U.S. patent application number 14/371935 was filed with the patent office on 2014-12-04 for compositions and methods for treating viral infections.
This patent application is currently assigned to Variation Biotechnologies, Inc.. The applicant listed for this patent is Variation Biotechnologies, Inc.. Invention is credited to David E. Anderson.
Application Number | 20140356399 14/371935 |
Document ID | / |
Family ID | 48782009 |
Filed Date | 2014-12-04 |
United States Patent
Application |
20140356399 |
Kind Code |
A1 |
Anderson; David E. |
December 4, 2014 |
COMPOSITIONS AND METHODS FOR TREATING VIRAL INFECTIONS
Abstract
The present disclosure provides compositions and methods useful
for treating viral infections. As described herein, the
compositions and methods are based on the development of
immunogenic compositions that include an inactivated virus in
combination with a non-ionic surfactant vesicle (NISV). In certain
embodiments at least a portion of the antigen present in the
composition is physically associated with the NISV. In certain
embodiments the compositions are lyophilized and subsequently
rehydrated after a period of storage. In certain embodiments the
rehydrated compositions exhibit greater potency as compared to
otherwise equivalent compositions that lack the NISV. In certain
embodiments the lyophilized compositions are stored at temperatures
in excess of 8.degree. C. prior to rehydration. In certain
embodiments the rehydrated compositions exhibit greater potency as
compared to otherwise equivalent compositions that lack the NISV
and that were also stored at temperatures in excess of 8.degree. C.
prior to rehydration. In certain embodiments the antigen is taken
from a licensed vaccine and the administered dose of antigen is
less than the standard human dose for the licensed vaccine.
Inventors: |
Anderson; David E.; (Boston,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Variation Biotechnologies, Inc. |
Gatineu |
|
CA |
|
|
Assignee: |
Variation Biotechnologies,
Inc.
Gatineu
CA
|
Family ID: |
48782009 |
Appl. No.: |
14/371935 |
Filed: |
January 11, 2013 |
PCT Filed: |
January 11, 2013 |
PCT NO: |
PCT/IB13/00453 |
371 Date: |
July 11, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61585971 |
Jan 12, 2012 |
|
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|
Current U.S.
Class: |
424/400 ;
424/224.1 |
Current CPC
Class: |
A61K 39/39 20130101;
Y02A 50/30 20180101; A61K 47/28 20130101; A61K 39/205 20130101;
A61K 47/14 20130101; A61K 2039/55555 20130101; A61K 2039/55566
20130101; A61K 39/12 20130101; A61K 47/24 20130101; C12N 2760/20171
20130101; A61P 37/04 20180101; C12N 2760/20134 20130101; A61P 31/12
20180101; C12N 7/00 20130101; Y02A 50/466 20180101 |
Class at
Publication: |
424/400 ;
424/224.1 |
International
Class: |
A61K 47/28 20060101
A61K047/28; C12N 7/00 20060101 C12N007/00; A61K 47/24 20060101
A61K047/24; A61K 39/205 20060101 A61K039/205; A61K 47/14 20060101
A61K047/14 |
Claims
1. A thermostable lyophilized immunogenic composition comprising:
an inactivated viral antigen; and a vesicle which comprises a
non-ionic surfactant.
2. The composition of claim 1, where the composition comprises an
inactivated polio virus, an inactivated rabies virus, an
inactivated hepatitis A virus, or a combination thereof.
3. The composition of claim 1 or 2, wherein the non-ionic
surfactant is an ester-linked surfactant.
4. The composition of claim 3, wherein the non-ionic surfactant is
a glycerol ester.
5. The composition of claim 3, wherein the non-ionic surfactant is
1-monopalmitoyl glycerol.
6. The composition of claim 1 or 2, wherein the non-ionic
surfactant is an ether-linked surfactant.
7. The composition of claim 6, wherein the non-ionic surfactant is
a glycol or glycerol monoether.
8. The composition of claim 6, wherein the non-ionic surfactant is
1-monocetyl glycerol ether or diglycolcetyl ether.
9. The composition of claim 1 or 2, where the vesicle further
comprises an ionic amphiphile.
10. The composition of claim 9, where the ionic amphiphile is an
alkanoic acid or an alkenoic acid.
11. The composition of claim 9, where the ionic amphiphile is a
phosphate.
12. The composition of claim 9, where the ionic amphiphile is
dicetylphospate, phosphatidic acid or phosphatidyl serine.
13. The composition of claim 9, where the ionic amphiphile is a
sulphate monoester.
14. The composition of claim 9, where the ionic amphiphile is
cetylsulphate.
15. The composition of claim 1 or 2, where the vesicle further
comprises a steroid.
16. The composition of claim 15, where the steroid is
cholesterol.
17. The composition of claim 1 or 2, wherein the vesicle comprises
1-monopalmitoyl glycerol, dicetylphospate and cholesterol.
18. The composition of any one of the preceding claims, wherein at
least a portion of the virus is associated with the vesicle.
19. The composition of any one of the preceding claims, wherein the
virus is encapsulated within an aqueous core of the vesicle.
20. A method of treating an individual suffering from, or at risk
for, infection from a viral infection, the method comprising:
providing a composition of any one of claim 1-19 or 28-39, wherein
the composition has been stored for a period of time at a
temperature in excess of 8.degree. C.; rehydrating the composition
with an aqueous solution; and administering to the individual a
therapeutically effective amount of the rehydrated composition.
21. The method of claim 20, wherein the individual is suffering
from, or at risk for, infection from a polio virus, a rabies virus,
a hepatitis A virus, or combination thereof.
22. The method of claim 20, wherein the composition has been stored
for a period of time at a temperature in excess of 25.degree.
C.
23. The method of claim 20, wherein the composition has been stored
for a period of time at a temperature in excess of 30.degree.
C.
24. The method of claim 20, wherein the composition has been stored
for a period of time at a temperature in excess of 35.degree.
C.
25. The method of any one of claims 20-24, wherein the composition
is administered by intramuscular injection.
26. The method of any one of claims 20-24, wherein the composition
is administered by subcutaneous injection.
27. The method of claim 20, wherein the composition elicits an
immune response in the individual at a first level that is higher
than a second level of an immune response elicited by a second
composition comprising the antigen and lacking the vesicle.
28. The composition of any one of claims 1-19, wherein the
composition is prepared by a method that includes: melting lipids
that include the non-ionic surfactant to produce molten lipids;
combining the molten lipids with an aqueous solution that includes
the virus antigen; and homogenizing the resulting product, wherein
the molten lipids and aqueous solution are combined in relative
amounts and volumes that achieve a lipid concentration of at least
about 5 mg/ml in the resulting product.
29. The composition of claim 28, wherein the molten lipids and
aqueous solution are combined in relative amounts and volumes that
achieve a lipid concentration in a range of about 5 mg/ml to about
100 mg/ml in the resulting product.
30. A composition comprising an inactivated viral antigen and
vesicles, wherein the vesicles are comprised of lipids that include
a non-ionic surfactant and the composition is prepared by a method
that includes: melting the lipids to produce molten lipids;
combining the molten lipids with an aqueous solution that includes
the inactivated viral antigen; and homogenizing the resulting
product, wherein the molten lipids and aqueous solution are
combined in relative amounts and volumes that achieve a lipid
concentration of at least about 5 mg/ml in the resulting
product.
31. The composition of claim 30, wherein the molten lipids and
aqueous solution are combined in relative amounts and volumes that
achieve a lipid concentration in a range of about 5 mg/ml to about
100 mg/ml in the resulting product.
32. The composition of claim 30 or 31, wherein the molten lipids
are added to the aqueous solution that includes the inactivated
viral antigen.
33. The composition of claim 30 or 31, wherein the aqueous solution
that includes the inactivated viral antigen is added to the molten
lipids.
34. The composition of any one of claim 1-19 or 28-33, wherein the
composition was prepared by a method that involved storing the
composition in dried form.
35. The composition of claim 34, wherein the composition in dried
form was not stored under temperature-controlled conditions.
36. The composition of claim 34, wherein the composition in dried
form was stored at a temperature that at least temporarily exceeded
8.degree. C.
