U.S. patent application number 14/642480 was filed with the patent office on 2015-09-03 for compositions, methods and uses for poxvirus elements in vaccine constructs against influenza virus subtypes or strains.
The applicant listed for this patent is Takeda Vaccines, Inc.. Invention is credited to Joseph N. Brewoo, Jeremy C. Jones, Jorge E. Osorio, Timothy D. Powell, Dan T. Stinchcomb.
Application Number | 20150246109 14/642480 |
Document ID | / |
Family ID | 48948499 |
Filed Date | 2015-09-03 |
United States Patent
Application |
20150246109 |
Kind Code |
A1 |
Stinchcomb; Dan T. ; et
al. |
September 3, 2015 |
COMPOSITIONS, METHODS AND USES FOR POXVIRUS ELEMENTS IN VACCINE
CONSTRUCTS AGAINST INFLUENZA VIRUS SUBTYPES OR STRAINS
Abstract
Embodiments of the present invention generally disclose methods,
compositions and uses for generating and expressing poxvirus
constructs. In some embodiments, constructs may contain an
influenza virus gene segment. In certain embodiments, methods
generally relate to making and using compositions of constructs
including, but not limited to, poxvirus vaccine compositions having
two or more influenza gene segments. In other embodiments, vaccine
compositions are reported of use in a subject.
Inventors: |
Stinchcomb; Dan T.; (Fort
Collins, CO) ; Osorio; Jorge E.; (Mount Horeb,
WI) ; Powell; Timothy D.; (Fort Collins, CO) ;
Jones; Jeremy C.; (Memphis, TN) ; Brewoo; Joseph
N.; (Madison, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Takeda Vaccines, Inc. |
Deerfield |
IL |
US |
|
|
Family ID: |
48948499 |
Appl. No.: |
14/642480 |
Filed: |
March 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13555026 |
Jul 20, 2012 |
9005632 |
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14642480 |
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13510601 |
Oct 29, 2012 |
9011874 |
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PCT/US10/57682 |
Nov 22, 2010 |
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13555026 |
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61263327 |
Nov 20, 2009 |
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Current U.S.
Class: |
424/199.1 ;
435/320.1 |
Current CPC
Class: |
A61K 2039/58 20130101;
A61K 39/00 20130101; A61K 39/12 20130101; A61K 2039/5256 20130101;
C12N 2710/24143 20130101; A61P 31/16 20180101; C07K 2319/02
20130101; C12N 2760/16034 20130101; C12N 2760/16122 20130101; A61K
2039/70 20130101; A61K 2039/545 20130101; A61K 2039/5254 20130101;
C07K 14/005 20130101; C12N 2760/16134 20130101; C12N 15/863
20130101; A61K 39/145 20130101 |
International
Class: |
A61K 39/145 20060101
A61K039/145; C07K 14/005 20060101 C07K014/005; A61K 39/12 20060101
A61K039/12 |
Goverment Interests
FEDERALLY FUNDED RESEARCH
[0002] Some embodiments disclosed herein were supported in part by
grant number 1R43A1061940-01 from the National Institutes of Health
and grant number 5R41A1074308-02 also from the National Institutes
of Health. The U.S. Government has certain rights to practice the
subject invention.
Claims
1. A construct comprising: a modified vaccinia virus construct
having at least one poxvirus secretory signal sequence associated
with a first influenza gene segment or fragment thereof, and at
least a second influenza gene segment or fragment thereof, wherein
the influenza gene segments in the construct are capable of
producing an immune response to more than one influenza subtype
when introduced to a subject.
2. The construct of claim 1, wherein the influenza gene segment
comprises hemagglutinin (HA), neuraminidase (NA), nucleoprotein
(NP), matrix (M) polymerase (P) or fragment thereof or a
combination thereof.
3. The construct of claim 1, wherein the fragment comprises at
least 6, or at least 8, or at least 10 contiguous amino acids of
the influenza gene segment.
4. The construct of claim 1, wherein the poxvirus secretory signal
sequence comprises, C13L, B8R or any other poxvirus secretory
signal sequence.
5. The construct of claim 1, wherein the poxvirus secretory signal
sequence is fused to the influenza gene segment.
6. The construct of claim 1, further comprising one or more
translational control sequences.
7. The construct of claim 1, wherein the vaccinia virus comprises
modified vaccinia Ankara (MVA).
8. The construct of claim 1, wherein at least one of the influenza
gene segments comprises a naturally-occurring influenza gene
segment comprising a naturally-occurring secretory signal
sequence.
9. The construct of claim 1, wherein the influenza gene segments
are from different influenza subtypes or different influenza
strains.
10. The construct of claim 1 wherein at least one influenza gene
segment comprises an influenza gene segment from a seasonal
influenza virus subtype.
11. The construct of claim 1, wherein at least one influenza gene
segment comprises an influenza gene segment from a pandemic
influenza virus subtype.
12. The construct of claim 1, wherein the first influenza gene
segment comprises the influenza gene segment nucleoprotein (NP);
and the second influenza gene segment comprises the influenza gene
segment comprises hemagglutinin (HA).
13. A pharmaceutical composition comprising the construct of claim
1; and a pharmaceutically acceptable excipient.
14. The pharmaceutical composition of claim 13, further comprising
one or more additional poxvirus constructs having non-poxvirus
antigens or fragments thereof.
