'-GTCTTCGCCAACGATG GTGATGATTG-3') and probe (5'
JOE-CCATGCAGGCGGTCACATTCCGACAA CTGC-TAMRA 3') were used (Kubar et
al., 2004). All primers and probes were obtained from Sigma-Aldrich
(St. Louis, Mo.). PCR reaction conditions were 2 min at 50.degree.
C., 10 min at 95.degree. C., and then 50 cycles at 95.degree. C.
for 15 s and 60.degree. C. for 1 min
[0454] In Vitro Vaccine Release.
[0455] In vitro vaccine release studies were carried out at
37.degree. C. The release studies for the silk films and
lyophilized silk films were carried out in a gelatin hydrogel
model. The gelatin hydrogel was prepared by mixing 4.5 g of
Knox.TM. original unflavored gelatin powder with 40 mL of boiled DI
water to obtain a 0.112 g/mL hydrogel. The solution was poured into
a 35 mm Petri dish and allowed to cool. To begin a release study, a
film was placed between two slabs of the hydrogel. Once a time
point was reached, the film was removed from the gel to stop the
release. The hydrogels were then digested in 400 .mu.L of 1 mg/mL
collagenase (Sigma Aldrich) for 2 hours at 37.degree. C.
Subsequently, the MMR released was quantified by the Vero cell
infectivity assay.
[0456] The in vitro release of the silk hydrogels and microspheres
were carried out in 2 mL Eppendorf tubes. Each hydrogel or
microsphere solution was placed in an Eppendorf tube with the
addition of 1.5 mL of sterile PBS. At each time point, the PBS
inside the Eppendorf tube was removed and transferred to another
Eppendorf tube and stored at -80.degree. C. Fresh PBS was then
added to the tubes to replenish the supply. Once all the samples
were taken at the set time points, the solutions were run through
the Vero cell infectivity assay to quantify the amount of MMR
released. The release values were reported as cumulative MMR
released.
[0457] Residual Moisture Determination.
[0458] Residual moisture of the lyophilized vaccine powder,
MMR-silk films, and lyophilized MMR-silk films was measured by the
thermo-gravimetric method, modified from Worrall et al., 2001, that
estimates the mean weight of three samples of each vaccine system
after drying for one hour at 80.degree. C. The weight of water lost
from the dried vaccine system is expressed as a percentage.
[0459] Differential Scanning Calorimetry (DSC).
[0460] Five mg of were encapsulated in Al pans and heated in a TA
Instrument Q100 DSC (New Castle, Del.) with a purged dry nitrogen
gas flow of 50 mL/min Tg was recorded as the onset temperature of
the discontinuity curve of the heat flow versus temperature. All
measurements were made at 10.degree. C./min. The samples were
initially equilibrated at -20.degree. C. for 5 minutes and then
heated to 200.degree. C., held at 200.degree. C. for 5 minutes,
followed by cooling to 20.degree. C. Nano-DSC measurements were
taken on a CSC Model 6100 Nano II Differential Scanning Calorimeter
(Lindon, Utah). Samples were prepared at a concentration of 1
mg/mL. The scanning rate was set at 1.degree. C./min for both the
heating and cooling runs from 0 to 100.degree. C.
[0461] Dynamic Light Scattering (DLS).
[0462] The size of the measles, mumps and rubella viral particles
as a function of temperature was monitored by DLS. A 400 .mu.L
aliquot of 2 mg/mL sample solution was filtered through a 0.45
.mu.m syringe filter (GE, Fairfield, Conn.). DLS was conducted
using the DynaPro DLS system (Wyatt Technology, Santa Barbara,
Calif.) with parameters set at 60 sec acquisition time, 10 number
of acquisition and laser power of 75 mW. A 100 .mu.L aliquot of the
sample was transferred into an RNAse-free, DNAse-free, protein-free
UVette Eppendorf cuvette to be inserted into the DLS. The effective
hydrodynamic diameter was calculated from the diffusion coefficient
by the Stokes-Einstein equation using the method of cumulants
(Koppel, 1972).
Example 3
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Sequence CWU 1
1
9124DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1ccctgaggga ttcaacatga ttct 24223DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2atccaccttc ttagctccga atc 23324DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 3tcttgctcgc aaaggcggtt acgg
24422DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4tgatacccag acctgtgttc ac 22520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5ggtcgatgag gacgtgtagg 20622DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 6gatcacccag cactccacgc aa
22718DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7cataggagat atgtgggg 18826DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8gtcttcgcca acgatggtga tgattg 26930DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
9ccatgcaggc ggtcacattc cgacaactg c 30
* * * * *