37. The composition of claim 34, wherein the composition in dried
form was stored at a temperature that at least temporarily exceeded
15.degree. C.
38. The composition of claim 34, wherein the composition in dried
form was stored at a temperature that at least temporarily exceeded
20.degree. C.
39. The composition of claim 34, wherein the composition in dried
form was stored at a temperature that at least temporarily exceeded
25.degree. C.
40. A method of preparing a composition comprising an inactivated
viral antigen and vesicles, wherein the lipid vesicles are
comprised of lipids that include a non-ionic surfactant, the method
comprising: melting the lipids to produce molten lipids; combining
the molten lipids with an aqueous solution that includes the
inactivated viral antigen; and homogenizing the resulting product,
wherein the molten lipids and aqueous solution are combined in
relative amounts and volumes that achieve a lipid concentration of
at least about 5 mg/ml in the resulting product.
41. The method of claim 40, wherein the molten lipids and aqueous
solution are combined in relative amounts and volumes that achieve
a lipid concentration in a range of about 5 mg/ml to about 100
mg/ml in the resulting product.
42. The method of claim 40 or 41, wherein the molten lipids are
added to the aqueous solution that includes the inactivated viral
antigen.
43. The method of claim 36 or 37, wherein the aqueous solution that
includes the inactivated viral antigen is added to the molten
lipids.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
provisional application Ser. No. 61/585,971, filed on Jan. 12,
2012, the contents of which are herein incorporated by reference in
their entirety.
BACKGROUND
[0002] Many viral infections cause severe health problems and may
ultimately lead to death of infected individuals. One strategy for
vaccination against such viral infections involves inactivating (or
"killing") a previously virulent virus and administering it to the
individual. The immune system may then later recognize a virulent
version of the infectious agent and can respond by neutralizing the
infectious agent or by destroying cells infected by the agent.
Several such inactivated vaccines have been developed e.g., for
polio virus, rabies virus, and hepatitis A.
[0003] Polio virus infection can lead to minor illness which does
not involve the central nervous system. However, in major illness
caused by polio infection, polio virus can enter the central
nervous system of an infected individual, where it infects and
destroys motor neurons and may lead to muscle weakness and acute
flaccid paralysis. Infection with rabies virus causes acute
encephalitis in warm-blooded animals and is almost always fatal if
treatment is not administered prior to the onset of severe
symptoms. Hepatitis A is a serious liver disease caused by the
hepatitis A virus (HAV), a virus which is transmitted from person
to person, primarily by the fecal-oral route. Hepatitis A may cause
symptoms including fatigue, fever, abdominal pain, jaundice, etc.,
which can last for as long as 6 months.
[0004] Several inactivated polio, rabies, and hepatitis A vaccines
are currently licensed and have been successful in reducing the
incidence of infection. However, all vaccines, including
inactivated antigen vaccines, lose potency over time and the rate
of potency loss is temperature-dependent. Therefore, cold-chain
systems have been established to ensure that the potency of
vaccines is maintained by storing them under refrigerated
conditions (in most cases between 2 and 8.degree. C.) until the
point of use. Establishing a cold chain for vaccine storage and
distribution is a major undertaking and maintenance is difficult.
It is also apparent that, despite best efforts, cold chains do not
always function as intended for many reasons, such as improperly
maintained or outdated refrigeration equipment, power outages
resulting in equipment failure, poor compliance with cold-chain
procedures and inadequate monitoring. The result is that vaccines
in the cold chain are often subjected to temperature excursions
(i.e., temperatures outside of the target range).
[0005] While inactivated polio, rabies, and hepatitis A vaccines
have been successful in reducing the incidence of disease
worldwide, there remains a need in the art for improved vaccines
that are stable and retain potency when exposed to high
temperatures.
SUMMARY
[0006] The present disclosure provides compositions and methods
useful for treating infections (e.g., those caused by polio virus,
rabies virus, and/or hepatitis A virus). As described herein, the
compositions and methods are based on the development of
immunogenic compositions that include an inactivated virus in
combination with a non-ionic surfactant vesicle (NISV). In certain
embodiments at least a portion of the antigen present in the
composition is physically associated with the NISV. In certain
embodiments the compositions are lyophilized and subsequently
rehydrated after a period of storage. In certain embodiments the
rehydrated compositions exhibit greater potency as compared to
otherwise equivalent compositions that lack the NISV. In certain
embodiments the lyophilized compositions are stored at temperatures
in excess of 8.degree. C. prior to rehydration. In certain
embodiments the rehydrated compositions exhibit greater potency as
compared to otherwise equivalent compositions that lack the NISV
and that were also stored at temperatures in excess of 8.degree. C.
prior to rehydration. In certain embodiments the antigen is taken
from a licensed vaccine and the administered dose of antigen is
less than the standard human dose for the licensed vaccine.
DEFINITIONS
[0007] Throughout the present disclosure, several terms are
employed that are defined in the following paragraphs.
[0008] As used herein, the term "antigen" or "viral antigen" refers
to a substance containing one or more epitopes that can be
recognized by an antibody. In certain embodiments, an antigen can
be a virus. The term "antigen" encompasses inter alia killed, but
previously virulent viruses. In certain embodiments, an antigen may
be an "immunogen."
[0009] As used herein, the term "immune response" refers to a
response elicited in an animal. An immune response may refer to
cellular immunity, humoral immunity or may involve both. An immune
response may also be limited to a part of the immune system. For
example, in certain embodiments, an immunogenic composition may
induce an increased IFN.gamma. response. In certain embodiments, an
immunogenic composition may induce a mucosal IgA response (e.g., as
measured in nasal and/or rectal washes). In certain embodiments, an
immunogenic composition may induce a systemic IgG response (e.g.,
as measured in serum).
[0010] As used herein, the term "immunogenic" means capable of
producing an immune response in a host animal against a non-host
entity (e.g., a viral antigen). In certain embodiments, this immune
response forms the basis of the protective immunity elicited by a
vaccine against a specific infectious organism (e.g., a virus).
[0011] As used herein, the terms "therapeutically effective amount"
refer to the amount sufficient to show a meaningful benefit in a
subject being treated. The therapeutically effective amount of an
immunogenic composition may vary depending on such factors as the
desired biological endpoint, the nature of the composition, the
route of administration, the health, size and/or age of the subject
being treated, etc.
[0012] As used herein, the term "treat" (or "treating", "treated",
"treatment", etc.) refers to the administration of a composition to
a subject who has a disease, a symptom of a disease or a
predisposition toward a disease, with the purpose to alleviate,
relieve, alter, ameliorate, improve or affect the disease, a
symptom or symptoms of the disease, or the predisposition toward
the disease. In certain embodiments, the term "treating" refers to
the vaccination of a subject.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0013] The present disclosure provides compositions and methods
useful for treating infections (e.g., infections by polio virus,
rabies virus, and/or hepatitis A virus). As described herein, the
compositions and methods are based on the development of
immunogenic compositions that include an inactivated virus in
combination with a non-ionic surfactant vesicle (NISV). In certain
embodiments at least a portion of the antigen present in the
composition is physically associated with the NISV. In certain
embodiments the compositions are lyophilized and subsequently
rehydrated after a period of storage. In certain embodiments the
rehydrated compositions exhibit greater potency as compared to
otherwise equivalent compositions that lack the NISV. In certain
embodiments the lyophilized compositions are stored at temperatures
in excess of 8.degree. C. prior to rehydration. In certain
embodiments the rehydrated compositions exhibit greater potency as
compared to otherwise equivalent compositions that lack the NISV
and that were also stored at temperatures in excess of 8.degree. C.
prior to rehydration. In certain embodiments the antigen is taken
from a licensed vaccine and the administered dose of antigen is
less than the standard human dose for the licensed vaccine.
I. Inactivated Antigens
[0014] In some embodiments, the compositions and methods of the
present disclosure may be used with one or more antigens included
in a vaccine that is licensed or under development. In certain
embodiments, inactivated refers to a whole killed virus. Table 1 is
a non-limiting list of vaccines that are licensed or under
development for polio, rabies, and Hepatitis A infections.