15. A method for inducing an immune response to more than one
influenza virus infection in a subject comprising: administering a
pharmaceutical composition comprising one or more construct of
claim 1 to the subject, wherein administration induces an immune
response to more than one influenza virus subtype in the
subject.
16. The method of claim 15, wherein the influenza gene segment
comprises hemagglutinin (HA), neuraminidase (NA), nucleoprotein
(NP), matrix (M) or a combination thereof.
17. The method of claim 15, wherein at least one influenza gene
segment is obtained from a seasonal influenza virus.
18. The method of claim 15, wherein at least one influenza gene
segment is obtained from a pandemic influenza virus.
19. The method of claim 15, wherein the subject has an influenza
virus infection and the composition treats the influenza virus
infection in the subject.
20. The method of claim 15, wherein the composition reduces the
onset of more than one influenza virus infection in the
subject.
21. The method of claim 15, further comprising administering a
composition comprising: one or more poxvirus constructs prior to or
at the same time as administering a composition comprising one or
more construct of claim 1 to the subject, wherein the composition
is capable of inducing an immune response in the subject.
22. The method of claim 21, wherein the composition comprising one
or more poxvirus constructs is administered to the subject between
6 months and immediately prior to the second composition.
23. The method of claim 15, wherein administration of the
pharmaceutical composition comprises administering the composition
intradermally, subcutaneously, intravenously, orally, intranasally,
by inhalation or other mode of administration.
24. A vaccine kit comprising; one or more pharmaceutical
compositions of claim 13; and at least one container.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application is a divisional application of U.S.
Non-Provisional application Ser. No. 13/555,026, filed Jul. 20,
2012, which is a continuation-in-part application of U.S.
Non-Provisional application Ser. No. 13/510,601, filed as a 371
application on May 17, 2012, which claims priority to PCT
Application No. PCT/US10/057682 filed on Nov. 22, 2010 (Nov. 20,
2010 fell on a Saturday), which claims the benefit of U.S.
Provisional Patent Application Ser. No. 61/263,327, filed on Nov.
20, 2009. These applications are incorporated herein in their
entirety by reference for all purposes.
FIELD
[0003] Embodiments of the present invention report methods,
compositions and uses for generating vaccine compositions. In some
embodiments, poxvirus elements can be used in viral constructs, for
example, a construct of use in vaccines. In some embodiments, a
poxvirus element may be a secretory signal. In certain embodiments,
methods for making and using constructs for vaccine preparations
that include, but are not limited to, using attenuated or modified
vaccinia virus vectors that can express peptides derived from
different organisms or different subtypes of organisms. In other
embodiments, constructs may be generated for use in vaccination
against influenza. In yet other embodiments, compositions and
methods herein report pre-exposing a subject to a construct
composition prior to administering a vaccine to the subject. In yet
other embodiments, compositions and methods herein concern
generating constructs of use in vaccinations for cross-protection
against more than one subtype or strain of influenza virus.
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 amongst the most successful vaccines used in public
health.
[0005] Influenza is an orthomyxovirus with three genera, types A,
B, and C. The types are distinguished by the nucleoprotein
antigenicity. Influenza B is a human virus and does not appear to
be present in an animal reservoir. Type A viruses exist in both
human and animal populations, with significant avian and swine
reservoirs.
[0006] Annual influenza A virus infections have a significant
impact in terms of human lives, between 500,000 and 1,000,000 die
worldwide each year, and economic impact resulting from direct and
indirect loss of productivity during infection. Of even greater
concern is the ability of influenza A viruses to undergo natural
and engineered genetic change that could result in the appearance
of a virus capable of rapid and lethal spread within the
population.
[0007] One of the most dramatic events in influenza history was the
so-called "Spanish Flu" pandemic of 1918-1919. In less than a year,
between 20 and 40 million people died from influenza, with an
estimated one fifth of the world's population infected. The US
military was devastated by the virus near the end of World War I,
with 80% of US army deaths between 1918 and 1919 due to influenza
infection. Because it is a readily transmitted, primarily airborne
pathogen, and because the potential exists for the virus to be
genetically engineered into novel forms, influenza A represents a
serious biodefense concern.
[0008] The past two decades have seen the emergence of highly
virulent avian H5N1 influenza viruses. H5N1 avian influenza first
emerged in the human population when the virus crossed the avian to
human species barrier in 1997. These viruses are currently endemic
to poultry populations in South East Asia, where they initially
emerged. Over the past decade, they have significantly broadened
their host and geographic range with a current human mortality rate
of approximately 60%. Compounding this threat is the resistance
displayed by H5N1 viruses to both classes of influenza
antivirals.
[0009] Current public and scientific concern over the possible
emergence of a pandemic strain of influenza, poxviruses or other
pathogenic or non-pathogenic viruses requires effective
preventative measures. Another challenge regarding generating
vaccines has been to generate vaccines that protects against more
than one strain of a virus
SUMMARY
[0010] Embodiments of the present invention report methods,
compositions and uses for generating novel vaccine compositions. In
some embodiments, poxvirus elements can be used in vaccine
constructs. In other embodiments, compositions and methods for
administering poxvirus elements prior to receiving a vaccine can be
used, for example, to circumvent interference from pre-exposure to
poxvirus elements. In some embodiments, a poxvirus element may be a
secretory signal or other poxvirus element. In certain embodiments,
methods for making and using constructs for vaccine preparations
including, but not limited to, using attenuated or modified
vaccinia virus vectors expressing viral-bacterial, protozoal,
fungal, or mammalian peptides to induce an immune response in a
subject. In other embodiments, constructs may be generated for use
in vaccines that protect against infectious diseases or in vaccines
used as therapies (e.g. for cancer, diabetes, Alzheimer's disease,
etc.). Some embodiments are of use as a therapeutic or as a
prophylactic against a medical condition in a subject. In other
embodiments, constructs may be generated for use in vaccination
against viral diseases. In further embodiments, constructs may be
generated for use in vaccines to protect from influenza.
