U.S. patent application number 13/300217 was filed with the patent office on 2014-10-09 for methods and compositions for live attenuated viruses.
The applicant listed for this patent is Jorge E. Osorio, Dan T. Stinchcomb, O'Neil Wiggan. Invention is credited to Jorge E. Osorio, Dan T. Stinchcomb, O'Neil Wiggan.
Application Number | 20140302091 13/300217 |
Document ID | / |
Family ID | 39827288 |
Filed Date | 2014-10-09 |
United States Patent
Application |
20140302091 |
Kind Code |
A1 |
Stinchcomb; Dan T. ; et
al. |
October 9, 2014 |
METHODS AND COMPOSITIONS FOR LIVE ATTENUATED VIRUSES
Abstract
Embodiments herein relate to compositions of and methods for
live viruses. In certain embodiments, a live, attenuated virus
composition includes, but is not limited to, one or more live,
attenuated viruses and compositions to reduce inactivation and/or
degradation of the live, attenuated virus. In other embodiments,
the live, attenuated virus composition may be a vaccine
composition. In yet other compositions, a live, attenuated virus
composition may include at least one carbohydrate, at least one
protein and at least one high molecular weight surfactants for
reducing inactivation and/or degradation of the live, attenuated
virus.
Inventors: |
Stinchcomb; Dan T.; (US)
; Osorio; Jorge E.; (Mount Horeb, WI) ; Wiggan;
O'Neil; (Fort Collins, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Stinchcomb; Dan T.
Osorio; Jorge E.
Wiggan; O'Neil |
Mount Horeb
Fort Collins |
WI
CO |
US
US
US |
|
|
Family ID: |
39827288 |
Appl. No.: |
13/300217 |
Filed: |
November 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12098077 |
Apr 4, 2008 |
8084039 |
|
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13300217 |
|
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|
60910579 |
Apr 6, 2007 |
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Current U.S.
Class: |
424/218.1 ;
435/236 |
Current CPC
Class: |
A61K 2039/55555
20130101; A61K 2039/70 20130101; C12N 2760/16061 20130101; A61K
47/36 20130101; C12N 2770/24151 20130101; Y02A 50/39 20180101; A61P
31/20 20180101; Y02A 50/396 20180101; Y02A 50/30 20180101; C08G
2650/58 20130101; A61K 47/42 20130101; Y02A 50/394 20180101; A61K
47/26 20130101; C12N 2710/24161 20130101; C08L 71/02 20130101; C12N
7/00 20130101; C12N 2760/16051 20130101; C12N 2770/36161 20130101;
A61K 39/12 20130101; C08L 2203/02 20130101; C12N 2710/10051
20130101; C12N 2710/24151 20130101; C12N 2770/24161 20130101; A61P
31/14 20180101; A61K 2039/5254 20130101; A61P 31/12 20180101; C12N
2770/36151 20130101; Y02A 50/386 20180101; C12N 2710/10061
20130101; C12N 2770/24134 20130101; Y02A 50/388 20180101; C12N
2760/18411 20130101 |
Class at
Publication: |
424/218.1 ;
435/236 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61K 47/36 20060101 A61K047/36; A61K 47/42 20060101
A61K047/42; C12N 7/00 20060101 C12N007/00; A61K 47/26 20060101
A61K047/26 |
Goverment Interests
FEDERALLY FUNDED RESEARCH
[0002] Embodiments disclosed herein were supported in part by grant
number U54 AI-06537-03 from Rocky Mountain Regional Center for
Biodefense and Emerging Infectious Diseases and grant number 5 U01
AI070443-01 from National Institutes of Allergy and Infectious
Diseases. The U.S. government may have certain rights to practice
the subject invention.
Claims
1-32. (canceled)
33. A live attenuated virus composition comprising: one or more
live, attenuated viruses; one or more poly(ethylene oxide) and
polypropylene oxide) (EO-PO) block copolymers, the one or more
EO-PO block copolymers include one or more of poloxamer 338
(Pluronic P108.RTM.), poloxamer 335 (Pluronic P105.RTM.) and
poloxamer 238 (Pluronic F88.RTM.), one or more proteins agents, the
one or more protein agents are one or more albumins selected from
the group consisting of lactalbumins and serum albumins, and one or
more carbohydrate agents, the one or more carbohydrate agents
include trehalose, wherein the composition is capable of reducing
the inactivation of the live attenuated virus.
34. The virus composition of claim 33, wherein the live, attenuated
viruses are selected from the group consisting of Flavivirus,
Togavirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus,
Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus,
Herpesvirus, Poxvirus families and combinations thereof.
35. The virus composition of claim 33, wherein the live, attenuated
viruses are Flaviviruses.
36. The virus composition of claim 33, wherein the composition is
in aqueous form.
37. The virus composition of claim 33, wherein the composition is
partially or wholly dehydrated.
38. The virus composition of claim 33, wherein the one or more
albumins include serum albumins from a vertebrate species.
39. The virus composition of claim 33, wherein the one or more
EO-PO block copolymer is poloxamer 338, and at least one protein
agent is serum albumin.
40. The virus composition of claim 33, wherein the EO-PO block
copolymer concentration is from 0.1 to 4% (w/v).
41. A method for decreasing inactivation of a live, attenuated
virus composition comprising, combining one or more live attenuated
viruses with a composition comprising one or more EO-PO block
copolymers, the one or more EO-PO block copolymers include one or
more of poloxamer 338 (Pluronic P108.RTM.), poloxamer 335 (Pluronic
P105.RTM.) and poloxamer 238 (Pluronic F88.RTM.), one or more
proteins agents, the one or more protein agents are one or more
albumins selected from the group consisting of lactalbumins and
serum albumins, and one or more carbohydrate agents, the one or
more carbohydrate agents include trehalose, wherein the composition
is capable of reducing the inactivation of the live attenuated
virus.
42. The method of claim 41, wherein the live, attenuated viruses
are selected from the group consisting of Flavivirus, Togavirus,
Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus,
Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Herpesvirus,
Poxvirus families and combinations thereof.
43. The method of claim 41, further comprising partially or wholly
dehydrating the combination.
44. The method of claim 43, further comprising partially or wholly
re-hydrating the composition prior to administration.
45. The method of claim 41, wherein the composition increases the
shelf life of an aqueous virus composition.
46. The method of claim 41, wherein the composition decreases
inactivation of an aqueous live, attenuated virus for 24 hours or
greater.
47. The method of claim 41, wherein the composition decreases
inactivation of an aqueous live, attenuated virus during one or
more freeze and thaw cycles.
48. The method of claim 41, wherein the one or more EO-PO block
copolymer is poloxamer 338, and the one or more protein agents is
serum albumin.
49. The method of claim 41, wherein the virus composition is
administered to a subject to reduce the onset of or prevent a
health condition.
50. The method of claim 49, wherein the virus is selected from the
group consisting of West Nile, Dengue, Japanese encephalitis, St.
Louis encephalitis, Tick-borne encephalitis, and Yellow fever.
51. A kit for decreasing the inactivation of a live, attenuated
virus composition comprising: at least one container; and a
composition comprising one or more EO-PO block copolymers, the one
or more EO-PO block copolymers include one or more of poloxamer 338
(Pluronic P108.RTM.), poloxamer 335 (Pluronic P105.RTM.) and
poloxamer 238 (Pluronic F88.RTM.), one or more albumins, the one or
more albumins selected from the group consisting of lactalbumins
and serum albumins, one or more carbohydrate agents, the one or
more carbohydrate agents include trehalose.
52. The kit of claim 51, wherein at least one albumin is serum
albumin.
53. The kit of claim 51, wherein the EO-PO block copolymer
concentration is from 0.1 to 4% (w/v).
54. The kit of claim 52, wherein the serum albumin concentration is
from 0.001 to 3% (w/v).
55. The kit of claim 51, wherein the composition further comprises
one or more live, attenuated viruses.
56. The kit of claim 51, wherein the live, attenuated viruses are
selected from the group consisting of Flavivirus, Togavirus,
Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus, Orthomyxovirus,
Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus, Herpesvirus,
Poxvirus families and combinations thereof.
Description
PRIORITY
[0001] This application claims priority under 35 U.S.C. 121 as a
divisional application of U.S. patent application Ser. No.
12/098,077, filed Apr. 4, 2008 which claims the benefit under 35
USC .sctn.119(e) of provisional U.S. patent application Ser. No.
60/910,579 filed on Apr. 6, 2007, which are incorporated herein in
their entirety for all purposes.
FIELD
[0003] Embodiments herein relate to compositions and methods for
stabilizing live, attenuated viruses. Other embodiments relate to
compositions and methods for reducing degradation of live,
attenuated viruses. Still other embodiments relate to uses of these
compositions in kits for portable applications and methods.
BACKGROUND
[0004] Vaccines to protect against viral infections have been
effectively used to reduce the incidence of human disease. One of
the most successful technologies for viral vaccines is to immunize
animals or humans with a weakened or attenuated strain of the virus
(a "live, attenuated virus"). Due to limited replication after
immunization, the attenuated strain does not cause disease.
However, the limited viral replication is sufficient to express the
full repertoire of viral antigens and generates potent and
long-lasting immune responses to the virus. Thus, upon subsequent
exposure to a pathogenic strain of the virus, the immunized
individual is protected from disease. These live, attenuated viral
vaccines are among the most successful vaccines used in public
health.
[0005] Ten of the sixteen viral vaccines approved for sale in the
U.S. are live, attenuated viruses. Highly successful live viral
vaccines include the yellow fever 17D virus, Sabin poliovirus types
1, 2 and 3, measles, mumps, rubella, varicella and vaccinia
viruses. Use of the vaccinia virus vaccine to control smallpox
outbreaks led to the first and only eradication of a human disease.