TABLE-US-00001 TABLE 1 Vaccine Disease Polio (Ipol .RTM., Imovax
.RTM. Polio ) Polio DTaP/IPV/HepB (Pediarix .RTM.) Polio Rabies
(BioRab .RTM., Imovax .RTM. Rabies, Rabies RabAvert .RTM.) HepA
(Havrix .RTM., Vaqta .RTM.) Hepatitis A HepA (Aimmugen) Hepatitis A
HepA/HepB (Twinrix .RTM.) Hepatitis A
[0015] In the following sections we discuss these and other
exemplary antigens that could be used in compositions or methods of
the present disclosure.
[0016] Polio Virus
[0017] In one aspect, the present application provides immunogenic
compositions that include an inactivated poliomyelitis (also called
"polio") virus. The first effective polio vaccine was first tested
by Jonas Salk and is an inactivated poliovirus vaccine based on
three wild virulent reference strains: [0018] Mahoney (type 1
poliovirus) [0019] MEF-1 (type 2 poliovirus) [0020] Saukett (type 3
poliovirus)
[0021] The reference poliovirus strains are generally cultured in
Vero cells, purified and then inactivated. It will be appreciated
that any method may be used to prepare an inactivated polio virus.
In general however, these methods may involve propagating a polio
virus in a culture vessel containing appropriate cells (e.g., Vero
cells), nutrient medium, isolating and then inactivating the
antigen. While heat and formalin are commonly used to inactivate
licensed polio vaccines it is to be understood that other
techniques could be used, e.g., treatment with chlorine, exposure
to high temperatures, etc.
[0022] Several poliovirus vaccines are currently licensed. For
example, each 0.5 ml dose of Imovax.RTM. Polio contains a
suspension of purified formaldehyde-inactivated polio vaccine,
including Mohoney (Type 1; 40 D antigen units), MEF1 (Type 2; 8 D
antigen units), and Saukett (Type 3; 32 D antigen units). Primary
immunization with Imovax.RTM. Polio is usually administered as
three doses, the first two doses administered 4-8 weeks apart and
the third dose following 6-12 months later. A booster is currently
recommended for adults and adolescents who are at greater risk of
exposure to poliovirus than the general population or if more than
10 years have elapsed since the last dose of their complete
vaccination series.
[0023] It will be appreciated that any poliovirus strain may be
used, e.g., without limitation any of the strains described herein.
In some embodiments, a single strain (e.g., subtype, serotype,
and/or biotype) of poliovirus may be used in accordance with the
present disclosure. In some embodiments, more than one strain
(e.g., subtype, serotype and/or biotype) of poliovirus may be used
in accordance with the present disclosure.
[0024] Rabies Virus
[0025] In one aspect, the present application provides immunogenic
compositions that include an inactivated rabies virus. Several
rabies virus vaccines are currently licensed. For example,
Imovax.RTM. Rabies vaccine is a freeze-dried suspension of rabies
virus prepared from WISTAR Rabies PM.WI 38 1503-3M strain. The
virus is harvested from infected MRC-5 human diploid cells,
concentrated by ultracentrifugation and inactivated by treatment
with beta-propiolactone.
[0026] It will be appreciated that any method may be used to
prepare an inactivated rabies virus. In general however, these
methods may involve propagating a rabies virus in a culture vessel
containing appropriate cells, nutrient medium, isolating and then
inactivating the antigen. For example, heat, formalin,
formaldehyde, treatment with chlorine, exposure to high
temperatures, etc. may be used to inactivate rabies virus.
[0027] Each 1.0 ml dose of Imovax.RTM. Rabies contains a
.gtoreq.2.5 IU rabies virus (WISTAR Rabies PM/WI 38 1503-3M
strain). Primary immunization with Imovax.RTM. Rabies for
individuals who have not been exposed to rabies is usually
administered as three doses, the first two doses administered 7
days apart and the third dose 21 days later. A booster is currently
recommended for individuals who may be repeatedly exposed to rabies
virus (e.g., laboratory workers and veterinarians). Primary
immunization with Imovax.RTM. Rabies for individuals who have been
exposed to rabies is usually administered as five doses, one dose
right immediately after exposure, followed by additional doses on
the 3.sup.rd, 7.sup.th, 14.sup.th, and 28.sup.th days.
[0028] It will be appreciated that any rabies virus strain may be
used, e.g., without limitation any of the strains described herein.
In some embodiments, a single strain (e.g., subtype, serotype,
and/or biotype) of rabies virus may be used in accordance with the
present disclosure. In some embodiments, more than one strain
(e.g., subtype, serotype and/or biotype) of rabies virus may be
used in accordance with the present disclosure.
[0029] Hepatitis A Virus
[0030] In one aspect, the present application provides immunogenic
compositions that include an inactivated hepatitis A virus (also
called "hepatitis A antigen", "HAV antigen" or "antigen" herein).
All known hepatitis A vaccines include an inactivated hepatitis A
virus.
[0031] It will be appreciated that any method may be used to
prepare an inactivated hepatitis A virus. In general however, these
methods may involve propagating a hepatitis A virus in a host cell,
lysing the host cell to release the virus, isolating and then
inactivating the antigen. For example, in preparing HAVRIX.RTM.,
hepatitis A virus strain HM175 is propagated in MRC-5 human diploid
cells. After removal of the cell culture medium, the cells are
lysed to form a suspension. This suspension is purified through
ultrafiltration and gel permeation chromatography procedures. The
purified lysate is then treated with formalin to ensure viral
inactivation (e.g., see Andre et al., Prog. Med. Virol. 37:72-95,
1990).
[0032] In preparing AIMMUGEN.RTM., hepatitis A virus strain KRM0003
(established from a wild-type HAV, which had been isolated from the
feces of a hepatitis A patient) is propagated in GL37 cells (a cell
strain established for vaccine production from a parent cell strain
of African green monkey kidney). The GL37 cells are inoculated with
HAV strain KRM0003 and antigen is harvested, extensively purified
and inactivated with formaldehyde.
[0033] Another example of an inactivated hepatitis A virus that is
commercially available but is not a licensed vaccine is hepatitis A
antigen (HAV-ag) from Meridian Life Sciences. Like HAVRIX.RTM. the
Meridian HAV-ag also derives from hepatitis A virus strain HM175
but it is propagated in FRhK-4 (fetal rhesus kidney) cells. After
removal of cell culture medium, the cells are lysed to form a
suspension and the suspension is partially purified by gradient
centrifugation and inactivated by treatment with formalin.
[0034] It will be appreciated that any hepatitis A virus strain may
be used, e.g., without limitation any of the following strains
which have been described in the art (and other non-human
variants):
[0035] Human hepatitis A virus Hu/Arizona/HAS-15/1979
[0036] Human hepatitis A virus Hu/Australia/HM175/1976
[0037] Human hepatitis A virus Hu/China/H2/1982
[0038] Human hepatitis A virus Hu/Costa Rica/CR326/1960
[0039] Human hepatitis A virus Hu/France/CF-53/1979
[0040] Human hepatitis A virus Hu/Georgia/GA76/1976
[0041] Human hepatitis A virus Hu/Germany/GBM/1976
[0042] Human hepatitis A virus Hu/Japan/HAJ85-1/1985
[0043] Human hepatitis A virus Hu/Los Angeles/LA/1975
[0044] Human hepatitis A virus Hu/Northern Africa/MBB/1978
[0045] Human hepatitis A virus Hu/Norway/NOR-21/1998
[0046] Human hepatitis A virus Hu/Sierra Leone/SLF88/1988
[0047] Human hepatitis A virus MSM1
[0048] Human hepatitis A virus Shanghai/LCDC-1/1984
[0049] In addition, while formalin and formaldehyde are commonly
used to inactivate licensed hepatitis A vaccines it is to be
understood that other techniques could be used, e.g., treatment
with chlorine, exposure to high temperatures, etc.
[0050] In certain embodiments it may prove advantageous to add
additional steps to the traditional method for preparing an
inactivated hepatitis A virus. For example, U.S. Pat. No. 6,991,929
describes including a protease treatment step (e.g., trypsin) after
the virus has been propagated. This step was found to improve the
removal of host cell material and yield a purer
[0051] It is to be understood that any one of these licensed
hepatitis A vaccines may be combined with another antigen to
produce an immunogenic composition.