[0011] Embodiments of the present invention generally relate to
methods, compositions and uses for expressing peptides (e.g.
poxvirus associated peptides and non-poxvirus peptides) to
stimulate immune responses. In some embodiments, viral peptide
formulations presented herein can be used to boost an immune
response in a subject before, during and/or after vaccination of
the subject or to overcome pre-existing immunity (e.g. previous
poxvirus exposure) in the subject. Certain embodiments report
making and using constructs of the present invention for treating
or protecting a subject having been exposed or likely to be exposed
to a pathogen. In accordance with these embodiments a pathogen can
include a bacterial, viral, protozoal or fungal pathogen. In some
embodiments, a pathogen can be influenza virus.
[0012] In accordance with embodiments disclosed herein, constructs
may include, but are not limited to, attenuated or modified
vaccinia virus vectors expressing bacterial-, viral-, fungal-,
protozoal-associated gene segments (e.g. non-poxvirus peptides).
For example, certain methods and compositions report making and
using compositions having constructs including, but not limited to,
attenuated or modified vaccinia virus vectors expressing
influenza-associated gene segments in order to induce an immune
response in a subject against the influenza. Certain compositions
report constructs having antigens or peptides derived from
influenza and associated with or combined with poxviruses related
elements. Influenza gene or gene segments can include, but are not
limited to, hemagglutinin (HA gene segment), neuraminidase (NA gene
segment), nucleoprotein (NP gene segment), matrix protein (M gene
segment), polymerase (P) and a combination thereof. Some
embodiments report vaccine compositions capable of reducing or
preventing infection in a subject caused by exposure to a poxvirus
and/or influenza virus. Some embodiments concern using a fragment
of one or more influenza gene segments for example, a fragment can
include at least 6, or at least 8, or at least 10, or at least 15,
or at least 20 contiguous etc amino acids of an influenza gene
segment, up to the full length of the gene segment.
[0013] In other embodiments, influenza fragments in a single
construct can be from more than one influenza subtype or strain. In
certain examples, a construct of use in a vaccine can contain one
or more of the same gene segments of different strains or one or
more different gene segments from different strains of influenza
(e.g. HA, NA, NP and M). Some embodiments include constructs having
one or more influenza gene segments from different strains where
each segment has an associated signal sequence (e.g.
naturally-occurring or artificial (e.g. from a different
organism)). Some embodiments concern constructs that require a
signal sequence in close proximity to one or more gene segments of
the construct (e.g. C13L next to NP). Other embodiments concern
secretory signal sequences fused to one or more influenza gene
segments of constructs described herein (e.g. fused to either end
of the gene segment). In other embodiments, certain
naturally-occurring influenza gene segments used in constructs
described are already associated with a secretory signal (e.g. HA).
Constructs contemplated herein can further include a modified viral
vector (e.g. MVA). In accordance with these embodiments,
pharmaceutical compositions containing one or more of these
constructs can be use to generate a vaccine capable of conferring
protection in a subject from more than one influenza strain or
variety. For example, a subject may be conferred protection from
H1NI, H5N1, H3N2 or any other influenza strain or variety in a
single composition vaccine.
[0014] In some aspects, constructs of use as vaccine compositions,
can include a secretory signal sequence alone or in combination
with a translation control region sequence. In accordance with
these embodiments, the secretory signal sequence can be one or more
signal sequences from a poxvirus. In other embodiments, the
secretory signal sequence can include, but are not limited to,
tissue plasminogen activator (tPA) leader sequence, the co-factor
leader sequence, the pre-proinsulin leader sequence, the invertase
leader sequence, the immunoglobulin A leader sequence, the
ovalbumin leader sequence, and the P-globin leader sequence or
other proleader sequences and combinations thereof.
[0015] In certain embodiments, a pre-boost of a construct may be
used to induce a greater immune response in a subject to a
subsequent vaccination. In some embodiments, a vaccinia virus
derived gene sequence may be used to pre-boost a subject. In
accordance with these embodiments, a pre-boost construct can
contain modified vaccinia Ankara (MVA). It is contemplated that
these pre-boosts can be administered to a subject by any method.
For example, the pre-boost can be introduced intramuscularly or
intradermally or by another method. In addition, a pre-boost may be
administered to a subject followed by introduction of a construct
having one or more elements derived from a pathogen or associated
with a condition to boost an immune response in the subject. In
certain examples, a pre-boost could be 6 months or less prior to a
vaccination, or 5 months or less, or 4 months or less, or 3 months
or less, or 1 month or less or a few weeks or immediately prior to
administering a vaccine to a subject. Administration regimens are
readily determinable by one skilled in the art for pre-boosts,
boost and post-boosts related to vaccinating a subject against an
infection or a condition.