The Sabin poliovirus vaccine has helped prevent crippling disease
throughout the world and is being used in the efforts to eradicate
polio. Childhood vaccination with measles, mumps, rubella and
varicella vaccines prevent millions of deaths and illnesses
internationally.
[0006] Recent technical advances, such as reassortment, reverse
genetics and cold adaptation, have led to the licensure of live,
attenuated viruses for influenza and rotavirus. A number of live,
viral vaccines developed with recombinant DNA technologies are in
human clinical testing, including vaccines for West Nile disease,
dengue fever, malaria, tuberculosis and HIV. These recombinant
viral vaccines rely on manipulation of well-characterized
attenuated viral vaccines, such as adenovirus, vaccinia virus,
yellow fever 17D or the dengue virus, DEN-2 PDK-53. The safe,
attenuated viruses are genetically engineered to express protective
antigens for other viral or bacterial pathogens. Several
recombinant viral vaccines have been approved for animal use,
including a canarypox/feline leukemia recombinant virus, a
canarypox/canine distemper recombinant virus, a canarypox/West Nile
recombinant virus and a yellow fever/West Nile recombinant virus.
As a group, the live attenuated virus vaccines are amongst the most
successful medical interventions in human history, second only to
the advent of antibiotics and hold the promise to improve public
health throughout the world.
[0007] In order for live, attenuated viral vaccines to be
effective, they must be capable of replicating after immunization.
Thus, any factors that inactivate the virus can cripple the
vaccine. For example, widespread distribution and use of the
smallpox vaccine prior to World War II was limited because the
virus was inactivated after only a few days at ambient
temperatures. In the 1920s, French scientists demonstration that
freeze-dried vaccine provided long term stability and techniques
for large-scale manufacture of freeze-dried vaccine were developed
in the 1940s (see for example Collier 1955). In addition to
freeze-drying, various additives have been identified that can help
stabilize the viruses in live, attenuated viral vaccines (See for
example Burke, Hsu et al 1999). These stabilizers typically include
one or more of the following components: divalent cations, buffered
salt solutions, chelators, urea, sugars (e.g. sucrose, lactose,
trehalose), polyols (e.g., glycerol, mannitol, sorbitol,
polyethylene glycol), amino acids, protein hydro lystates (e.g.
casein hydrolysate, lactalbumin hydrolysate, peptone), proteins
(e.g. gelatin, human serum albumin) or polymers (e.g. dextran).
[0008] However, even with these stabilizing agents, many of the
commonly used vaccines still require refrigeration for
stabilization. Other commonly used vaccines are sensitive to
temperature extremes; either excessive heat or accidental freezing
can inactivate the vaccine. Maintaining this "cold chain"
throughout distribution is particularly difficult in the developing
world. Thus, there remains a need for improving the stability of
both existing and newly developed live, attenuated viral
vaccines.
[0009] Flaviviruses are amongst the most labile viruses. They are
enveloped viruses with a RNA genome of approximately 11,000 bases.
Most of the flaviviruses are transmitted by an arthropod vector,
commonly mosquitoes. There are over 70 different flaviviruses that
are grouped into three major categories based on serology: the
dengue group, the Japanese encephalitis group and the yellow fever
group. Amongst the known flaviviruses, 40 are transmitted by
mosquitoes, 16 are transmitted by ticks and 18 viruses have no
identified insect vector. Thus, most flaviviruses have evolved to
replicate in both their arthropod vector and their vertebrate host
species (often birds or mammals). Expanding urbanization, worldwide
travel and environmental changes (such as deforestation or rain
patterns) have lead to the emergence of several flaviviruses as
threats to human public health. Such viruses include, but are not
limited to, yellow fever virus, the dengue viruses, West Nile
virus, Japanese encephalitis virus, and tick-borne encephalitis
viruses.
[0010] Through intensive mosquito control and vaccination efforts,
yellow fever was eliminated from much of North, Central and South
America, the Caribbean and Europe. However, in the last 20 years,
the number of countries reporting cases has increased. Yellow fever
virus is now endemic in major portions of Africa and South America
and some Caribbean islands. The World Health Organization (WHO)
estimates that 200,000 cases of yellow fever occur annually leading
to 30,000 deaths. Since World War II, dengue flaviviruses have
spread to tropical and subtropical regions throughout the world and
now threaten over 3.5 billion people, about half of the world's
population. The WHO estimates that 50-100 million cases of dengue
fever occur annually. 500,000 of these are the more sever,
life-threatening form of the disease, termed dengue hemorrhagic
fever, that leads to more than 25,000 deaths per year. A
particularly virulent form of West Nile virus was introduced into
the Western hemisphere, presumably by travel, in New York in 1999.
The mosquito-transmitted virus infected birds as the primary host,
but also caused disease and mortality in humans and horses. West
Nile virus spread throughout the United States and into Canada and
Mexico. Since its introduction, West Nile virus has caused over
20,000 reported cases of West Nile disease leading to 950 deaths in
the United States. Japanese encephalitis virus causes 30,000 to
50,000 cases of neurological disease annually, primarily in eastern
and southern Asia. 25-30% of the reported cases are fatal. The
tick-borne encephalitis viruses are endemic to parts of Europe and
Asia and continue to cause episodic outbreaks affecting thousands
of individuals. Related viruses with more limited geographical
spread include Kunjin virus (a close relative of West Nile) and
Murray Valley encephalitis virus in Australia and New Guinea, St.
Louis encephalitis virus in North and South America, the Usutu,
Koutango, and Yaonde viruses in Africa, and Cacipacore virus in
South American.
[0011] Live, attenuated viral vaccines have been developed that are
safe and protect against flavivirus diseases, such as yellow fever
and Japanese encephalitis. The live, attenuated viral vaccine, 17D,
has been widely used to prevent yellow fever. The current
flavivirus vaccines are lyophilized in the presence of stabilizers.
Nonetheless, the vaccines require storage and shipment at
2-8.degree. C., a requirement that is difficult to achieve in the
developing world and more remote regions of developed nations.
Furthermore, upon reconstitution, the vaccines rapidly lose potency
even when stored at 2-8.degree. C.
[0012] The measles vaccine is another example of a labile
attenuated virus that is used worldwide to prevent disease. Measles
virus is an enveloped, non-segmented negative strand RNA virus of
the Paramyxovirus family. Measles is a highly contagious, seasonal
disease that can affect virtually every child before puberty in the
absence of vaccination. In developing countries, mortality rates in
measles-infected children can by as high as 2 to 15%. Indeed,
despite efforts to institute worldwide immunization, measles still
causes greater than 7,000 deaths in children per year. The measles
vaccine is a live, attenuated virus that is manufactured in primary
chicken fibroblast cells. The vaccine is stabilized with gelatin
and sorbitol and is then lyophilized. The stabilized, lyophilized
vaccine has a shelf life of 2 years or more if stored at 2 to
8.degree. C. However, the lyophilized vaccine still requires a cold
chain that is difficult to maintain in the developing world.
Furthermore, upon reconstitution, the vaccine loses 50% of its
potency within 1 hour at room temperature (20 to 25.degree.
C.).
[0013] Thus, a need exists in the art for improved vaccine
formulations.
SUMMARY
[0014] Embodiments herein concern methods and compositions to
reduce or prevent deterioration or inactivation of a live
attenuated virus composition. Certain compositions disclosed can
include combinations of components that reduce deterioration of a
live attenuated virus. Other embodiments herein concern
combinations of excipients that greatly enhance the stability of
live attenuated viruses. Yet other compositions and methods herein
are directed to reducing the need for lower temperatures (e.g.
refrigerated or frozen storage) while increasing the shelf life of
aqueous and/or reconstituted live attenuated virus.
[0015] In accordance with these embodiments, certain live
attenuated viruses are directed to flaviviruses. Some embodiments,
directed to compositions, can include, but are not limited to, one
or more live, attenuated viruses, such as one or more live,
attenuated flaviviruses in combination with one or more high
molecular weight surfactants, proteins, and carbohydrates.
[0016] Compositions contemplated herein can increase the
stabilization and/or reduce the inactivation and/or degradation of
a live attenuated virus including, but not limited to, a live
attenuated Flavivirus, To gavirus, Coronavirus, Rhabdovirus,
Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus,
Retrovirus, Hepadnavirus, Pestivirus, Picornavirus, Calicivirus,
Reovirus, Parvovirus, Papovavirus, Adenovirus, Herpes virus, or
Poxvirus.
[0017] Other embodiments concern live, attenuated virus
compositions and methods directed to a vaccine compositions capable
of reducing or preventing onset of a medical condition caused by
one or more of the viruses contemplated herein. In accordance with
these embodiments, medical conditions may include, but are not
limited to, West Nile infection, dengue fever, Japanese
encephalitis, Kyasanur forest disease, Murray valley encephalitis,
Alkhurma hemorrhagic fever, St. Louis encephalitis, tick-borne
encephalitis, yellow fever and hepatitis C virus infection.
[0018] In certain embodiments, compositions contemplated herein can
be partially or wholly dehydrated or hydrated. In other
embodiments, protein agents contemplated of use in compositions
herein can include, but are not limited to, lactalbumin, human
serum albumin, a recombinant human serum albumin (rHSA), bovine
serum albumin (BSA), other serum albumins or albumin gene family
members. Saccharides or polyol agents can include, but are not
limited to, monosaccharides, disaccharides, sugar alcohols,
trehalose, sucrose, maltose, isomaltose, cellibiose, gentiobiose,
laminaribose, xylobiose, mannobiose, lactose, fructose, sorbitol,
mannitol, lactitol, xylitol, erythritol, raffinose, amylse,
cyclodextrins, chitosan, or cellulose. In certain embodiments,
surfactant agents can include, but are not limited to, a nonionic
surfactant such as alkyl poly(ethylene oxide), copolymers of
poly(ethylene oxide) and polypropylene oxide) (EO-PO block
copolymers), poly(vinyl pyrroloidone), alkyl polyglucosides (such
as sucrose monostearate, lauryl diglucoside, or sorbitan
monolaureate, octyl glucoside and decyl maltoside), fatty alcohols
(cetyl alcohol or olelyl alcohol), or cocamides (cocamide MEA,
cocamide DEA and cocamide TEA).