II. Vesicles
[0052] In general, immunogenic compositions of the present
disclosure include a non-ionic surfactant vesicle (NISV). As is
well known in the art, vesicles generally have an aqueous
compartment enclosed by one or more bilayers which include
amphipathic molecules. Any non-ionic surfactant with appropriate
amphipathic properties may be used to form such a vesicle. In some
embodiments, at least a portion of the antigen present in the
composition is associated with the vesicle (i.e., encapsulated
within an aqueous core of the vesicle and/or associated with a
vesicle bilayer). These embodiments are encompassed by the term
"antigen-containing vesicle." In certain embodiments an immunogenic
composition may also include amounts or components of the antigen
that are not associated with a vesicle.
[0053] Without limitation, examples of suitable surfactants include
ester-linked surfactants based on glycerol. Such glycerol esters
may comprise one of two higher aliphatic acyl groups, e.g.,
containing at least ten carbon atoms in each acyl moiety.
Surfactants based on such glycerol esters may comprise more than
one glycerol unit, e.g., up to 5 glycerol units. Glycerol
monoesters may be used, e.g., those containing a
C.sub.12-C.sub.20alkanoyl or alkenoyl moiety, for example caproyl,
lauroyl, myristoyl, palmitoyl, oleyl or stearoyl. An exemplary
surfactant is 1-monopalmitoyl glycerol.
[0054] Ether-linked surfactants may also be used as the non-ionic
surfactant. For example, ether-linked surfactants based on glycerol
or a glycol having a lower aliphatic glycol of up to 4 carbon
atoms, such as ethylene glycol, are suitable. Surfactants based on
such glycols may comprise more than one glycol unit, e.g., up to 5
glycol units (e.g., diglycolcetyl ether and/or
polyoxyethylene-3-lauryl ether). Glycol or glycerol monoethers may
be used, including those containing a C.sub.12-C.sub.20alkanyl or
alkenyl moiety, for example capryl, lauryl, myristyl, cetyl, oleyl
or stearyl. Ethylene oxide condensation products that can be used
include those disclosed in PCT Publication No. WO88/06882 (e.g.,
polyoxyethylene higher aliphatic ether and amine surfactants).
Exemplary ether-linked surfactants include 1-monocetyl glycerol
ether and diglycolcetyl ether.
[0055] It is also to be understood that vesicles may also
incorporate an ionic amphiphile, e.g., to cause the vesicles to
take on a negative charge. For example, this may help to stabilize
the vesicles and provide effective dispersion. Without limitation,
acidic materials such as higher alkanoic and alkenoic acids (e.g.,
palmitic acid, oleic acid) or other compounds containing acidic
groups including phosphates such as dialkyl phosphates (e.g.,
dicetylphospate, or phosphatidic acid or phosphatidyl serine) and
sulphate monoesters such as higher alkyl sulphates (e.g.,
cetylsulphate), may all be used for this purpose. The ionic
amphiphile, if present, will typically comprise, between 1 and 50%
by weight of the non-ionic surfactant (e.g., 1-5%, 1-10%, 1-15%,
1-20, 1-25%, 1-30%, 1-35%, 1-40%, 1-45%, 5-10%, 5-15%, 5-20%,
5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 10-15%, 10-20%, 10-25%,
10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 15-20%, 15-25%, 15-30%,
15-35%, 15-40%, 15-45%, 15-50%, 20-25%, 20-30%, 20-35%, 20-40%,
20-45%, 20-50%, 25-30%, 25-35%, 25-40%, 25-45%, 25-50%, 30-35%,
30-40%, 30-45%, 30-50%, 35-40%, 35-45%, 35-50%, 40-45%, 40-50%, or
45-50%).
[0056] To form vesicles, the components may be admixed with an
appropriate hydrophobic material of higher molecular mass capable
of forming a bi-layer (such as a steroid, e.g., a sterol such as
cholesterol). The presence of the steroid assists in forming the
bi-layer on which the physical properties of the vesicle depend.
The steroid, if present, will typically comprise between 20 and
120% by weight of the non-ionic surfactant (e.g., 20-30%, 20-40%,
20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-110%, 30-40%,
30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-100%, 30-110%, 30-120%,
40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-100%, 40-110%, 40-120%,
50-60%, 50-70%, 50-80%, 50-90%, 50-100%, 50-110%, 50-120%, 60-70%,
60-80%, 60-90%, 60-100%, 60-110%, 60-120%, 70-80%, 70-90%, 70-100%,
70-110%, 70-120%, 80-90%, 80-100%, 80-110%, 80-120%, 90-100%,
90-110%, 90-120%, 100-110%, 100-120%, or 110-120%).
[0057] In certain embodiments, the vesicles comprise a non-ionic
surfactant, an ionic amphiphile and a steroid. In certain
embodiments, the vesicles comprise 1-monopalmitoyl glycerol,
dicetylphospate and cholesterol.
[0058] In certain embodiments, the vesicles consist essentially of
a non-ionic surfactant, an ionic amphiphile and a steroid. In
certain embodiments, the vesicles consist essentially of
1-monopalmitoyl glycerol, dicetylphospate and cholesterol.
[0059] In certain embodiments, the vesicles do not comprise a
transport enhancing molecule which facilitates the transport of
lipid-like molecules across mucosal membranes. In some embodiments,
the vesicles do not comprise a "bile acid" such as cholic acid and
chenodeoxycholic acid, their conjugation products with glycine or
taurine such as glycocholic and taurocholic acid, derivatives
including deoxycholic and ursodeoxycholic acid, and salts of each
of these acids. In some embodiments, the vesicles do not comprise
acyloxylated amino acids, such as acylcarnitines and salts thereof,
and palmitoylcarnitines.
Methods for Making Vesicles
[0060] It will be appreciated that there are known techniques for
preparing vesicles comprising non-ionic surfactants, such as those
referred to in PCT Publication No. WO93/019781. An exemplary
technique is the rotary film evaporation method, in which a film of
non-ionic surfactant is prepared by rotary evaporation from an
organic solvent, e.g., a hydrocarbon or chlorinated hydrocarbon
solvent such as chloroform, e.g., see Russell and Alexander, J.
Immunol. 140:1274, 1988. The resulting thin film is then rehydrated
in bicarbonate buffer optionally in the presence of antigen.
[0061] Another method for the production of vesicles is that
disclosed by Collins et al., J. Pharm. Pharmacol. 42:53, 1990. This
method involves melting a mixture of the non-ionic surfactant,
steroid (if used) and ionic amphiphile (if used) and hydrating with
vigorous mixing in the presence of aqueous buffer.
[0062] Another method involves hydration in the presence of
shearing forces. An apparatus that can be used to apply such
shearing forces is a well-known, suitable equipment (see, e.g., PCT
Publication No. WO88/06882). Sonication and ultra-sonication are
also effective means to form the vesicles or to alter their
particle size.
[0063] In certain embodiments, at least a portion of the viral
antigen is associated with lipid vesicles (where, as used herein,
the term "association" encompasses any form of physical
interaction). In certain embodiments, at least a portion of the
viral antigen is entrapped within lipid vesicles. Association and
entrapment may be achieved in any manner. For example, in the
rotary film evaporation technique, this can be achieved by
hydration of the film in the presence of antigen. In other methods,
the viral antigen may be associated with preformed vesicles by a
dehydration-rehydration method in which viral antigen present in
the aqueous phase is entrapped by flash freezing followed by
lyophilization, e.g., see Kirby and Gregoriadis, Biotechnology
2:979, 1984. Alternatively a freeze thaw technique may be used in
which vesicles are mixed with the viral antigen and repeatedly
flash frozen in liquid nitrogen, and warmed to a temperature of the
order of, e.g., 60.degree. C. (i.e., above the transition
temperature of the relevant surfactant), e.g., see Pick, Arch.
Biochem. Biophys. 212:195, 1981.
[0064] In certain embodiments, vesicles for use in accordance with
the present invention are prepared by a method that includes:
melting the non-ionic surfactant (optionally with a steroid and/or
an ionic amphiphile, collectively the "lipids") to produce a molten
mixture; combining the molten mixture with an aqueous solution that
includes a viral antigen; and homogenizing the resulting product.