[0016] Other embodiments concern kits for making or using
compositions disclosed. It is reported that a kit may include
constructs having a modified vaccinia viral vector and one or more
enterobacterial antigen.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The following drawings form part of the present
specification and are included to further demonstrate certain
embodiments. Some embodiments may be better understood by reference
to one or more of these drawings alone or in combination with the
detailed description of specific embodiments presented.
[0018] FIGS. 1A and 1B represent exemplary plots of parameters in
mice after intramuscular (IM) or intradermal (ID) introduction of
various constructs of some embodiments described herein to the mice
followed by challenge with influenza, FIG. 1A) Weight loss and FIG.
1B) Viral Lung titers on day 4 post-challenge.
[0019] FIGS. 2A-2C represent exemplary plots of experiments where
mice were vaccinated intradermal (ID) with various influenza
challenge concentration and then examined on day 63
post-vaccination. Weight loss curves are displayed for some of the
constructs FIG. 2A) MVA/IRES/tPA/HA, FIG. 2B) MVA/IRES/C13L/HA, and
FIG. 2C) MVA/HA native.
[0020] FIG. 3 represents an exemplary plot that illustrates various
constructs and effects on long-term immune protection in mice
against a viral insult.
[0021] FIG. 4 represents an exemplary plot that illustrates
cross-clade protection using various viral-derived antigens.
[0022] FIG. 5 represents an exemplary plot of mice tested with
certain constructs described herein, described herein to the mice
followed by a viral challenge.
[0023] FIGS. 6A and 6B represent exemplary plots of parameters in
mice after intramuscular (IM) introduction of various constructs of
some embodiments described herein to the mice followed by challenge
with influenza, FIG. 6A) Weight loss and FIG. 6B) Survival
post-challenge.
[0024] FIGS. 7A and 7B represent exemplary plots of parameters in
mice after intradermal (ID) introduction of various constructs of
some embodiments described herein to the mice followed by challenge
with influenza, FIG. 7A) Weight loss and FIG. 7B) Survival
post-challenge.
[0025] FIGS. 8A and 8B represent exemplary plots of parameters in
mice after (FIG. 8A) intramuscular (IM) or (FIG. 8B) intradermal
(ID) introduction of various constructs of some embodiments
described herein to the mice followed by challenge with
influenza.
[0026] FIG. 9 represents an exemplary plot that illustrates viral
titers in lung after introduction of various constructs of some
embodiments described herein to the mice followed by challenge with
influenza.
[0027] FIGS. 10A and 10B represent exemplary plots of percent
weight change in mice after introduction of 2 different constructs
of some embodiments described herein having the HA gene segment in
each construct followed by challenge with influenza. FIG. 10A
represents construct MVA/HA and FIG. 10B represents construct
MVA/IRES/tpa/HA.
[0028] FIG. 10C illustrates Table 1 which represents MVA influenza
transfer vectors and constructs.
[0029] FIGS. 11A and 11B represent exemplary plots of percent
weight change in mice after introduction of 2 different constructs
of some embodiments described herein followed by challenge with
influenza. FIG. 11A represents construct MVA/IRES/C13L/HA and FIG.
11B represents construct MVA-GFP.
[0030] FIGS. 12A and 12B represent exemplary plots of percent
weight change in mice after introduction of 2 different constructs
at various concentrations of some embodiments described herein
followed by challenge with influenza. FIG. 12A represents construct
MVA/IRES/tpa/HAt and FIG. 12B represents construct
MVA/IRES/C13L/HAt.
[0031] FIGS. 13A and 13B represent exemplary plots of survival in
mice after introduction of 2 different constructs at various
concentrations of some embodiments described herein followed by
challenge with influenza. FIG. 13A represents construct MVA/HA and
FIG. 13B represents construct MVA/IRES/tpa/HA.
[0032] FIGS. 14A and 14B represent exemplary plots of survival in
mice after introduction of 2 different constructs at various
concentrations of some embodiments described herein followed by
challenge with influenza. FIG. 14A represents construct
MVA/IRES/tpa/HA and FIG. 14B represents construct
MVA/IRES/C13L/HA.
[0033] FIGS. 15A and 15B represent exemplary plots of survival in
mice after introduction of 2 different constructs at various
concentrations of some embodiments described herein followed by
challenge with influenza (dpi represents days post infection). FIG.
15A represents construct MVA/IRES/tpa/HAt and FIG. 15B represents
construct MVA/IRES/C13L/HAt.
[0034] FIGS. 16A and 16B represent exemplary plots of clinical
scores (e.g. physical and physiological parameters) in mice after
introduction of 2 different constructs at various concentrations of
some embodiments described herein followed by challenge with
influenza. FIG. 16A represents construct MVA/HA and FIG. 16B
represents construct MVA/IRES/tpa/HA.
[0035] FIGS. 17A and 17B represent exemplary plots of clinical
scores (e.g. physical and physiological parameters) in mice after
introduction of 2 different constructs at various concentrations of
some embodiments described herein followed by challenge with
influenza. FIG. 17A represents construct MVA/IRES/C13L/HA and FIG.
17B represents construct MVA/IRES/tpa/HAt.
[0036] FIG. 18 represents an exemplary plot of clinical scores
(e.g. physical and physiological parameters) in mice after
introduction of a construct at various concentrations of some
embodiments described herein followed by challenge with
influenza.