[0019] In other embodiments, the surfactants can include, but are
not limited to, the Pluronic F127, Pluronic F68, Pluronic P123, or
other EO-PO block copolymers of greater than 3,000-4,000 MW.
[0020] In some embodiments, vaccine compositions can include, but
are not limited to, one or more protein agent that is serum
albumin; one or more saccharide agent that is trehalose; and one or
more surfactant polymer agent that is the EO-PO block copolymer
Pluronic F127.
[0021] Some embodiments herein concern partially or wholly
dehydrated live, attenuated viral compositions. In accordance with
these embodiments, a composition may be 20% or more; 30% or more;
40% or more; 50% or more; 60% or more; 70% or more; 80% or more; or
90% or more dehydrated.
[0022] Other embodiments concern methods for decreasing
inactivation of a live attenuated viruses including, but not
limited to, combining one or more live attenuated viruses with a
composition capable of reducing inactivation of a live, attenuated
virus including, but not limited to, one or more protein agents;
one or more saccharides or polyols agents; and one or more high
molecular weight surfactants, wherein the composition decreases
inactivation of the live attenuated virus. In accordance with these
embodiments, the live attenuated virus may include, but is not
limited to, a Flavivirus, Togavirus, Coronavirus, Rhabdovirus,
Filovirus, Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus,
Retrovirus, Hepadnavirus, Pestivirus, Picornavirus, Calicivirus,
Reovirus, Parvovirus, Papovavirus, Adenovirus, Herpes virus, or a
Poxvirus. Additionally, methods and compositions disclosed herein
can include freeze drying or other dehydrating methods for the
combination. In accordance with these methods and compositions, the
methods and compositions decrease inactivation of the freeze dried
or partially or wholly dehydrated live attenuated virus. In other
methods, compositions for decreasing inactivation of a live
attenuated virus may comprise an aqueous composition or may
comprise a rehydrated composition after dehydration. Compositions
described herein are capable of increasing the shelf life of an
aqueous or rehydrated live attenuated virus.
[0023] In certain particular embodiments, a live attenuated virus
for use in a vaccine composition contemplated herein may include,
but is not limited to, one or more live, attenuated flavivirus
vaccines, including but not limited to, attenuated yellow fever
viruses (such as 17D), attenuated Japanese encephalitis viruses,
(such as SA 14-14-2), attenuated dengue viruses (such as
DEN-2/PDK-53 or DEN-4A30) or recombinant chimeric flaviviruses.
[0024] In certain embodiments, compositions contemplated herein are
capable of decreasing inactivation and/or degradation of a hydrated
live attenuated virus for greater than 24 hours at room
temperatures (e.g. about 20.degree. to about 25.degree. C.) or
refrigeration temperatures (e.g. about 0.degree. to about
10.degree. C.). In more particular embodiments, a combination
composition is capable of maintaining about 100 percent of the live
attenuated virus for greater than 24 hours. In addition,
combination compositions contemplated herein are capable of
reducing inactivation of a hydrated live attenuated virus during at
least 2 freeze and thaw cycles. Other methods concern combination
compositions capable of reducing inactivation of a hydrated live
attenuated virus for about 24 hours to about 50 days at
refrigeration temperatures (e.g. about 0.degree. to about
10.degree. C.). Compositions contemplated in these methods, can
include, but are not limited to, one or more protein agent of serum
albumin; one or more saccharide agent of trehalose; and one or more
EO-PO block copolymer agent of Pluronic F127. In certain
embodiments, the live, attenuated virus composition remains at
about 100% viral titer after 7 days at approximately 21.degree. C.
and about 100% viral titer after 50 days at refrigeration
temperatures around 4.degree. C. Other embodiments herein may
include live, attenuated virus composition remaining at about 90%,
or about 80% viral titer after 7 days at approximately 21.degree.
C. and about 90%, or about 80% viral titer after 50 days at
refrigeration temperatures around 4.degree. C. Other embodiments
contemplated include live, attenuated virus compositions remaining
at about 3.times. to about 10.times. the concentration of viral
titer after several hours (e.g. 20 hours) at approximately
37.degree. C. compared to other compositions known in the art. (see
for example, FIGS. 4 and 5). Compositions disclosed herein reduce
degradation of the live, attenuated virus when the composition is
stored at approximately 37.degree. C.
[0025] Other embodiments concern kits for decreasing the
inactivation of a live, attenuated virus composition including, but
not limited to, a container; and a composition including, but not
limited to, one or more protein agents, one or more saccharide or
polyol agents, and one or more EO-PO block copolymer agents,
wherein the composition decreases inactivation and/or degradation
of a live, attenuated virus. In accordance with these embodiments,
a kit composition may include one or more one protein agent of
serum albumin; one or more saccharide agent of trehalose; and one
or more EO-PO block copolymer agent. Additionally, a kit
contemplated herein may further include one or more live,
attenuated viruses including, but not limited to, a Flavivirus,
Togavirus, Coronavirus, Rhabdovirus, Filovirus, Paramyxovirus,
Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus, Hepadnavirus,
Pestivirus, Picornavirus, Calicivirus, Reovirus, Parvovirus,
Papovavirus, Adenovirus, Herpes virus, or Poxvirus. In certain
embodiments, compositions herein can include trehalose as a
saccharide agent. In accordance with these embodiments, trehalose
concentration may be equal to or greater than 5% (w/v). In certain
embodiments, compositions herein can include polymer F127 as an
EO-PO block copolymer agent. In accordance with these embodiments,
polymer F127 concentration may be about 0.1 to about 4 percent
(w/v).
[0026] In other embodiments, compositions contemplated herein may
contain trace amounts or no divalent cations. For example,
compositions contemplated herein may have trace amounts or no
calcium/magnesium (Ca.sup.+2/Mg.sup.+2).
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The following drawings form part of the instant
specification and are included to further demonstrate certain
aspects of particular embodiments herein. The embodiments may be
better understood by reference to one or more of these drawings in
combination with the detailed description presented herein.
[0028] FIG. 1 represents an exemplary histogram of experiments
using various compositions for testing the stability of an
exemplary virus, DEN-2 PDK 53 flavivirus, in the compositions.
[0029] FIG. 2 represents an exemplary graph of a kinetic analysis
of an exemplary virus, DEN-2 PDK 53 flavivirus, for viral
inactivation at 37.degree. C. in various exemplary
compositions.
[0030] FIG. 3 represents an exemplary histogram of an analysis of
an exemplary virus, DEN-2 PDK 53 virus, stored at 37.degree. C. for
21 hours. Values are expressed as a percentage of the viral titer
remaining after incubation relative to the input titer. Formulation
percentages refer to (w/v) of the respective excipient.
[0031] FIG. 4 represents an exemplary histogram of an analysis of
an exemplary virus, DEN-2 PDK 53 virus, stored at 37.degree. C. for
23 hours in different compositions. Values are expressed as a
percentage of the viral titer remaining after incubation relative
to the input titer.
[0032] FIG. 5 represents an exemplary histogram of an analysis of
an exemplary virus, DEN-2 PDK 53 virus, stored at 37.degree. C. for
23 hours in different compositions. Values are expressed as a
percentage of the viral titer remaining after incubation relative
to the input titer. The two bars for each formulation represent
duplicates in the experiment.
[0033] FIG. 6 represents an exemplary histogram analysis of an
exemplary virus, DEN-2 PDK 53 virus, after two freeze-thaw cycles
when stored in different formulations. Values are expressed as a
percentage of the viral titer remaining after freeze-thaw cycles
relative to the input titer.
[0034] FIG. 7 represents an exemplary graph of a kinetic analysis
of an exemplary virus, DEN-2 PDK 53/WN recombinant flavivirus, in
various exemplary compositions for viral inactivation at 25.degree.
C. over several weeks of time.
[0035] FIG. 8 represents an exemplary graph of a kinetic analysis
of an exemplary virus, DEN-2 PDK 53/WN recombinant flavivirus, in
various exemplary compositions for viral inactivation at 4.degree.
C. over several weeks of time.
[0036] FIG. 9 represents an exemplary histogram analysis of an
exemplary virus, DEN-2 PDK-53 virus, after lyophilization in
various exemplary compositions. Viral inactivation was assessed as
described above after two weeks at different temperatures.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Definitions
[0037] As used herein, "a" or "an" may mean one or more than one of
an item.
[0038] As used herein, "about" may mean up to and including plus or
minus five percent, for example, about 100 may mean 95 and up to
105.
[0039] As used herein, "saccharide" agents can mean one or more
monosaccharides, (e.g. glucose, galactose, ribose, mannose,
rhamnose, talose, xylose, or allose arabinose.), one or more
disaccharides (e.g. trehalose, sucrose, maltose, isomaltose,
cellibiose, gentiobiose, laminaribose, xylobiose, mannobiose,
lactose, or fructose.), trisaccharides (e.g. acarbose, raffinose,
melizitose, panose, or cellotriose) or sugar polymers (e.g.
dextran, xanthan, pullulan, cyclodextrins, amylose, amylopectin,
starch, celloologosaccharides, cellulose, maltooligosaccharides,
glycogen, chitosan, or chitin).
[0040] As used herein, "polyol" agents can mean any sugar alcohol
(e.g. mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol,
glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene
glycol, or glycerol). As used herein, "high molecular weight
surfactants" can mean a surface active, amphiphilic molecule
greater than 1500 molecular weight.
[0041] As used herein, "EO-PO block copolymer" can mean a copolymer
consisting of blocks of poly(ethylene oxide) and poly(propylene)
oxide. In addition, as used herein, "Pluronic" can mean EO-PO block
copolymers in the EOx-POy-EOx. This configuration of EO-PO block
copolymer is also referred to as "Poloxamer" or "Synperonic".