In certain embodiments, the molten mixture is are added to the
aqueous solution that includes the viral antigen. In certain
embodiments, aqueous solution that includes the viral antigen is
added to the molten mixture.
[0065] In certain embodiments, the molten mixture and aqueous
solution are combined in relative amounts and volumes that achieve
a lipid concentration of at least about 2 mg/ml in the resulting
product. Indeed, through experimentation and as described in the
Examples, we have found that when the lipids and viral antigen are
homogenized with a lipid concentration in excess of 5 mg/ml the
resulting compositions tend to be more thermostable than when a
lower lipid concentration is used (see Examples). In some
embodiments, therefore, the present invention provides desirable
compositions (specifically including thermostable compositions)
comprising a viral antigen and vesicles, which compositions contain
a specified lipid concentration established herein to impart
particular characteristics (e.g., improved thermostability) to the
compositions.
[0066] In certain embodiments, a lipid concentration of at least
about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or
95 mg/ml is achieved. In certain embodiments, the lipid
concentration is in a range of about 5 mg/ml to about 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, or 25 mg/ml. In certain
embodiments, the lipid concentration is in a range of about 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15 mg/ml to about 30 mg/ml. In certain
embodiments, the lipid concentration is in a range of about 2 mg/ml
to about 5 mg/ml, about 5 mg/ml to about 50 mg/ml, about 5 mg/ml to
about 25 mg/ml, about 10 mg/ml to about 50 mg/ml, about 10 mg/ml to
about 30 mg/ml, or about 10 mg/ml to about 50 mg/ml.
[0067] In some embodiments, the non-ionic surfactant (optionally
with other components such as a steroid and/or an ionic amphiphile)
is melted at a temperature range between 120.degree. C. and
150.degree. C. (e.g., between 120.degree. C. and 125.degree. C.,
between 120.degree. C. and 130.degree. C., between 120.degree. C.
and 140.degree. C., between 130.degree. C. and 140.degree. C.,
between 135.degree. C. and 145.degree. C., or between 140.degree.
C. and 145.degree. C.). In some embodiments, the non-ionic
surfactant (optionally with other components such as a steroid
and/or an ionic amphiphile) is melted at about 120.degree. C., at
about 125.degree. C., at about 130.degree. C., at about 135.degree.
C., at about 140.degree. C., at about 145.degree. C. or at about
150.degree. C.
[0068] In some embodiments, the aqueous solution comprising a viral
antigen is temperature controlled. In some embodiments, the aqueous
solution comprising a viral antigen is kept at a temperature of
less than about 50.degree. C. during the step of adding (e.g., less
than about 45.degree. C., less than about 40.degree. C., less than
about 35.degree. C., less than about 30.degree. C., less than about
25.degree. C., etc.). In some embodiments, the aqueous solution
comprising a viral antigen is kept at a temperature range between
about 25.degree. C. and about 50.degree. C. In some embodiments,
the aqueous solution comprising a viral antigen is kept at room
temperature.
[0069] In certain embodiments the vesicles are made by a process
which includes steps of providing a lyophilized non-ionic
surfactant (optionally with other components such as a steroid
and/or an ionic amphiphile) and rehydrating the lyophilized
non-ionic surfactant with an aqueous solution comprising a antigen
such that antigen-containing vesicles are formed. The lyophilized
non-ionic surfactant is prepared by melting the non-ionic
surfactant (optionally with other components such as a steroid
and/or an ionic amphiphile) to produce a molten mixture and then
lyophilizing the molten mixture.
[0070] As described in more detail herein, in some embodiments, an
immunogenic composition that includes a antigen formulated with
vesicles may be lyophilized for future use and subsequently
hydrated prior to use.
Vesicle Size and Processing
[0071] It will be appreciated that a vesicle composition will
typically include a mixture of vesicles with a range of sizes. It
is to be understood that the diameter values listed below
correspond to the most frequent diameter within the mixture. In
some embodiments >90% of the vesicles in a composition will have
a diameter which lies within 50% of the most frequent value (e.g.,
1000.+-.500 nm). In some embodiments the distribution may be
narrower, e.g., >90% of the vesicles in a composition may have a
diameter which lies within 40, 30, 20, 10 or 5% of the most
frequent value. In some embodiments, sonication or ultra-sonication
may be used to facilitate vesicle formation and/or to alter vesicle
particle size. In some embodiments, filtration, dialysis and/or
centrifugation may be used to adjust the vesicle size
distribution.
[0072] In general, vesicles produced in accordance with the methods
of the present disclosure may be of any size. In certain
embodiments, the composition may include vesicles with diameter in
range of about 10 nm to about 10 .mu.m. In certain embodiments,
vesicles are of diameters between about 100 nm to about 5 .mu.m. In
certain embodiments, vesicles are of diameters between about 500 nm
to about 2 .mu.m. In certain embodiments, vesicles are of diameters
between about 800 nm to about 1.5 .mu.m. In some embodiments, the
compositions may include vesicles with a diameter in the range of
about 150 nm to about 15 .mu.m. In certain embodiments, the
vesicles may have a diameter which is greater than 10 .mu.m, e.g.,
about 15 .mu.m to about 25 .mu.m. In certain embodiments, the
vesicles may have a diameter in the range of about 0.1 .mu.m to
about 20 .mu.m, about 0.1 .mu.m to about 15 .mu.m, about 0.1 .mu.m
to about 10 .mu.m, about 0.5 .mu.m to about 20 .mu.m, about 0.5
.mu.m to about 15 .mu.m, about 0.5 .mu.m to about 10 .mu.m, about 1
.mu.m to about 20 .mu.m, about 1 .mu.m to about 15 .mu.m, or about
1 .mu.m to about 10 .mu.m. In certain embodiments, the vesicles may
have a diameter in the range of about 2 .mu.m to about 10 .mu.m,
e.g., about 1 .mu.m to about 4 .mu.m. In certain embodiments, the
vesicles may have a diameter which is less than 150 nm, e.g., about
50 nm to about 100 nm.
Lyophilization
[0073] Liquid formulation of vaccines has been the default
presentation since the introduction of vaccines. Most of the
existing liquid vaccine compositions have been developed for
storage under refrigeration, but not at higher temperatures, with
the result that their stability may not be optimal. In some cases,
licensed vaccines are currently formulated and stored as liquids.
In the aqueous environment the antigens are subjected to physical
and chemical degradation that may lead to inactivation and loss of
potency.
[0074] As discussed above, the methods of the present disclosure
may include a step of lyophilizing a solution of a non-ionic
surfactant (optionally with other components such as a steroid
and/or an ionic amphiphile). Lyophilization is an established
method used to enhance the long-term stability of products.
Enhancement of physical and chemical stability is thought to be
accomplished by preventing degradation and hydrolysis.
Lyophilization involves freezing the preparation in question and
then reducing the surrounding pressure (and optionally heating the
preparation) to allow the frozen solvent(s) to sublime directly
from the solid phase to gas (i.e., drying phase). In certain
embodiments, the drying phase is divided into primary and secondary
drying phases.
[0075] The freezing phase can be done by placing the preparation in
a container (e.g., a flask, eppendorf tube, etc.) and optionally
rotating the container in a bath which is cooled by mechanical
refrigeration (e.g., using dry ice and methanol, liquid nitrogen,
etc.). In some embodiments, the freezing step involves cooling the
preparation to a temperature that is below the eutectic point of
the preparation. Since the eutectic point occurs at the lowest
temperature where the solid and liquid phase of the preparation can
coexist, maintaining the material at a temperature below this point
ensures that sublimation rather than evaporation will occur in
subsequent steps.
[0076] The drying phase (or the primary drying phase when two
drying phases are used) involves reducing the pressure and
optionally heating the preparation to a point where the solvent(s)
can sublimate. This drying phase typically removes the majority of
the solvent(s) from the preparation. It will be appreciated that
the freezing and drying phases are not necessarily distinct phases
but can be combined in any manner. For example, in certain
embodiments, the freezing and drying phases may overlap.
[0077] A secondary drying phase can optionally be used to remove
residual solvent(s) that was adsorbed during the freezing phase.