[0037] FIGS. 19A and 19B represent exemplary plots of percent
weight change (FIG. 19A) and assessed clinical scores (FIG. 19B) in
mice after introduction of different constructs in mice pre-exposed
to vaccinia followed by challenge with influenza.
[0038] FIG. 20 represents an exemplary plot of survival of mice
challenged above after exposure to the same constructs as in FIGS.
19A and 19B.
[0039] FIG. 21 is a schematic representation of a recombinant
plasmid construct.
[0040] FIGS. 22A-22E represent gel electrophoresis separation of
constructs of certain embodiments contemplated herein. FIG. 22A
represents construct MVA/HA1; FIG. 22B represents construct
MVA/HA1/C13L/NP; FIG. 22C represents HA5 and construct MVA/HA5;
FIG. 22D represents construct MVA/HA5/C13L/NP and FIG. 22E
represents constructs MVA/NP and MVA/C13L/NP.
[0041] FIGS. 23A-23H represent vaccine efficacy against homologous
challenge of influenza virus. FIGS. 23A and 23B represent histogram
plots of construct MVA/HA1 and MVA/GFP; FIGS. 23C and 23D represent
days post challenge of various constructs; FIGS. 23E and 23F
represent lung viral titer post challenge with various constructs
and FIGS. 23G and 23H represent weight loss in an animal model post
challenge after exposure to various constructs.
[0042] FIG. 24 represents lung viral titers in vaccinated and
control animals following challenge with an influenza virus.
[0043] FIGS. 25A-25D represents grafts representing exemplary
immune responses following vaccinations of various constructs
contemplated herein. FIGS. 25A, 25B, 25C and 25D represent weight
loss in an animal model post challenge after exposure to various
constructs.
[0044] FIGS. 26A-26B represent effects of vector immunity on
vaccine efficacy. FIG. 26A represents various vaccination regimens
in an animal model. FIG. 26B represents percent survival post
challenge using various vaccination regimens in the animal
model.
[0045] FIG. 27 is a graphic representation of examination of safety
of vectored vaccine compositions of some embodiments herein.
[0046] FIG. 28 represents an exemplary plasmid construct of some
embodiments herein.
[0047] FIG. 29 represents a histogram plot of exemplary immune
responses following prime and booster vaccinations of various
constructs contemplated herein.
DEFINITIONS
[0048] As used herein, "a" or "an" can mean one or more than one of
an item.
[0049] As used herein the specification, "subject" or "subjects"
can include, but are not limited to, mammals such as humans or
mammals, domesticated or wild, for example dogs, cats, other
household pets (e.g. hamster, guinea pig, mouse, rat), ferrets,
rabbits, pigs, horses, cattle, prairie dogs, wild rodents, or zoo
animals. A subject can be an adult or a child.
[0050] As used herein, "about" can mean plus or minus ten
percent.
[0051] As used herein, "attenuated virus" can mean a virus that
demonstrates reduced or no clinical signs of disease when
administered to a subject such as a mammal (e.g. human or an
animal).
[0052] As used herein, "MSC" can mean multiple cloning site.
[0053] As used herein, "dSP" can mean divergent vaccinia
promoter.
[0054] As used herein, "MVA" can mean modified vaccinia Ankara.
[0055] As used herein, "EMCV" can mean encephalomyocarditis
virus.
[0056] As used herein, "IRES" can mean internal ribosome entry site
from encephalomyocarditis virus or other viruses.
[0057] As used herein, "IRES(A7)" can mean IRES from
encephalomyocarditis virus with 7 adenosine residues in bifurcation
loop; source--pCITE-1.
[0058] As used herein, "IRES(A6)" can mean IRES from
encephalomyocarditis virus mutated to have 6 adenosine residues in
bifuraction loop.
[0059] As used herein, "pDIIIgfp" can mean MVA del III gfp marker
transfer plasmid.
[0060] As used herein, "pI*" can mean transfer vector plasmids.
[0061] As used herein, "tPA" can mean secretory signal from tissue
plaminogen activator.
[0062] As used herein, "se/l" can mean synthetic optimized early
late poxvirus promoter.
[0063] As used herein, "H6" can mean the vaccinia gene H6
early/late native poxvirus promoter.
[0064] As used herein, "del III" can mean modified vaccinia Ankara
deletion region III.
[0065] As used herein, "GFP" can mean enhanced green fluorescent
protein.
[0066] As used herein, "CEF" can mean chicken embryo
fibroblasts.
[0067] As used herein, "RCN" can mean raccoon pox virus.
DESCRIPTION
[0068] 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 details outlined herein, but rather that
concentrations, times and other details may be modified through
routine experimentation. In some cases, well-known methods or
components have not been included in the description.