[0042] As used herein, "attenuated virus" can mean a virus that
demonstrates reduced or no clinical signs of disease when
administered to an animal.
DETAILED DESCRIPTIONS
[0043] In the following sections, various exemplary compositions
and methods are described in order to detail various embodiments.
It will be obvious to one skilled in the art that practicing the
various embodiments does not require the employment of all or even
some of the specific details outlined herein, but rather that
concentrations, times and other specific details may be modified
through routine experimentation. In some cases, well known methods
or components have not been included in the description.
[0044] Stability of flavivirus vaccines has been assessed for both
the existing yellow fever and Japanese encephalitis live,
attenuated viruses. When tested in 1987, only five of the twelve
yellow fever vaccines manufactured at that time met minimal
standards of stability. Subsequently, addition of a mixture of
sugars, amino acids and divalent cations was demonstrated to
stabilize the lyophilized vaccine, so that the vaccine lost less
than 1 log of potency after incubation at 37.degree. C. for 14
days. Stabilizing lyophilized formulations for the yellow fever
vaccine have been described (see for example U.S. Pat. No.
4,500,512). U.S. Pat. No. 4,500,512, describes a combination of
lactose, sorbitol, the divalent cations, calcium and magnesium, and
at least one amino acid. While this formulation may help to
stabilize the lyophilized vaccine, it fails to provide stability to
the vaccine in aqueous form. Another study examined the ability of
several different formulations including the compositions described
above and the effect of sucrose, trehalose and lactalbumin on the
stability of the lyophilized yellow fever vaccine. Formulations
consisting of 10% sucrose alone, 2% sorbitol with 4% inositol, or
10% sucrose with 5% lactalbumin, 0.1 g/l CaCl2 and 0.076 g/l MgSO4
were found to provide the best stability (see for example Adebayo,
Sim-Brandenburg et al. 1998). However, in all cases after
resuspension, yellow fever vaccine is still very unstable and must
be discarded after only about one hour (see for example Monath
1996; Adebayo, Sim-Brandenburg et al. 1998). This leads to vaccine
wastage and the potential to cause administration of ineffective
vaccine under field conditions, if an unstable vaccine is used.
[0045] Another live, attenuated flavirus vaccine for protection
against Japanese encephalitis has been licensed and is in
widespread use in China (see for example Halstead and Tsai 2004).
The Japanese encephalitis vaccine strain, SA 14-14-2, is grown on
primary hamster kidney cells and the cell supernatant is harvested
and coarsely filtered. One previous composition included 1% gelatin
and 5% sorbitol added as stabilizers. Using these stabilizers, the
vaccine is lyophilized and then is stable at 2 to 8.degree. C. for
at least 1.5 years, but for only 4 months at room temperature and
10 days at 37.degree. C. As with the yellow fever vaccine, the
reconstituted vaccine is very labile and is stable for only 2 hours
at room temperature (see for example Wanf, Yang et al 1990). In
certain embodiments herein, live, attenuated flavirus virus
compositions for stabilizing or reducing the degradation of
Japanese encephalitis are contemplated.
[0046] No formulation for a live, attenuated flavivirus vaccine has
been identified that provides long term stability of lyophilized
formulations at temperatures greater than 2-8.degree. C. In
addition, no formulation has been described that prevents loss of
titer, stabilizes or reduces degradation of aqueous vaccines for
greater than a few hours.
[0047] Formulations for other live, attenuated viruses have also
been described (see for example Burke, Hsu et al. 1999). One common
stabilizer, referred to as SPGA is a mixture of 2 to 10% sucrose,
phosphate, potassium glutamate and 0.5 to 2% serum albumin (see for
example Bovarnick, Miller et al. 1950). Various modifications of
this basic formulation have been identified with different cations,
with substitutions of starch hydrolysate or dextran for sucrose,
and with substitutions of casein hydrolysate or poly-vinyl
pyrrolidone for serum albumin. Other formulations use hydrolyzed
gelatin instead of serum albumin as a protein source (Burke, Hsu et
al 1999). However, gelatin can cause allergic reactions in
immunized children and could be a cause of vaccine-related adverse
events. U.S. Pat. No. 6,210,683 describes the substitution of
recombinant human serum albumin for albumin purified from human
serum in vaccine formulations.
[0048] Embodiments herein disclose compositions that enhance the
stability of and/or reduce deterioration of live, attenuated virus
vaccines compared to those in the prior art. Certain compositions
disclosed herein provide stability of aqueous viruses for up to 2
hours; up to 3 hours; up to 4 hours and greater than 4 hours at or
about 37.degree. C. Certain compositions disclosed herein provide
stability of aqueous viruses for up to 1 day to about 1 week or
more, at or about room temperature (e.g. 25.degree. C.).
Embodiments contemplated herein provide increased protection of a
live, attenuated virus from for example, freezing and/or thawing,
and/or elevated temperatures. In certain embodiments, compositions
herein can stabilize, reduce deterioration and/or prevent
inactivation of dehydrated live, attenuated viral products in room
temperature conditions (e.g. about 25.degree. C.). In other
embodiments, compositions contemplated herein can stabilize, reduce
deterioration and/or prevent inactivation of aqueous live,
attenuated viral products at about 25.degree. C. or up to or about
37.degree. C. Compositions and methods disclosed herein can
facilitate the storage, distribution, delivery and administration
of viral vaccines in developed and under developed regions.
[0049] Other embodiments can include compositions for live
attenuated virus vaccines including, but not limited to,
Picornaviruses (e.g., polio virus, foot and mouth disease virus),
Caliciviruses (e.g., SARS virus, and feline infectious peritonitis
virus), Togaviruses (e.g., sindbis virus, the equine encephalitis
viruses, chikungunya virus, rubella virus, Ross River virus, bovine
diarrhea virus, hog cholera virus), Flaviviruses (e.g., dengue
virus, West Nile virus, yellow fever virus, Japanese encephalitis
virus, St. Louis encephalitis virus, tick-borne encephalitis
virus), Coronaviruses (e.g., human coronaviruses (common cold),
swine gastroenteritis virus), Rhabdoviruses (e.g., rabies virus,
vesicular stomatitis viruses), Filoviruses (e.g., Marburg virus,
Ebola virus), Paramyxoviruses (e.g., measles virus, canine
distemper virus, mumps virus, parainfluenza viruses, respiratory
syncytial virus, Newcastle disease virus, rinderpest virus),
Orthomyxoviruses (e.g., human influenza viruses, avian influenza
viruses, equine influenza viruses), Bunyaviruses (e.g., hantavirus,
LaCrosse virus, Rift Valley fever virus), Arenaviruses (e.g., Lassa
virus, Machupo virus), Reoviruses (e.g., human reoviruses, human
rotavirus.), Birnaviruses (e.g., infectious bursal virus, fish
pancreatic necrosis virus), Retroviruses (e.g., HIV 1, HIV 2,
HTLV-1, HTLV-2, bovine leukemia virus, feline immunodeficiency
virus, feline sarcoma virus, mouse mammary tumor virus),
Hepadnaviruses (e.g., hepatitis B virus), Parvoviruses (e.g., human
parvovirus B, canine parvovirus, feline panleukopenia virus)
Papovaviruses (e.g., human papillomaviruses, SV40, bovine
papillomaviruses), Adenoviruses (e.g., human adenovirus, canine
adenovirus, bovine adenovirus, porcine adenovirus), Herpes viruses
(e.g., herpes simplex viruses, varicella-zoster virus, infectious
bovine rhinotracheitis virus, human cytomegalovirus, human
herpesvirus 6), and Poxviruses (e.g., vaccinia, fowlpoxviruses,
raccoon poxvirus, skunkpox virus, monkeypoxvirus, cowpox virus,
musculum contagiosum virus).
[0050] Those skilled in the art will recognize that compositions or
formulas herein relate to viruses that are attenuated by any means,
including but not limited to, cell culture passage, reassortment,
incorporation of mutations in infectious clones, reverse genetics,
other recombinant DNA or RNA manipulation. In addition, those
skilled in the art will recognize that other embodiments relate to
viruses that are engineered to express any other proteins or RNA
including, but not limited to, recombinant flaviviruses,
recombinant adenoviruses, recombinant poxviruses, recombinant
retroviruses, recombinant adeno-associated viruses and recombinant
herpes viruses. Such viruses may be used as vaccines for infectious
diseases, vaccines to treat oncological conditions, or viruses to
introduce express proteins or RNA (e.g., gene therapy, antisense
therapy, ribozyme therapy or small inhibitory RNA therapy) to treat
disorders.
[0051] In some embodiments, compositions herein can contain one or
more viruses with membrane envelopes (e.g., enveloped viruses) of
the Togavirus, Flavivirus, Coronavirus, Rhabdovirus, Filovirus,
Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, Retrovirus,
Hepadnavirus, Herpesvirus or Poxvirus families. In certain
embodiments compositions contain one or more enveloped RNA viruses
of the Togavirus, Flavivirus, Coronavirus, Rhabdovirus, Filovirus,
Paramyxovirus, Orthomyxovirus, Bunyavirus, Arenavirus, or
Retrovirus families. In other embodiments, compositions herein can
contain one or more enveloped, positive strand RNA virus of the
Togavirus, Flavivirus, Coronavirus, or Retrovirus families. In
certain embodiments, compositions can contain one or more live,
attenuated Flaviviruses (e.g., dengue virus, West Nile virus,
yellow fever virus, or Japanese encephalitis virus).