Without wishing to be bound to any theory, this phase involves
raising the temperature to break any physico-chemical interactions
that have formed between the solvent molecules and the frozen
preparation. Once the drying phase is complete, the vacuum can be
broken with an inert gas (e.g., nitrogen or helium) before the
lyophilized product is optionally sealed.
[0078] In some embodiments, the lyophilized product is
substantially free of organic solvent(s).
[0079] Excipients such as sucrose, amino acids or proteins such as
gelatin or serum albumin may be used to protect the antigen during
the drying process and storage. In some embodiments, a
lyoprotectant may be used to protect antigens during
lyophilization. Exemplary lyoprotectants include sucrose,
trehalose, polyethylene glycol (PEG), dimethyl-succinate buffer
(DMS), bovine serum albumin (BSA), mannitol, sorbitol, and dextran.
Any suitable amount and/or combination of lyoprotectant(s) may be
used to protect the antigen. For example, as demonstrated in U.S.
Pat. No. 6,290,967, the dual presence of a disaccharide (e.g.,
sucrose) and a 6-carbon polyhydric alcohol (e.g., a sorbitol)
enhanced the stability of a vaccine composition compared to control
compositions. Sucrose was added in an amount ranging from 10 to 70
grams per liter of vaccine, and sorbitol was added in an amount
ranging from about 15 to 90 grams per liter of vaccine.
Rehydration
[0080] Once a solution has been lyophilized, the methods of the
present disclosure may include a step of rehydrating the
lyophilized product to form antigen-containing vesicles. In some
embodiments, this is achieved by mixing the lyophilized product
with an aqueous solution comprising a antigen. In some embodiments,
this involves adding the aqueous solution to the lyophilized
product.
[0081] In some embodiments, the antigen-containing vesicles contain
at least about 10% of the antigen added in the step of rehydrating.
In some embodiments, the antigen-containing vesicles contain at
least about 20% of the antigen added in the step of rehydrating. In
some embodiments, the antigen-containing vesicles contain at least
about 30% of the antigen added in the step of rehydrating. In some
embodiments, the antigen-containing vesicles contain at least about
40% of the antigen added in the step of rehydrating. In some
embodiments, the antigen-containing vesicles contain at least about
50% of the antigen added in the step of rehydrating. In some
embodiments, the antigen-containing vesicles contain at least about
60% of the antigen added in the step of rehydrating. In some
embodiments, the antigen-containing vesicles contain at least about
70% of the antigen added in the step of rehydrating. In some
embodiments, the antigen-containing vesicles contain at least about
80% of the antigen added in the step of rehydrating. In some
embodiments, the antigen-containing vesicles contain at least about
90% of the antigen added in the step of rehydrating.
[0082] In some embodiments, the aqueous solution includes a buffer.
The buffer used will typically depend on the nature of the antigen
or antigens in the aqueous solution. For example, without
limitation, a PCB buffer, an Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4
buffer, a PBS buffer, a bicine buffer, a Tris buffer, a HEPES
buffer, a MOPS buffer, etc. may be used. PCB buffer is produced by
mixing sodium propionate, sodium cacodylate, and bis-Tris propane
in the molar ratios 2:1:2. Varying the amount of HCl added enables
buffering over a pH range from 4-9. In some embodiments, a
carbonate buffer may be used.
[0083] In some embodiments, a composition of antigen-containing
vesicles may be lyophilized for future use and subsequently
hydrated (e.g., with sterile water or an aqueous buffer) prior to
use. In some embodiments, a composition of antigen-containing
vesicles may be stored at -80.degree. C. prior to
lyophilization.
[0084] In certain embodiments, the rehydrated immunogenic
composition exhibits substantially the same potency as the
immunogenic composition prior to lyophilization.
[0085] In some embodiments, the rehydrated immunogenic composition
exhibits at least about 50% of the potency as the immunogenic
composition prior to lyophilization (e.g., at least about 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%). In some embodiments, the
level of potency is based on measurements obtained using an ELISA.
In some embodiments, the level of potency is based on a plaque
assay measurement.
[0086] In some embodiments, the rehydrated immunogenic composition
exhibits at least 1.5 fold greater potency as compared to an
otherwise equivalent immunogenic composition that was formulated
without NISV (e.g., at least about 2 fold, 2.5 fold, 3 fold, 3.5
fold, 4 fold or 5 fold). In some embodiments, the level of potency
is based on measurements obtained using an ELISA. In some
embodiments, the level of potency is based on a plaque assay
measurement.
Storage
[0087] In certain embodiments, the lyophilized immunogenic
composition may be stored for a period of time (e.g., days, weeks
or months) prior to rehydration and administration to a subject in
need thereof. In certain embodiments, the lyophilized immunogenic
composition is exposed to temperatures in excess of 8.degree. C.
during storage (e.g., temperatures in excess of 10.degree. C.,
15.degree. C., 20.degree. C., 25.degree. C., 30.degree. C.,
35.degree. C., or 40.degree. C., temperatures in the range of
10.degree. C. to 40.degree. C., temperatures in the range of
20.degree. C. to 40.degree. C., temperatures in the range of
30.degree. C. to 40.degree. C., temperatures in the range of
10.degree. C. to 30.degree. C., temperatures in the range of
20.degree. C. to 30.degree. C., room temperature, etc.). In certain
embodiments, the lyophilized immunogenic composition is stored
under conditions that are not temperature controlled.
[0088] In certain embodiments, the lyophilized immunogenic
compositions are thermostable in that the potency of the
immunogenic composition remains substantially unchanged during
storage despite being exposed to temperatures in excess of
8.degree. C. (e.g., temperatures in excess of 10.degree. C.,
15.degree. C., 20.degree. C., 25.degree. C., 30.degree. C.,
35.degree. C., or 40.degree. C., temperatures in the range of
10.degree. C. to 40.degree. C., temperatures in the range of
20.degree. C. to 40.degree. C., temperatures in the range of
30.degree. C. to 40.degree. C., temperatures in the range of
10.degree. C. to 30.degree. C., temperatures in the range of
20.degree. C. to 30.degree. C., room temperature, etc.) for a
period of Ito 36 months or longer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 14, 16, 18, 20, 22, 24, 28, 36, or more months).
[0089] In certain embodiments, storage of the lyophilized
immunogenic composition at these elevated temperatures destroys
less than 20% of the potency of the antigen (e.g., less than 15%,
less than 10%, less than 5%, less than 1%) as measured in an ELISA
and as compared to an equivalent lyophilized immunogenic
composition that was stored between 2 and 8.degree. C. for the same
time period.
[0090] In certain embodiments, the potency of the antigen
post-storage is at least 1.5 fold greater than in an otherwise
equivalent lyophilized immunogenic composition that was stored
under the same elevated temperatures but that was formulated
without NISV (e.g., at least about 2 fold, 2.5 fold, 3 fold, 3.5
fold, 4 fold or 5 fold). In some embodiments, the level of potency
is based on measurements obtained using an ELISA. In some
embodiments, the level of potency is based on plaque assay
measurements.
[0091] In some embodiments, one or more of these potency results
are obtained when the lyophilized immunogenic composition is stored
at 25.degree. C. for 1, 2, 3, 4, 5 or 6 months. In some
embodiments, these results are obtained when the lyophilized
immunogenic composition is stored at 15.degree. C., 20.degree. C.,
30.degree. C., 35.degree. C. or 40.degree. C. for 1 month. In some
embodiments, these results are obtained when the lyophilized
immunogenic composition is stored at 15.degree. C., 20.degree. C.,
30.degree. C., 35.degree. C. or 40.degree. C. for 2 months. In some
embodiments, these results are obtained when the lyophilized
immunogenic composition is stored at 15.degree. C., 20.degree. C.,
30.degree. C., 35.degree. C. or 40.degree. C. for 3 months. In some
embodiments, these results are obtained when the lyophilized
immunogenic composition is stored at 15.degree. C., 20.degree. C.,
30.degree. C., 35.degree. C. or 40.degree. C. for 4 months. In some
embodiments, these results are obtained when the lyophilized
immunogenic composition is stored at 15.degree. C., 20.degree. C.,
30.degree. C., 35.degree. C. or 40.degree. C. for 5 months. In some
embodiments, these results are obtained when the lyophilized
immunogenic composition is stored at 15.degree. C., 20.degree. C.,
30.degree. C., 35.degree. C. or 40.degree. C. for 6 months. In some
embodiments, these results are obtained when the lyophilized
immunogenic composition is stored at 15.degree. C., 20.degree. C.,
30.degree. C., 35.degree. C. or 40.degree. C. for 6 months, 7
months, 8 months, 9 months, 10 months, 11 months, 12 months, 14
months, 16 months, 18 months, 20 months, 22 months, 24 months, 28
months, 36 months, or longer. In certain embodiments these
temperatures may be allowed to vary within a range, e.g.,
.+-.2.degree. C.