[0069] Embodiments of the present invention concern methods,
compositions and uses for generating novel vaccine compositions. In
some embodiments, poxvirus elements can be used in vaccine
constructs or in pre-immunization constructs for introduction to a
subject. In certain embodiments, poxvirus elements can be used to
pre-immunize a subject prior to receiving a vaccine. In some
embodiments, a poxvirus element can be a secretory signal or other
poxvirus element. Other embodiments concern methods for making and
using constructs including, but not limited to, attenuated or
modified vaccinia virus vectors expressing viral, bacterial,
protozoal fungal, or mammalian derived peptides. In other
embodiments, constructs may be generated for use in vaccines that
protect against infectious diseases or in vaccines used as
therapies (e.g. for cancer, diabetes, Alzheimer's disease, etc.) to
boost an immune response in a subject. Some embodiments are of use
as a therapeutic or as a prophylactic against a medical condition
in a subject. In other embodiments, constructs may be generated for
use in vaccination against viral diseases. In further embodiments,
constructs may be generated for use in vaccines to protect from a
pathogen. Some embodiments described herein concern constructs to
protect against and/or treat a subject exposed to or having an
influenza infection.
Influenza Virus
[0070] Influenza is an orthomyxovirus. Three genera, types A, B,
and C of influenza currently exist. Types A and B are the most
clinically significant, causing mild to severe respiratory illness.
Type A viruses exist in both human and animal populations, with
significant avian and swine reservoirs. Although relatively
uncommon, it is possible for nonhuman influenza A strains to infect
humans by jumping from their natural host. In one specific example,
the highly lethal Hong Kong avian influenza outbreak in humans in
1997 was due to an influenza A H5N1 virus that was an epidemic in
the local poultry population at that time. In this case, the virus
killed six of the 18 patients shown to have been infected.
[0071] Annual seasonal influenza A or B virus infections have a
significant impact on humanity both in terms of death, between
500,000 and 1,000,000 worldwide each year and economic impact
resulting from direct and indirect loss of productivity during
infection.
[0072] In 2009, an influenza pandemic erupted. The virus isolated
from patients in the United States was found to be made up of
genetic elements from four different flu viruses--North American
Mexican influenza, North American avian influenza, human influenza,
and swine influenza virus typically found in Asia and Europe. This
new strain appears to be a result of reassortment of human
influenza and swine influenza viruses, in all four different
strains of subtype H1N1.
[0073] In certain embodiments, a virus can include an influenza
virus infection, for example, influenza type A, B or C or subtype
or strain thereof. Some embodiments include, but are not limited
to, influenza A, H1N1 subtype or H1N1 of swine origin and strains.
Other influenza A viruses may include, but are not limited to,
H2N2, which caused Asian Flu in 1957; H3N2, which caused Hong Kong
Flu in 1968; H5N1, a current pandemic threat; H7N7, which has
unusual zoonotic potential; H1N2, endemic in humans and pigs; H9N2;
H7N2; H7N3, H10N7 or combinations thereof. Other varieties can
include, but are not limited to, A/Vietnam/1203/04 (H5N1),
A/Norway/3487-2/09 (pandemic H1N1), A/Influenza/Puerto Rico/8/34
(seasonal H1N1) and seasonal H3N2 virus (A/Aichi/68) or others
[0074] In certain embodiments, constructs having two or more
influenza gene segments from different subtypes or strains can be
directed toward prevention or treatment of seasonal influenza virus
outbreaks. In other embodiments, construct having two or more
influenza gene segments from different subtypes or strains can be
directed toward prevention or treatment of pandemic influenza virus
outbreaks. Other constructs can be used to target both seasonal and
pandemic outbreaks of different subtypes or strains of influenza
virus by using influenza gene segments specific for a given subtype
or strain.
[0075] Influenza A and B each contain 8 segments of negative sense
ssRNA. Type A viruses can also be divided into antigenic subtypes
on the basis of two viral surface glycoproteins, hemagglutinin (HA)
and neuraminidase (N). There are currently 15 identified HA
subtypes (designated H1 through H15) and 9 NA subtypes (N1 through
N9) all of which can be found in wild aquatic birds. Embodiments of
the present invention can include constructs having one or more of
any influenza gene segment subtype or strain known in the art. Of
all the possible (e.g. over 135) combinations of HA and NA, four
(H1N1, H1N2, H2N2, and H3N2) have widely circulated in the human
population since the virus was first isolated in 1933. Two of the
more common subtypes of influenza A currently circulating in the
human population are H3N2 and H1N1.
[0076] Some embodiments include constructs having one or more
influenza gene segments from different subtypes or strains where
each gene segment has an associated signal sequence (e.g.
naturally-occurring or artificial (e.g. from a different
organism)). Some embodiments concern constructs that require a
signal sequence in close proximity to one or more gene segments of
the construct (e.g. C13L next to an NP influenza gene segment).
Other embodiments concern secretory signal sequences fused to one
or more influenza gene segments of constructs described herein
(e.g. fused to either end of the gene segment). In other
embodiments, certain naturally-occurring influenza gene segments
used in constructs described are already associated with a
secretory signal (e.g. HA).
[0077] New type influenza A strains emerge due in part to genetic
drift that can result in slight changes in the antigenic sites on
the surface of the virus. This shift can lead to the human
population experiencing epidemics of influenza infection every
year. More drastic genetic changes can result in an antigenic shift
(a change in the subtype of HA and/or NA) resulting in a new
subtype capable of rapidly spreading in a susceptible
population.