[0052] Some embodiments herein relate to compositions for live,
attenuated viruses in aqueous or lyophilized form. Those skilled in
the art will recognize that formulations that improve thermal viral
stability and prevent freeze-thaw inactivation will improve
products that are liquid, powdered, freeze-dried or lyophilized and
prepared by methods known in the art. After reconstitution, such
stabilized vaccines can be administered by a variety routes,
including, but not limited to intradermal administration,
subcutaneous administration, intramuscular administration,
intranasal administration, pulmonary administration or oral
administration. A variety of devices are known in the art for
delivery of the vaccine including, but not limited to, syringe and
needle injection, bifurcated needle administration, administration
by intradermal patches or pumps, needle-free jet delivery,
intradermal particle delivery, or aerosol powder delivery.
[0053] Embodiments can include compositions consisting of one or
more live attenuated viruses (as described above) and a mixture of
one or more high molecular weight surfactants and one or more
proteins in a physiological acceptable buffer. In certain
embodiments, compositions include, but are not limited to one or
more live attenuated viruses, one or more high molecular weight
surfactants, one or more proteins, and one or more carbohydrates,
in a physiological acceptable buffer.
[0054] In other embodiments, compositions can contain one or more
high molecular weight surfactants that increase the thermal
stability of live, attenuated viruses. Surfactants have been
incorporated into vaccine formulations to prevent material loss to
surfaces such as glass vials (see for example Burke, Hsu et al.
1999). However, certain embodiments herein include high molecular
weight surfactants with some unusual biochemical properties of
utility for compositions and methods disclosed herein. The EO-PO
block copolymers can include blocks of polyethylene oxide
(--CH.sub.2CH.sub.2O-- designated EO) and polypropylene oxide
(--CH.sub.2CHCH.sub.3O-- designated PO). The PO block can be
flanked by two EO blocks in a EO.sub.x--PO.sub.y-EO.sub.x
arrangement. Since the PO component is hydrophilic and the EO
component is hydrophobic, overall hydrophilicity, molecular weight
and the surfactant properties can be adjusted by varying x and y in
the EO.sub.x--PO.sub.y-EO.sub.x block structure. In aqueous
solutions, the EO-PO block copolymers will self-assemble into
micelles with a PO core and a corona of hydrophilic EO groups.
EO-PO block copolymer formulations have been investigated as
potential drug delivery agents for a variety of hydrophobic drugs
and for protein, DNA or inactivated vaccines (e.g. Todd, Lee et al.
1998; Kabanov, Lemieux et al. 2002). At high concentrations (for
example: >than 10%) certain of the higher molecular weight EO-PO
block copolymers will undergo reverse gelation, forming a gel as
the temperature increases. Gel formation at body temperatures
permits use of the EO-PO block copolymer gels to act as a depot in
drug and vaccine delivery applications (see for example Coeshott,
Smithson et al. 2004). In addition, due to their surfactant
properties, these polymers have been used in adjuvant formulations,
and as an emulsifier in topically applied creams and gels. The
EO-PO block copolymers have also been shown to accelerate wound and
burn healing and to seal cell membranes after radiation or
electroporation-mediated damage.
[0055] In other embodiments, vaccine compositions can include one
or more surfactants with molecular weight of 1500 or greater. In a
certain embodiment, the surfactant is a non-ionic, hydrophilic,
polyoxyethylene-polyoxypropylene block copolymer (or EO-PO block
copolymer). While EO-PO block copolymers have been used as
adjuvants and delivery vehicles for inactivated vaccines, protein
vaccines or DNA vaccines, their use to prevent inactivation of a
live virus is not anticipated in the art. In a particular
embodiment, a formulation can contain one or more EO-PO polymers
with a molecular weight of 3,000 or greater. In further
embodiments, compositions can include in part an EO-PO block
copolymer Pluronic F127 or Pluronic P123. Those skilled in the art
will recognize that modifications of the surfactants can be
chemically made. It is contemplated herein any essentially
equivalent surfactant polymers are considered.
[0056] Embodiments herein can include compositions of one or more
live, attenuated viruses, one or more surfactants and one or more
proteins. In certain embodiments, a protein can be an albumin.
Serum albumin is one of the most common proteins in vertebrate
blood and has multiple functions. The protein is 585 amino acids
with a molecular weight of 66500. Human serum albumin is not
glycosylated and has a single free thiol group implicated in some
of its myriad binding activities. Serum albumin is predominantly
.alpha.-helix with three structural domains, each subdivided into
two subdomains. Albumin is known to specifically bind a variety of
molecules, including drugs such as aspirin, ibuprofen, halothane,
propofol and warfarin as well as fatty acids, amino acids,
steroids, glutathione, metals, bilirubin, lysolecithin, hematin,
and prostaglandins. The different structural domains are implicated
in drug binding; most small molecule drugs and hormones bind to one
of two primary sites located in subdomains IIA and IIIA. Due to its
lack of immunogenicity, albumin is commonly used as a carrier
protein in biological products. Since the protein dose contained in
a live, attenuated viral vaccine can be fractions of a microgram
(derived from 10.sup.3 to 10.sup.5 viral particles), an inert
carrier protein is used to prevent loss due to absorption and
non-specific binding to glass, plastic or other surfaces. However,
as demonstrated herein, an unexpected improvement in stability was
observed with the combination of an albumin and EO-PO block
copolymers suggesting interactions between the two components
and/or between the components and the viral particles. In addition,
enhanced stabilization of viruses in the presence of albumin is not
likely due to function as a general carrier protein: other proteins
such as gelatin and lactoferrin fail to improve virus
stability.
[0057] In certain embodiments, serum albumin may be from a human or
other mammalian source. For vaccines intended for human use,
particular embodiments can include human albumin or other human
products as needed in order to reduce or eliminate adverse immune
responses. Those skilled in the art will recognize that albumins
specific for each species may be used in animal vaccines (e.g.
canine albumin for canine products, bovine albumin for bovine
products). In further embodiments, the protein is a recombinant
human albumin. Standard methods exist for expressing recombinant
human albumin or portions thereof in a variety of expression
systems including bacteria, yeast, algae, plant, mammalian cell or
transgenic animal systems. In addition, serum albumin or portions
thereof can be produced in cell-free systems or chemically
synthesized. Recombinant human albumin produced in these or in any
similar system is incorporated herein. Those skilled in the art
will recognize that other proteins can substitute for albumin. For
example, albumin is a member of a multi-gene family. Due to their
structural and sequence similarities, other members of the family
(e.g. .alpha.-fetoprotein, vitamin D binding protein, or afamin)
may substitute for albumin in compositions and methods contemplated
herein. Those skilled in the art will also recognize that
modifications can be made to albumin by any means known in the art,
for example, by recombinant DNA technology, by post-translational
modification, by proteolytic cleavage and/or by chemical means.
Those substitutions and alterations to albumin that provide
essentially equivalent stabilizing function to serum albumin
without substitutions and alterations are contemplated herein.
[0058] In certain embodiments, compositions having a high molecular
weight surfactant, a protein and a carbohydrate in a
pharmaceutically acceptable buffer are described. In some
embodiments, the carbohydrate is a sugar or a polyol. Sugars can
include, but are not limited to, monosaccharides, (e.g. glucose,
galactose, ribose, mannose, rhamnose, talose, xylose or allose
arabinose), disaccharides (e.g. trehalose, sucrose, maltose,
isomaltose, cellibiose, gentiobiose, laminaribose, xylobiose,
mannobiose, lactose, or fructose.), trisaccharides (e.g. acarbose,
raffinose, melizitose, panose, or cellotriose) or sugar polymers
(e.g. dextran, xanthan, pullulan, cyclo dextrins, amylose,
amylopectin, starch, celloologosaccharides, cellulose,
maltooligosaccharides, glycogen, chitosan, or chitin). Polyols can
include, but are not limited to, mannitol, sorbitol, arabitol,
erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol,
polyethylene glycol, polypropylene glycol, and glycerol.
[0059] In a particular embodiment, formulations can contain a
combination of one or more EO-PO block copolymers, one or more
proteins, and trehalose in a pharmacologically acceptable buffer.
In certain embodiments, trehalose can be present at concentrations
ranging from 5 to 50% (w/v). Trehalose has been used to enhance the
stability of protein formulations. It is widely known in the art as
a cryopreservative and is used in nature to protect organisms from
stress. Anhydrobiotic organisms that can tolerate low water
conditions contain large amounts of trehalose. Trehalose has been
shown to prevent both membrane fusion events and phase transitions
that can cause membrane destabilization during drying. Structural
analysis suggests that trehalose fits well between the polar head
groups in lipid bylayers. Trehalose also prevents denaturation of
labile proteins during drying. It is thought that trehalose
stabilizes proteins by hydrogen bonding with polar protein
residues. Trehalose is a disaccharide consisting of two glucose
molecules in a 1:1 linkage. Due to the 1:1 linkage, trehalose has
little or no reducing power and is thus essentially non-reactive
with amino acids and proteins. This lack of reducing activity may
improve the stabilizing affect of trehalose on proteins. In certain
embodiments, trehalose provides stability to live, attenuated
viruses. This activity of trehalose may be due to its ability to
stabilize both the membranes and coat proteins of the viruses.
[0060] In further embodiments, compositions can include one or more
EO-PO block copolymers, one or more proteins and one or more
carbohydrates, where one of the carbohydrates is chitosan, in a
physiological acceptable buffer to provide improved stability to
live, attenuated viruses. In certain embodiments, compositions can
include chitosan at concentrations ranging from 0.001 to 2% (e.g at
a pH of about 6.8). Chitosan is a cationic polysaccharide derived
by deacetylation of chitin, the structural polymer of crustacean
exoskeletons. It is a polymer of N-acetyl-glucosamine and
glucosamine; the content of the two carbohydrates depends on the
extent of deacetylation. Chitosan's positive charge allows it to
bind to negatively charged surfaces and molecules. Thus, it binds
musosal surfaces and is thought to promote mucosal absorption.
Chitosan also can bind and form nanoparticles with DNA, RNA and
other oligonucleotides and has been used in non-viral gene
delivery. Certain embodiments herein demonstrate that chitosan
increases live, attenuated virus stability.