IV. Dosage and Administration
[0092] The compositions and methods of this disclosure are useful
for treating infections in humans including adults and children. In
general, however, compositions and methods of the present
disclosure may be used with any animal. In certain embodiments,
compositions and methods herein may be used for veterinary
applications, e.g., canine and feline applications. If desired,
compositions and methods herein may also be used with farm animals,
such as ovine, avian, bovine, porcine and equine breeds.
[0093] Compositions described herein will generally be administered
in such amounts and for such a time as is necessary or sufficient
to induce an immune response. Dosing regimens may consist of a
single dose or a plurality of doses over a period of time. The
exact amount of an immunogenic composition to be administered may
vary from subject to subject and may depend on several factors.
Thus, it will be appreciated that, in general, the precise dose
used will be as determined by the prescribing physician and will
depend not only on the weight of the subject and the route of
administration, but also on the age of the subject and the severity
of the symptoms and/or the risk of infection.
[0094] In certain embodiments, the antigen is taken from a licensed
human viral vaccine and the immunogenic composition is administered
to a human at a dose that is less than the standard human dose
(e.g., in the range of 10-90%, 10-80%, 10-70%, 10-60%, 10-50%,
10-40%, 10-30%, 10-20%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%,
20-40%, 20-30%, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%,
40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-90%, 50-80%, 50-70%,
50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, or 80-90% of the
standard human dose).
[0095] In certain embodiments the immunogenic composition is
administered as a single dose. In certain embodiments the
immunogenic composition is administered as more than one dose
(e.g., 1-3 doses that are separated by 1-12 months).
[0096] In certain embodiments, the compositions may be formulated
for delivery parenterally, e.g., by injection. In such embodiments,
administration may be, for example, intravenous, intramuscular,
intradermal, or subcutaneous, or via by infusion or needleless
injection techniques. In certain embodiments, the compositions may
be formulated for intramuscular delivery. In certain embodiments,
the compositions may be formulated for subcutaneous delivery. For
such parenteral administration, the compositions may be prepared
and maintained in conventional lyophilized compositions and
reconstituted prior to administration with a pharmaceutically
acceptable saline solution, such as a 0.9% saline solution. The pH
of the injectable composition can be adjusted, as is known in the
art, with a pharmaceutically acceptable acid, such as
methanesulfonic acid. Other acceptable vehicles and solvents that
may be employed include Ringer's solution and U.S.P. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil can be
employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid are used in the preparation of
injectables. The injectable compositions can be sterilized, for
example, by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0097] In some embodiments, compositions described herein (e.g.,
antigen-containing vesicles described herein) elicit immune
responses that are higher than immune responses elicited by
corresponding compositions comprising antigens but lacking
vesicles. In some embodiments, compositions comprising
antigen-containing vesicles elicit immune responses that are at
least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
125%, 150%, 175%, 200%, 250%, 500%, 750%, 1000% or more, higher
than immune responses elicited by compositions comprising
corresponding antigens but lacking vesicles Immune responses can be
measured using known assays, such as, for example, an enzyme immune
assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a
radioimmune assay (RIA), a Western blot assay, or a slot blot
assay. These methods are described in, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, 2nd ed., 1988.
EXAMPLES
[0098] The following examples describe some exemplary modes of
making and practicing certain compositions that are described
herein. It should be understood that these examples are for
illustrative purposes only and are not meant to limit the scope of
the compositions and methods described herein.
Example 1
Inverted Melt Formulation Method for Preparing Antigen-Containing
Vesicles
[0099] This example describes an inverted melt formulation method
for preparing antigen-containing non-ionic surfactant vesicles
(NISV). In Step 1, a 5:4:1 molar ratio of the following lipids:
1-monopalmitoyl glycerol (MPG), cholesterol (CHO) and dicetyl
phosphate (DCP) was placed in a flat bottom 50 ml glass beaker,
ensuring none of the powder stuck to the side of the glass beaker.
The lipids were melted in a heated oil bath at about
120-125.degree. C. for 10 minutes, with occasional swirling in the
glass beaker covered with aluminum foil.
[0100] At this stage, a stock solution of inactivated antigen
vaccine (Imovax.RTM. Rabies vaccine reconstituted as per
manufacturer Sanofi Pasteur's instructions) was pre-incubated for
5-10 minutes at about 30-35.degree. C. in a heated water bath. In
Step 2, the resulting vaccine stock solution was homogenized at
8,000 rpm at 30-35.degree. C., and the molten lipid mixture was
added into the homogenizing vaccine stock solution (to give either
a 6.25 mg/ml--test article 1 (TA 1), 12.5 mg/ml--test article 2 (TA
2) or 25 mg/ml--test article 3 (TA 3) total lipid concentration
homogenate) and homogenization was continued for a further 30
seconds at about 30.degree. C. The resulting liposomal suspension
homogenate was transferred into a closed bottle and shaken for 30
minutes at 220.+-.10 rpm and about 30-35.degree. C. An equivalent
volume of a 400 mM sucrose solution in WFI water was added to the
shaken homogenate and the homogenate was further shaken for 5
minutes at 220.+-.10 rpm at about 30-35.degree. C. This mixture was
aliquoted (0.5 ml aseptically transferred into sterile 2 cc vials
sealed with a rubber stopper) and frozen at -78 to -82.degree. C.,
then lyophilized and reconstituted with sterile water for injection
(WFI) prior to use in thermostability studies or in vivo
immunogenicity studies in animals.
Example 2
Thermostability Studies of Inverted Melt Method Formulated
Antigen-Containing Vesicles
[0101] To assess thermostability, NISVs were prepared as described
in Example 1, and lyophilized aliquots were stored (prior to
reconstitution) at two different thermal storage temperatures
(5.+-.3.degree. C. and 40.+-.2.degree. C.). The freeze-dried
Imovax.RTM. Rabies vaccine, used in this Study, is stable if stored
in the refrigerator at 2.degree. C. to 8.degree. C.; while
reconstituted vaccine is not stable and should be used immediately.
The Imovax.RTM. Rabies vaccine is also not stable at elevated
temperatures in either lyophilized or reconstituted forms. At
specified times, stability samples were removed from the
temperature chambers, reconstituted in WFI and analyzed by
appearance, pH, microscopy, Zeta Potential, nanosizing and ELISA
(antigen content). Vaccine controls (Test article 7 (TA
7)--unformulated lyophilized Imovax.RTM. Rabies vaccine) were
stored as above but without addition of NISVs and were also
tested.
[0102] Rabies antigen content in NISV formulations was determined
by performing a sandwich ELISA assay. Prior to the ELISA analysis,
samples and standards were extracted by adding an equal volume of
100 mM carbonate-bicarbonate buffer (pH 9.5) with 0.5% Triton X-100
and pipetting up and down 10 times. Briefly, each well of 96 well
ELISA plates was coated overnight at 4.degree. C. with rabies virus
monoclonal antibody (Ms Mab to Rabies virus (4.2 mg/ml) ab1002,
Abcam) diluted 1/2000 in 25 mM bicarbonate buffer pH 9.7. The next
day the coating solution was removed and the plates were blocked
(1-3 hours at 37.degree. C.) with 5% FBS in 0.05% Tween 20 in PBS.