[0078] Subtypes are sufficiently different as to make them
non-crossreactive with respect to antigenic behavior; prior
infection with one subtype (e.g. H1N1) can lead to no immunity to
another (e.g. H3N2). It is this lack of crossreactivity that in
certain cases allows a novel subtype to become pandemic as it
spreads through an immunologically naive population. In some
embodiments, development of novel vaccine vectors and production
techniques are disclosed herein for generating effective vaccine
with broad cross-protective efficacy against various influenza
subtypes or strains. Modified vaccinia Ankara (MVA) vector offers
several advantages in such a vaccine such as; 1) safety, 2)
stability, 3) rapid induction of humoral and cellular responses,
and 4) multiple routes of inoculation. While several vaccines have
been created using an MVA vector, most of them have been tested
against homologous or closely related challenge viruses and
provided limited to no protection against genetically divergent
strains. In certain embodiments, vaccine constructs concern
recombinant MVA vaccines expressing antigens (e.g. influenza gene
segments) from two or more subtypes of influenza. Some embodiments
concern influenza gene segments that are the same or different
regions (e.g. HA, NA, M, NP) from two or more subtypes or strains.
In certain embodiments, use of an NP segment can require a
secretory signal sequence in close proximity to the gene segment in
a construct. For example, certain embodiments concern fusing a
secretory signal sequence to an influenza gene segment in a
construct having one or more additional influenza gene segments
within the same construct from different or the same influenza
subtype. In certain embodiments, the constructs can be plasmid
constructs.
[0079] Although relatively uncommon, it is possible for nonhuman
influenza A strains to transfer from their "natural" reservoir to
humans. In one example, the highly lethal Hong Kong avian influenza
outbreak in humans in 1997 was due to an influenza A H5N1 virus
that was an epidemic in the local poultry population at that time.
This virus transferred to other hosts (e.g. humans) from
contaminated chickens.
[0080] Some embodiments of the present invention report vaccine
compositions including, but not limited a poxvirus and one or more
poxvirus secretory signals associated with one or more non-poxvirus
peptides. In certain embodiments, a vaccine composition may include
a modified or attenuated poxvirus associated with one or more
secretory poxvirus secretory signals associated with one or more
non-poxvirus peptides. In other embodiments, recombinant modified
vaccinia Ankara (MVA) vector associated with one or more poxvirus
secretory signals associated with one or more non-poxvirus
peptides. In other embodiments, a vaccine composition may include a
recombinant modified vaccinia Ankara (MVA) vector associated with
one or more influenza-associated peptides where at least one of the
one or more influenza-associated peptides is associated with a
poxvirus secretory signal. For example, a vaccine composition can
include recombinant modified vaccinia Ankara (MVA) vector
expressing influenza virus components. In accordance with this
vaccine composition, an MVA construct expressing one or more
influenza-associated antigens may be generated (e.g. HA, NP, NA, M,
P, etc.) for use to vaccinate a subject against influenza. It is
contemplated that vaccine constructs can contain a more conserved
or highly conserved influenza genetic region or influenza
associated peptide alone or in combination with a more variable
influenza associated peptide. Alternatively, a vaccine construct
contemplated herein can contain a peptide or the entire segment of
an internal influenza gene region (e.g. M) or an externally (e.g.
HA) exposed gene region.
[0081] In one embodiment contemplated herein, a vaccine construct
can contain two influenza gene segments from different subtypes. In
accordance with this embodiment, the vaccine construct can contain
a hemagglutinin (HA) gene segment from H1N1 and a nucleoprotein
(NP) from H5N1 each associated with a secretory signal sequence
(e.g. MVA/HA.sub.1/C13L/NP).
[0082] In certain embodiments, influenza virus is selected from the
group consisting of any influenza A subtype or strain, influenza A
H3N2, influenza A H1N1, influenza A H1N1 swine-origin, avian
influenza A H5N1, and influenza B.
[0083] Certain embodiments of the present invention report
compositions having constructs directed against poxviruses. For
example, vaccine compositions may be directed to the prevention or
reduced incidence of conditions associated with poxvirus or
influenza viruses.
Poxviridae
[0084] Poxviruses (members of the family Poxviridae) are viruses
that can, as a family, infect both vertebrate and invertebrate
animals. There are four known genera of poxviruses that may infect
humans: orthopox, parapox, yatapox, molluscipox. Orthopox include,
but are not limited to, variola virus, vaccinia virus, cowpox
virus, monkeypox virus, and smallpox. Parapox include, but are not
limited to, orf virus, pseudocowpox, bovine papular stomatitis
virus; Yatapox: tanapox virus, yaba monkey tumor virus. Molluscipox
include, but are not limited to, molluscum contagiosum virus (MCV).
Some of the more common oixviruses are vaccinia and molluscum
contagiousum, but monkeypox infections seem to be on the rise.
[0085] Poxvirus family, vaccinia virus, has been used to
successfully vaccinate against smallpox virus. Vaccinia virus is
also used as an effective tool for foreign protein expression to
elicit strong host immune response. Vaccinia virus enters cells
mainly by cell fusion, although currently the receptor is not
known. Virus contains three classes of genes, early, intermediate
and late that are transcribed by viral RNA polymerase and
associated transcription factors. Diseases caused by pox viruses
have been known about for centuries.
Orthopoxviruses
[0086] Certain embodiments of the present invention may include
using modified or attenuated orthopoxviruses or orthopoxvirus
associated genetic elements or peptides in vaccine compositions.