[0061] In certain embodiments, compositions can be described that
typically include a physiologically acceptable buffer. Those
skilled in the art recognize that a variety of physiologically
acceptable buffers exist, including, but not limited to buffers
containing phosphate, TRIS, MOPS, HEPES, bicarbonate, other buffers
known in the art ad combinations of buffers. In addition, those
skilled in the art recognize that adjusting salt concentrations to
near physiological levels (e.g., saline or 0.15 M total salt) may
be optimal for parenteral administration of compositions to prevent
cellular damage and/or pain at the site of injection. Those skilled
in the art also will recognize that as carbohydrate concentrations
increase, salt concentrations can be decreased to maintain
equivalent osmolarity to the formulation. In certain embodiments, a
buffering media with pH greater than 6.8 is contemplated; some
live, attenuated viruses (e.g. flaviviruses) are unstable at low
pH. In another embodiment, physiologically acceptable buffer can be
phosphate-buffered saline (PBS).
[0062] Some live, attenuated viral vaccine compositions herein
concern compositions that increase stability and/or reduce
deterioration of live, attenuated virus in addition to having
reduced immunogenicity or are non-immunogenic. In accordance with
these embodiments, compositions can include one or more protein
agents; one or more saccharides or polyols agents; and one or more
high molecular weight surfactants, wherein the composition
decreases inactivation of the live attenuated virus. Therefore,
certain compositions contemplated herein have reduced adverse
reaction when administered to a subject. In some exemplary
compositions, the surfactant agent(s) consists of one or more EO-PO
block copolymers; the protein agent(s) are selected from the group
consisting of lactalbumin, serum albumin, .alpha.-fetoprotein,
vitamin D binding protein, afamin derived from a vertebrate
species; and the carbohydrate agent(s) is one or more of a
saccharide and/or a polyol. In certain embodiments, compositions
can include one or more of the carbohydrate agent(s) selected from
the group consisting of trehalose, sucrose, chitosan, sorbitol, and
mannitol. In certain more particular embodiments, in order to
reduce immune reaction to a vaccine, the serum albumin can be
derived from a vertebrate species or in other embodiments, from the
same source as the subject (e.g. human). In other embodiments, the
carbohydrate agent is trehalose. In certain embodiments, at least
one surfactant agent is the EO-PO block copolymer Pluonic F127. In
some live, attenuated viral vaccine compositions at least one
carbohydrate agent is trehalose. In certain live, attenuated viral
vaccine compositions include, the EO-PO block copolymer Pluronic
F127 where the concentration is from 0.1 to 4% (w/v); and/or serum
albumin concentration from 0.001 to 3% (w/v) and/or the trehalose
concentration can be from 5 to 50% (w/v).
Pharmaceutical Compositions
[0063] Embodiments herein provide for administration of
compositions to subjects in a biologically compatible form suitable
for pharmaceutical administration in vivo. By "biologically
compatible form suitable for administration in vivo" is meant a
form of the active agent (e.g. live, attenuated virus composition
of the embodiments) to be administered in which any toxic effects
are outweighed by the therapeutic effects of the active agent.
Administration of a therapeutically active amount of the
therapeutic compositions is defined as an amount effective, at
dosages and for periods of time necessary to achieve a desired
result. For example, a therapeutically active amount of a compound
may vary according to factors such as the disease state, age, sex,
and weight of the individual, and the ability formulations to
elicit a desired response in the individual. Dosage regima may be
adjusted to provide the optimum therapeutic response.
[0064] In some embodiments, composition (e.g. pharmaceutical
chemical, protein, peptide of an embodiment) may be administered in
a convenient manner such as subcutaneous, intravenous, by oral
administration, inhalation, transdermal application, intravaginal
application, topical application, intranasal or rectal
administration. In a more particular embodiment, the compound may
be orally or subcutaneously administered. In another embodiment,
the compound may be administered intravenously. In one embodiment,
the compound may be administered intranasally, such as
inhalation.
[0065] A compound may be administered to a subject in an
appropriate carrier or diluent, co-administered with the
composition. The term "pharmaceutically acceptable carrier" as used
herein is intended to include diluents such as saline and aqueous
buffer solutions. The active agent may also be administered
parenterally or intraperitoneally. Dispersions can also be prepared
in glycerol, liquid polyethylene glycols, and mixtures thereof and
in oils. Under ordinary conditions of storage and use, these
preparations may contain a preservative to prevent the growth of
microorganisms.
[0066] Pharmaceutical compositions suitable for injectable use may
be administered by means known in the art. For example, sterile
aqueous solutions (where water soluble) or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersion may be used. In all cases, the composition
can be sterile and can be fluid to the extent that easy
syringability exists. It may further be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The pharmaceutically acceptable carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyetheylene
glycol, and the like), and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants.
[0067] Sterile injectable solutions can be prepared by
incorporating active compound in an amount with an appropriate
solvent or with one or a combination of ingredients enumerated
above, as required, followed by sterilization.
[0068] Upon formulation, solutions can be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above. It is contemplated that slow release
capsules, timed-release microparticles, and the like can also be
employed for administering compositions herein. These particular
aqueous solutions are especially suitable for intravenous,
intramuscular, subcutaneous and intraperitoneal administration.
[0069] The active therapeutic agents may be formulated within a
mixture can include about 0.0001 to 1.0 milligrams, or about 0.001
to 0.1 milligrams, or about 0.1 to 1.0 or even about 1 to 10 gram
per dose. Single dose or multiple doses can also be administered on
an appropriate schedule for a predetermined situation. In some
embodiments, doses can be administered before, during and/or after
exposure to a virus contemplated herein.
[0070] In another embodiment, nasal solutions or sprays, aerosols
or inhalants may be used to deliver the compound of interest.
Additional formulations that are suitable for other modes of
administration include suppositories and pessaries. A rectal
pessary or suppository may also be used. In general, for
suppositories, traditional binders and carriers may include, for
example, polyalkylene glycols or triglycerides; such suppositories
may be formed from mixtures containing the active ingredient in the
range of 0.5% to 10%, preferably 1% 2%.
[0071] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. In certain embodiments, oral pharmaceutical
compositions can include an inert diluent or assimilable edible
carrier, or may be enclosed in hard or soft shell gelatin capsule,
or may be compressed into tablets, or may be incorporated directly
with the food of the diet. For oral therapeutic administration, the
active compounds may be incorporated with excipients and used in
the form of ingestible tablets, buccal tables, troches, capsules,
elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 0.1% of
active compound. The percentage of the compositions and
preparations may, of course, be varied and may conveniently be
between about 2 to about 75% of the weight of the unit, or
preferably between 25-60%. The amount of active compounds in such
therapeutically useful compositions is such that a suitable dosage
can be obtained.
Kits
[0072] Further embodiments concerns kits for use with methods and
compositions described herein. Compositions and live virus
formulations may be provided in the kit. The kits can also include
a suitable container, live, attenuated virus compositions detailed
herein and optionally one or more additional agents such as other
anti-viral agents, anti-fungal or anti-bacterial agents.
[0073] The kits may further include a suitably aliquoted
composition of use in a subject in need thereof. In addition,
compositions herein may be partially or wholly dehydrated or
aqueous. Kits contemplated herein may be stored at room
temperatures or at refrigerated temperatures as disclosed herein
depending on the particular formulation.
[0074] The container means of the kits will generally include at
least one vial, test tube, flask, bottle, syringe or other
container means, into which a composition may be placed, and
preferably, suitably aliquoted. Where an additional component is
provided, the kit will also generally contain one or more
additional containers into which this agent or component may be
placed. Kits herein will also typically include a means for
containing the agent, composition and any other reagent containers
in close confinement for commercial sale. Such containers may
include injection or blow-molded plastic containers into which the
desired vials are retained.
EXAMPLES
[0075] The following examples are included to demonstrate certain
embodiments presented herein. It should be appreciated by those of
skill in the art that the techniques disclosed in the Examples
which follow represent techniques discovered to function well in
the practices disclosed herein, and thus can be considered to
constitute preferred modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope herein.
Example 1
Base Stability of DEN-2 PDK 53 Flavivirus in Liquid Phase
[0076] In one illustrative method, the thermal stability for
flaviviruses in liquid phase was investigated. In accordance with
this method, the base stability of the DEN-2 PDK 53 parental
vaccine vector, stored in phosphate buffered saline (PBS), at
different temperatures was determined (Table 1). In one example,
1.times.10.sup.4 pfu of DEN-2 PDK 53 virus in a total volume of 0.5
ml PBS was incubated, in 2 ml screw capped vials at either
4.degree. C., room temperature (.about.21.degree. C.) or 37.degree.
C. After 24 hours of incubation viral titer and activity was
determined by a Neutral Red agarose overlay plaque titration assay
in Vero cells. As illustrated in Table 1, incubation of DEN-2 PDK
53 in PBS at 4.degree. C. results in an average four-fold decrease
in viral titer and complete loss in viral activity when incubated
at 37.degree. C. for the same period. These results demonstrate the
relatively poor stability of the DEN-2 PDK 53 flavivirus in the
absence of stabilizing excipients.
TABLE-US-00001 TABLE 1 Stability of Den-2 PDK53 virus stored for 24
hours at different temperatures. Percentage Viral Temperature
Formulation Titer Loss 4.degree. C. PBS 75 ~21.degree. C. PBS 78
37.degree. C. PBS 100
Example 2
Stabilizing Effects of Compositions
[0077] In certain exemplary compositions, pharmaceutically
acceptable excipients contemplated herein that aid in thermal
stability of live viral vaccines are known in the art. In one
exemplary method, PBS was used as a base composition to assess the
stabilizing effects of different excipients. In these examples, a
stock solution of each excipient was made in PBS and the pH
adjusted to approximately 7.1 with NaOH, except for chitosan where
the pH of the stock solution was adjusted to approximately 6.8.