After the incubation time, plates were washed six times in wash
buffer (0.05% Tween 20 in PBS). Then four to eight 2-fold serial
dilutions of each extracted sample and standard were prepared using
5% FBS in 0.05% Tween 20 in PBS. The extracted and diluted samples
and standards were added to the 96 well ELISA plates and were
incubated for 1.5 h at 37.degree. C. The plates were washed six
times in wash buffer and incubated for 1 h at 37.degree. C. with
primary antibody (1/500 dilution of ferret sera in blocking
solution). The plates were washed six times in wash buffer and
incubated for 1 h at 37.degree. C. with a 1/10,000 dilution of a
goat anti-ferret IgG-Fc HRP conjugated secondary antibody (Bethyl).
The plates were washed six times and developed using TMB substrate
for 10 min at room temperature. Stop solution was added to each
well and absorbance was read at 450 nm within 1 hour using an ELISA
plate reader (Bio-Rad).
[0103] In Table 2 in vitro antigen content results are shown for TA
1 (Imovax.RTM. Rabies vaccine formulated in 6.25 mg/ml total lipid
concentration NISVs), TA 2 (Imovax.RTM. Rabies vaccine in 12.5
mg/ml total lipid concentration NISVs), TA 3 (Imovax.RTM. Rabies
vaccine in 25 mg/ml total lipid concentration NISVs) and TA 7
(unformulated lyophilized Imovax.RTM. Rabies vaccine) stability
samples stored at either 4.degree. C. or 40.degree. C. for 0, 5 or
9 months. (Percent antigen content reflects the ratio of antigen
detected in extracts from NISVs relative to the initial amount of
inactivated antigen vaccine used in the preparation of NISVs).
TABLE-US-00002 TABLE 2 Test Article 0 months 5 months 9 months TA
1-4.degree. C. 71% 78% 79% TA 1-40.degree. C. NA 80% 78% TA
2-4.degree. C. 69% 64% 63% TA 2-40.degree. C. NA 65% 65% TA
3-4.degree. C. 65% 39% 43% TA 3-40.degree. C. NA 43% 47% TA
7-4.degree. C. 88% 81% 73% TA 7-40.degree. C. NA 75% 60%
[0104] As can be seen in Table 2 there is no difference in
thermostability between 4.degree. C. and 40.degree. C. stored
samples of the same test articles for up to 9 months but overall
the higher lipid concentration NISVs formulations stored at both
temperatures were found to have a lower in vitro antigen
content.
[0105] In Table 3 is shown the in vitro antigen content loss
between 4.degree. C. and 40.degree. C. stored samples for TA 1
(Imovax.RTM. Rabies vaccine formulated in 6.25 mg/ml total lipid
concentration NISVs) and TA 7 (unformulated lyophilized Imovax.RTM.
Rabies vaccine) stability samples stored at either 4.degree. C. or
40.degree. C. for 0, 5, 9 or 18 months.
TABLE-US-00003 TABLE 3 Test Article 0 months 5 months 9 months 18
months TA 1 0% 0% 1.3% 13.8% TA 7 0% 7.4% 17.8% 63.6%
[0106] As can be seen in Table 3 no appreciable loss in antigen
content occurred between the 4.degree. C. and 40.degree. C. stored
NISVs formulated Rabies Imovax.RTM. vaccine (TA 1-6.25 mg/ml total
lipid concentration NISVs) for up to 18 months indicative of
thermostability; while TA 7 (unformulated Rabies vaccine) loses
significant antigen content between the 4.degree. C. and 40.degree.
C. stored samples at the same time points which indicates lack of
thermostability.
[0107] In Table 4 is presented the physical-chemical data derived
on testing NISVs formulated Imovax.RTM. Rabies vaccine (TA 1-6.25
mg/ml total lipid concentration NISVs stored for 18 months at
4.degree. C. and 40.degree. C.) versus unformulated Imovax.RTM.
Rabies vaccine (TA 7 stored for 18 months at 4.degree. C. and
40.degree. C.).
TABLE-US-00004 TABLE 4 Z-Ave Zeta Potential Osmolality Test Article
(d, nm) PDI (mV) (mmol/kg) pH TA 1-4.degree. C. 1111 0.530 -76.2
692 9.18 TA 1-40.degree. C. 2126 0.790 -60.0 690 9.33 TA
7-4.degree. C. 18.83 0.741 -20.6 288 9.24 TA 7-40.degree. C. 17.42
0.508 -16.6 299 9.33
[0108] As expected the Z-average and zeta potential were different
between the two test articles as TA 1 was formulated to have
lipid-based antigen-containing vesicles and TA 7 was the
unformulated vaccine control that did not contain any vesicles.
Also as expected the Osmolality between TA 1 and TA 7 was different
due to TA 1 containing sucrose whereas TA 7 did not contain any
sucrose. Test Articles stored at the two different temperatures did
not show any significant differences in physical-chemical
parameters when compared to the other similarly formulated test
articles.
Example 3
In Vivo Animal Testing of Inverted Melt Method Formulated
Antigen-Containing Vesicles
[0109] Female Balb/C mice (6-8 weeks old; body weight 18 to 28
grams, Charles River Canada Inc.) were immunized (n=8)
intramuscularly once on day 0 (with 0.1 ml of indicated vaccine
samples). Pre-immunization and post-1st immunization blood samples
were collected to assess humoral immune responses to formulated and
unformulated Imovax.RTM. Rabies Vaccine. Humoral immune responses
were determined by performing an IgG ELISA Serological Assay. An
indirect ELISA was performed to assess anti-rabies specific IgG
titres in immune serum. Briefly, each well of 96 well ELISA plates
was coated overnight at 4.degree. C. with rabies antigen
(Imovax.RTM. Vaccine, Sanofi Pasteur) diluted 1/25 in 25 mM
bicarbonate buffer pH 9.7. The next day the plates were washed with
PBS containing 0.05% Tween 20 and then blocked (1 h at 37.degree.
C.) with 10% goat sera in PBS. After the incubation time, plates
were washed six times in wash buffer (0.05% Tween 20 in PBS). Then
four to eight 2-fold serial dilutions of each serum sample were
prepared using 10% goat sera in PBS. The sample and the controls
were added to the 96 well ELISA plates and were incubated for 1.5 h
at 37.degree. C. The plates were washed six times in wash buffer
and incubated for 1 h at 37.degree. C. with a 1/5000 dilution of a
goat anti-mouse IgG-Fc HRP conjugated secondary antibody (Bethyl).
The plates were washed six times and developed using TMB substrate
for 3 min at room temperature. Absorbance was read at 450 nm with
an ELISA plate reader (Bio-Rad). The inverted end point titre is
considered the highest sera dilution for which the OD450 reading is
higher or equal with 0.1. Results on Geometric Mean (GM) of OD450
reading of 1/800 dilution of serum samples are presented in Table 5
for Imovax.RTM. Rabies Vaccine formulated with lipids as described
previously versus unformulated Imovax.RTM. Rabies Vaccine.
TABLE-US-00005 TABLE 5 GM of OD450 of 1/800 Test Article Storage
Antigen Dose Formulation Total Serum Group (n = 8) Temp (IU/volume)
Method Lipid Homogenization Dilution) TA 1 4.degree. C. Imovax
.RTM. Inverted Melt 6.25 mg 30 sec at 0.77 Rabies (0.25 with
Sucrose 8,000 rpm IU/100 .mu.L) TA 7 4.degree. C. Imovax .RTM.
Commercial -- -- 0.43 Rabies (0.25 Formulation IU/100 .mu.L)
[0110] The GM of OD450 reading for a 1/800 serum dilution of TA 1
(Imovax.RTM. Rabies Vaccine formulated with 6.25 mg/ml total lipid
concentration NISVs stored at 4.degree. C. for 18 months) was
significantly higher than the GM of OD450 reading for a 1/800 serum
dilution of TA 7 (unformulated Imovax.RTM. Rabies Vaccine stored at
4.degree. C. for 18 months) indicating that the inverted melt
non-ionic surfactant (NISVs) lipid based formulation appeared to
have an adjuvant effect on the Rabies Vaccine.
Other Embodiments
[0111] Other embodiments of the disclosure will be apparent to
those skilled in the art from a consideration of the specification
or practice of the disclosure disclosed herein. It is intended that
the specification and examples be considered as exemplary only,
with the true scope of the disclosure being indicated by the
following claims. The contents of any reference that is referred to
herein are hereby incorporated by reference in their entirety.
* * * * *