Excipients were diluted in PBS to the final concentrations
indicated (w/v) (Table 2). In accordance with this method,
1.times.10.sup.4 pfu of DEN-2 PDK 53 virus, in serum-free medium,
was added to 0.5 ml of each composition and stored at 37.degree. C.
for 24 hours. Following incubation, viral activity and titer was
determined by plaque titration in Vero cells, as described above.
As illustrated in Table 2, the stabilizing effects of compositions
including a single excipient, at various concentrations comparable
to previous experimental examples, was minimal. However, some
excipients for example, trehalose and recombinant human serum
albumin (rHSA), were more effective than others at stabilizing
DEN-2 PDK 53 virus at 37.degree. C. Results of the study
represented in Table 2 also revealed that increased stabilizing
effects of several excipients, including rHSA and trehalose, can be
obtained within certain ranges of concentrations of these
excipients. In this particular example, trehalose was more
effective at concentrations above 15% (w/v) and F127 at
concentrations between 0.5 and 3%.
TABLE-US-00002 TABLE 2 Effects of different excipients on DEN-2
PDK53 stability when stored at 37.degree. C. for 24 hours
Percentage Viral Formulation Titer Loss PBS 100.0 10% Sucrose 99.9
15% Sucrose 98.3 20% Sucrose 96.4 25% Sucrose 93.4 2% Trehalose
98.3 5% Trehalose 97.0 10% Trehalose 93.3 15% Trehalose 83.3 2%
Mannitol 100.0 5% Mannitol 100.0 10% Mannitol 99.8 15% Mannitol
86.7 5% Sorbitol 100 10% Sorbitol 99.9 15% Sorbitol 99.9 1%
Polyvinyl Pyrrolidone 100.0 5% Polyvinyl Pyrrolidone 100.0 10%
Polyvinyl Pyrrolidone 100.0 0.2% F127 99.6 0.5% F127 99.6 1% F127
99.5 2% F127 99.5 10% F127 99.9 0.1% rHSA 91.2 0.5% rHSA 95.0 1.0%
rHSA 89.0 3.0% rHSA 89.0 5.0% rHSA 97.5 0.05% Chitosan 99.0 0.1%
Chitosan 99.0
Example 3
Stabilizing Effects of Compositions Including Specific Combinations
of Excipients
[0078] In the following illustrative procedure, compositions
including multiple excipients in differing combinations and
concentrations were tested for stabilizing effects on the parental
DEN-2 PDK 53 flaviviral vaccine. Excipients were diluted to the
indicated final concentrations in PBS from stock solutions as
described in Example 2. 1.times.10.sup.4 pfu of DEN-2 PDK 53
vaccine virus was incubated at 37.degree. C. in 0.5 ml of each
composition for 21 hours (FIG. 1) or over a 48 hour period (FIG.
2). At the specified time intervals viral titer and activity was
determined by a plaque titration assay as described in example 1.
FIG. 1 represents exemplary results of this demonstration expressed
as percentage of viral titer remaining after incubation, relative
to input, and as log.sub.10 titer loss in FIG. 2. Analysis of
different combinations of excipients, in this particular
illustration, revealed that formulations consisting of a
saccharide, a pluronic co-polymer non-ionic surfactant and a
protein were optimal at improving DEN-2 PDK 53 stability at
37.degree. C. Formulations including trehalose, F127 and rHSA had
the greatest stabilizing effects. Unexpectedly, the combined
stabilizing effect of these three excipients was much greater than
the sum of that observed with each individual component suggesting
synergism between the components. Improved thermal stability of the
DEN-2 PDK 53 flavivirus was obtained through the synergistic
activities of the combination of trehalose, F127 and rHSA could not
have been anticipated based on prior art examples. FIGS. 1 and 2
also illustrate that the stabilizing effect of the
trehalose/F127/rHSA mixture was further enhanced by the addition of
0.05% chitosan. FIG. 2 shows that the rate of viral inactivation
when stored over a 48 hour period at 37.degree. C. is significantly
reduced by compositions containing trehalose, F127 and rHSA.
Examples in the art suggest that the stability of flaviviruses can
be enhanced by formulations containing Ca.sup.2+ and Mg.sup.2+
divalent cations. However, as represented in FIGS. 1 and 2, the
addition of Ca.sup.2+ (0.0009M) and Mg.sup.2+ (0.0005M) to a
formulation confers no additional stabilizing benefits. The results
from FIG. 2 suggest that addition of divalent cations may have a
negative impact to long term liquid phase viral stability in the
context of particular embodiments.
[0079] In one exemplary method, a composition including trehalose,
F127 and rHSA was assessed for its stabilizing properties with
multiple flaviviruses. The stability of chimeric DEN-2 flaviviruses
expressing the membrane and envelope proteins from either West Nile
(DEN-2/WN), Dengue 1 (DEN-2/D1), Dengue 3 (DEN-2/D3, or Dengue 4
(DEN-2/D4) viruses was determined as described for Example 1.
Illustrative results in Table 3 reveal greatly improved liquid
phase stability of all the chimeric flaviviruses when stored in a
composition including trehalose, F127 and rHSA. The different
chimeras express different envelope and membrane proteins from five
serologically distinct flaviviruses. In addition, West Nile virus
and the dengue viruses are significantly divergent. This result
suggests that compositions herein may be useful for liquid phase
stabilization of diverse members of the family of Flaviviradae as
well as other virus families. The ability to stabilize flaviviruses
at room temperature (.about.21.degree. C.) and at 4.degree. C. was
examined by representative procedures as outlined for Example 1.
The exemplary results, illustrated in Table 4, reveal that a
composition including trehalose, F127 and rHSA effectively
preserves viral activity for 7 days at 21.degree. C. and for 48
days at 4.degree. C.
TABLE-US-00003 TABLE 3 Stability of different chimeric flaviviruses
stored at 37.degree. C. for 21 hours in PBS or a composition (F1)
including 15% trehalose, 2% F127 and 1% rHSA. % Viral Titer Virus
Formulation Remaining DEN-2/WN PBS 2 F1 45 DEN-2/D1 PBS 0.2 F1 22
DEN-2/D3 PBS 0.3 F1 30 DEN-2/D4 PBS 1 F1 28
TABLE-US-00004 TABLE 4 Stability of flaviviruses stored at
different temperatures for 7 or 48 days in PBS or a composition
(F1) including 15% trehalose, 2% F127 and 1% rHSA. Percentage Viral
Titer Remaining Virus Temperature Formulation 7 days 48 days DEN-2
PDK-53 21.degree. C. PBS 0 0 21.degree. C. F1 100 0 4.degree. C.
PBS 0 0 4.degree. C. F1 100 100 DEN-2/WN 21.degree. C. PBS 0 0
21.degree. C. F1 100 0 4.degree. C. PBS 0 0 4.degree. C. F1 100
100
Example 4
Use of Alternate Components
[0080] Another exemplary method was used to compare the stabilizing
effects of bovine serum albumin (BSA) and, gelatin, to that of rHSA
and of different pluronic co-polymers. DEN-2 PDK 53 viral stability
assays were conducted as outlined previously for Examples 1 and 2.
The previous examples suggested that formulations including
trehalose, F127 and rHSA optimally improved the thermal stability
of the DEN-2 PDK 53 parental vaccine virus. As shown by example in
FIG. 3, the stabilizing effects of bovine serum albumin are
comparable to those of rHSA either alone or in combination with
trehalose and F127. FIG. 3 also demonstrates that as isolated
excipients, gelatin is comparable to rHSA in stabilizing DEN-2 PDK
53 at 37.degree. C. However in this exemplary method, unlike BSA,
gelatin does not appear to be an effective substitute for rHSA in
compositions also containing trehalose and F127. Thus, while
proteins other than rHSA may be used in combination with trehalose
and F127 to aid in stabilization of flaviviral vaccines, the use of
a serum albumin or closely related proteins is more suitable in
accordance with this exemplary method. In addition, FIG. 3
illustrates that, as isolated excipients, the polymer Pluronic P123
is comparable to Pluronic F127 in its ability to stabilitze the
DEN-2 PDK-53 virus. However, in this exemplary method, P123 does
not appear to be an effective subsititute for F127 in compositions
also containing trehalose and serum albumin. As exemplified in FIG.
4, compositions containing trehalose, rHSA and other commonly used
pharmaceutical surfactants such as Polysorbate 20 (Tween 20),
instead of a pluronic co-polymer, are not effective in stabilizing
DEN-2 PDK 53 relative to formulations containing a pluronic
co-polymer. These exemplary methods suggest better stabilizing
efficiencies of formulations containing distinct high molecular
weight pluronic co-polymer surfactants.
[0081] Exemplary data is further illustrated in FIG. 4. FIG. 4.
represents stability of the DEN-2 PDK 53 virus in compositions
containing different surfactants. DEN-2 PDK 53 was stored at
37.degree. C. for 23 hours in each formulation. Surfactants
evaluated in this example include n-octyl-.beta.-D-glucopyranoside
(.beta.-OG), Polysorbate 20 (P 20), Polysorbate 80 (P 80) and F127
(F). Other formulation components include trehalose (T) and rHSA
(A). Values are expressed as a percentage of the viral titer
remaining after incubation relative to the input titer.
Example 5
Comparison of the Stabilizing Effects of Different Compositions
[0082] The stabilizing properties of one exemplary composition were
compared to that of compositions known in the art. A stabilizing
composition for live flaviviral vaccines, disclosed in the art
(U.S. Pat. No. 4,500,512), includes 4% lactose, 2% sorbitol, 0.1
g/L CaCl.sub.2, 0.076 MgSO.sub.4 and amino acids on the order of
0.0005M to 0.05M in PBS. Another composition reported by Adebayo et
al (1998) consists of 10% sucrose, 5% lactalbumin, 0.1 g/L
CaCl.sub.2, and 0.076 g/L MgSO.sub.4. In one exemplary method,
stabilizing properties of these formulations were compared to a
particular embodiment herein. In one example composition, F1, this
composition includes 15% trehalose, 2% F127 and 1% recombinant HSA.
F2 is the formulation of U.S. Pat. No. 4,500,512 without amino
acids and F3 is the same formulation with the amino acids histidine
and alanine. F4 is the composition of Adebayo, et al.
1.times.10.sup.4 pfu of DEN-2 PDK 53 vaccine virus were incubated
at 37.degree. C. in 0.5 ml of each composition for 23 hours, after
which viral activity and titer was assayed as described in Example
1. As exemplified in FIG. 5, some embodiments, for example
formulation F1, represents a significant improvement over those
previously described compositions. In the example shown, virtually
no viral activity was recovered after storage in the formulations
known in the art (formulations F3 and F4), whereas upwards of 50%
of the initial viral titer was recovered after storage in a
composition disclosed herein. These results reveal that previous
formulations are ineffective at promoting live viral vaccine
stability during liquid phase storage.
Example 6
Preservation of Viral Activity after Multiple Freeze-Thaws
[0083] In one exemplary method, the ability of select compositions
to preserve viral activity after freeze-thaw cycles was
demonstrated. 1.times.10.sup.4 pfu of DEN-2 PDK 53 vaccine virus
was suspended in 0.5 ml of each composition in screw cap vials. For
the first freeze-thaw cycle vials were frozen at -80.degree. C. for
24 hours and thawed rapidly at 37.degree. C. This was immediately
followed by a second freeze-thaw cycle where the vials were frozen
at -80.degree. C. for 1 hour and thawed rapidly at 37.degree. C.
Viral titer and activity was then assessed by a plaque titration
assay as described in Example 1. As illustrated in FIG. 6,
particular compositions that include trehalose, F127 and rHSA
effectively preserved full viral activity through two freeze-thaw
cycles. Additionally, compositions including these three excipients
were more effective than those containing just a single excipient.
The results of this particular illustrative experiment suggest the
compositions and methods disclosed herein are an effective
cryoprotectant for flaviviral vaccines and may facilitate viral
preservation during freeze-drying, spray-drying, or other
dehydration techniques.
Example 7
Stabilization of Other Live, Attenuated Viruses
[0084] Examples illustrated previously reveal effective liquid
phase stabilization of several live, attenuated flaviviruses in
compositions including trehalose, F127 and rHSA. It is anticipated
that embodiments disclosed herein may also be effective at
stabilizing other live, attenuated viruses. For example, a
formulation including trehalose, F127 and rHSA may be used to
stabilize live attenuated measles virus, an attenuated sindbis
virus, an attenuated influenza virus, a recombinant, attenuated
adenovirus or a recombinant, attenuated vaccinia virus. In one
exemplary method, these non-flaviviral viruses can be suspended and
maintained in liquid phase, in a composition including trehalose,
F127, and rHSA directly after harvesting from cell culture. In
another illustrative method, non-flaviviral viruses can be
suspended in a composition prior to, or subsequent to, freeze or
spray-drying. Statistically improved viral stability may
demonstrate that the formulation of this embodiment is applicable
to other attenuated viral vaccines outside of the Flavivirus
family. Those skilled in the art recognize that application may
then be extended to other live, attenuated viruses.
Example 8
Safety and In Vivo Immunogenicity
[0085] Molecular interactions between excipients and molecular or
cellular components may serve, not only to enhance stability of
viral vaccines, but also to cause increased cell or tissue damage
in vivo. The formulations may decrease the immunogenicity of these
viral vaccines in live animals. In this example, it is demonstrated
that exemplary compositions are safe after subcutaneous injection
and are essentially immunologically inert. Four different exemplary
compositions were selected for testing in mice as follows.
Formulation 1: 15% Trehalose, 2% F-127, 1% rHSA Formulation 2: 15%
Trehalose, 2% F-127, 1% rHSA, 1 mM CaCl.sub.2/0.5 mM MgSO.sub.4
Formulation 3: 15% Trehalose, 2% F-127, 1% rHSA, 0.5% chitosan
Formulation 4: 22.5% Trehalose, 3% F-127, 1.5% rHSA
Formulation 5: PBS
[0086] In certain methods described herein, groups of 8 or 9 NIH
Swiss mice were immunized by subcutaneous injection with
1.times.10.sup.5 pfu of a formulated DEN-2 PDK-53/WN recombinant
flavivirus vaccine at day 0 (d0), were boosted with the same
formulated vaccine at d29 and were then challenged with 10.sup.3
pfu on a pathogenic West Nile strain (NY99) on d45. Control mice
(four groups of 8) received formulations 1-4 alone with no virus.
No adverse events after administration in any of the immunized mice
were observed. Thus, in this example, no apparent adverse events
are caused by the exemplary formulations with or without vaccine
virus. Sera were collected prior to immunization at d0, prior to
boost at d28, prior to challenge at d44 and post-challenge at d75.
West Nile neutralizing antibody titers in the sera were determined
by plaque reduction neutralization test (PRNT). The results of the
study are represented in Table 5.
TABLE-US-00005 TABLE 5 Neutralizing antibody and protection induced
by formulated DEN2/WN vaccines Post-prime Post-boost Post-Challenge
(d28) (d44) (d75) Formulation Number GMT.sup.1 % SC.sup.2 GMT % SC
GMT % SC Survival % Survival 1 8 30 87.5 123 100 761 100 8/8 100 2
8 10 62.5 226 100 830 100 8/8 100 3 8 40 100 123 100 1810 100 8/8
100 4 9 10 66.7 137 100 1660 100 8/9 88.9 5 9 10 66.7 109 100 1742
100 9/9 100 Controls 32 1 0 1 0 1280 100 7/32 21.9 .sup.1GMT =
geometric mean titer; titers of <10 were arbitrarily assigned a
value of 1. .sup.2% SC = percentage of animals that sero-converted
with PRNT titers >10.
[0087] A majority of the animals receiving the DEN-2/WN vaccine
sero-converted after the first dose regardless of whether no
formulation (Formulation 5) or one of the exemplary formulations
(Formulations 1-4) was used. In addition, all of the vaccinated
animals sero-converted after the booster administration. Geometric
mean PRNT titers (GMT) demonstrate few differences between the
vaccine groups. Titers were low after the primary immunization,
increased 3-10 fold after the boost and then showed a dramatic
anamnestic response upon challenge. 100% of all the vaccinated
animals survived challenge, again independent of vaccine
formulation. Only 22% of the control animals survived; those that
did survive showed evidence of potent neutralizing antibody
responses after challenge. One advantage is that this example
demonstrates that the exemplary formulations do not reduce the
ability of an exemplary recombinant DEN-2/WN vaccine to prevent
West Nile disease in a mice.
Example 9
[0088] In another example, liquid compositions were used containing
trehalose, rHSA and F127 to stabilize a West Nile chemeric
flavivirus stored for various periods at either 25.degree. C. or
4.degree. C. 1.times.10.sup.4 pfu of chimeric DEN-2/WN vaccine
virus were incubated at each temperature and viral activity was
assessed at one or two week intervals as described in Example 1. As
illustrated in FIGS. 7 and 8, formulations containing trehalose,
rHSA and F127 significantly improved the thermal stability of the
DEN-2/WN vaccine virus during storage at 25.degree. C. and
4.degree. C., respectively. At 25.degree. C. loss of viral activity
was less than one log over 7 days. At 4.degree. C. viral
inactivation was negligible for periods up to 12 weeks when stored
in exemplary formulations including trehalose, F127 and rHSA.
Example 10
[0089] In another exemplary method, stabilizing effects of
compositions were demonstrated including trehalose, rHSA and a
pluronic co-polymer with dehydrated DEN-2 PDK 53 vaccines.
1.times.10.sup.4 pfu of DEN-2 PDK 53 vaccine virus formulated in
accordance with procedures disclosed herein. Formulated vaccines
were placed in serum vials and subjected to conventional
lyophilzation procedures. Dried vaccines were stoppered under
vacuum, stored at either 37.degree. C. or 4.degree. C. for 14 days
followed by reconstitution of the vaccine to its original liquid
volume by addition of sterile water. Viral activity of the
reconstituted vaccine was assessed as outlined earlier. At
37.degree. C., in the presence of compositions containing
trehalose, rHSA and a pluronic co-polymer formulated in phosphate
buffered saline, an average viral titer loss of 1 log was observed
(FIG. 9). No loss in viral activity was observed for formulated
dehydrated DEN-2 PDK 53 viral vaccines stored at 4.degree. C. for
14 days. These results demonstrate effective preservation of a
dehydrated viral vaccine utilizing compositions disclosed
herein.
[0090] FIG. 9. represents stability of lyophilized DEN-2 PDK 53 at
different temperatures. Log titer loss of formulated lyophilized
DEN-2 PDK 53 vaccine virus following incubation at 37.degree. C. or
4.degree. C. for 2 weeks as indicated. Formulations F1 (15%
trehalose, 2% F127, 1% rHSA) and F2 (15% trehalose, 2% F127, 0.01%
rHSA) were formulated in phosphate buffered saline. Formulation F3
(15% trehalose, 2% F127, 0.01% rHSA) was formulated in 10 mM Tris
base.
[0091] All of the COMPOSITIONS and METHODS disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
have been described in terms of preferred embodiments, it is
apparent to those of skill in the art that variations maybe applied
to the COMPOSITIONS and METHODS and in the steps or in the sequence
of steps of the methods described herein without departing from the
concept, spirit and scope herein. More specifically, certain agents
that are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept as defined by the appended
claims.
[0092] All documents, or portions of documents, cited in this
application, including but not limited to patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety.
* * * * *