Orthopoxvirus is a genus of the Poxviridae family, that includes
many agents isolated from mammals, including, but not limited to,
vaccinia, monkeypox, cowpox, camelpox, seal poxvirus, buffalo
poxvirus, raccoon poxvirus, skunk poxvirus, vole poxvirus and
ectromelia viruses. Members of Poxviridae have large linear
double-stranded DNA, with genome sizes ranging from 130 to 300 kbp.
One of the members of the genus is variola virus, which causes
smallpox. Smallpox was previously eradicated using another
orthopoxvirus, the vaccinia virus, as a vaccine.
Modified Vaccinia Virus Ankara (MVA)
[0087] Some embodiments in the present invention report
compositions and methods of use of recombinant vaccinia viruses
derived from attenuated poxviruses that are capable of expressing
predetermined or preconstucted genes or gene segments. Those
skilled in the art recognize that other attenuated poxviruses can
be generated by serial passage in cell culture or by deliberate
deletion of poxviral genes. In certain embodiments, predetermined
genes may be inserted at the site of a naturally occurring deletion
in the MVA genome. In other embodiments, recombinant MVA viruses
can be used, for example, for the production of polypeptides (e.g.
antigens) or for encoding antigens of use for vaccine compositions
capable of inducing an immune response in a subject administered
the vaccine compositions.
[0088] In certain embodiments, modified or attenuated poxviruses
(e.g. modified vaccinia Ankara (MVA), NYVAC, LC16m8, or CVI-78),
can be used in a subject (e.g. mammals such as humans) as a
delivery system for pre-boost, boost or post-boost vaccination in
order to induce immunity to a pathogen in the subject. It is
contemplated herein that a subject may benefit from having more
than one administration of a compositions disclosed herein.
Previously, MVA was administered to over 120,000 individuals and
proven to be a safe and effective vaccine against smallpox. In
certain embodiments, recombinant MVA vaccine candidates have been
shown to induce protective humoral and cellular immunity against
diseases caused by viruses, bacteria, parasites, or tumors from
which antigens or peptides were derived. Additional features that
make MVA a suitable vector include its ability to induce protective
immune responses when administered by different routes and its
genetic and physical stability properties.
Translational Control Sequences
[0089] Some embodiments may include an optional enhancer, for
example, a translation control sequence. In certain embodiments, a
translation control sequence may include an internal ribosomal
entry site (IRES) (e.g. EMCV-IRES). Viral IRESs are classified into
four groups: Group 1 (Cricket paralysis virus (CrPV), Plautia stali
intestine virus (PSIV) and Taura syndrome virus (TSV)); Group 2
(Hepatitis C virus, (HCV), classical swine fever virus (CSFV) and
porcine teschovirus 1 (PTV-1)); Group 3 (encephalomyocarditis virus
(EMCV), foot-and-mouth-disease virus (FMDV) and Theiler's Murine
Encephalomyelitis virus (TMEV)); and Group 4 (poliovirus (PV) and
rhinovirus (RV)). In other embodiments, viral untranslated regions
(UTRs) found 5' to viral coding sequences can be used to direct
translation. Any translation control sequence of use in viral
constructs known in the art is contemplated.
Secretory Signals
[0090] Alternatively, embodiments of the present invention may
include constructs having one or more poxvirus secretory signal
sequences in combination with other elements. Translation control
sequences and/or poxvirus secretory signals were demonstrated to
increase efficacy of certain vaccine constructs. In some
embodiments, one or more poxvirus secretory signal sequences of
constructs disclosed herein can include, but are not limited to,
secretory signal sequence in the poxvirus genes C13L(putative), B8R
(soluble interferon gamma receptor), B19R (interferon a/b
receptor), A39R(semaphoring), M2L(putative), C13L(putative), C19L
or other secretory signal sequences known in the art. Constructs
disclosed herein can contain one or more secretory signal
sequence.
[0091] In some embodiments, when designing a construct, such that a
protein is expressed, it may be necessary to incorporate into a
first nucleic acid region a DNA sequence encoding a signal
sequence, for example, in cleavable form, where the expressed
protein is desired to be secreted. Without limiting embodiments of
the present invention to any one theory or mode of action, a signal
sequence can be a peptide that is present on proteins destined
either to be secreted or to be membrane bound. These signal
sequences can be found at the N-terminus of the protein and are
generally cleaved from a mature form of a protein. The signal
sequence generally interacts with the signal recognition particle
and directs the ribosome to the endoplasmic reticulum where
co-translational insertion takes place. Where the signal sequence
is cleavable, it is generally removed by for example, a signal
peptidase. The choice of signal sequence which is to be utilized
may depend on the requirements of the particular situation and can
be determined by the person of skill in the art. In the context of
the exemplification provided herein, but without being limited in
that regard, tPA, a poxvirus signal sequences from C13L or B8R may
be used to facilitate secretion of a peptide, protein, gene segment
or construct of interest. If a membrane protein is desired, both a
5' cleavable signal sequence at the amino end of the protein and a
non-cleavable membrane anchor at the 3'(carboxy) end of the protein
may be needed. These could be provided within the vector or one or
both could be encoded by the DNA of the protein of interest.
[0092] Some embodiments of the present invention include, but are
not limited to, compositions including one or more constructs. A
construct may be designed to produce proteins that are
cytoplasmically retained, secreted or membrane bound. Deciding what
form a protein of interest may need to take can depend on the
functional requirement of the protein. For example, anchored cell
surface expression of a protein of interest can provide a
convenient way for screening for molecules that interact with a
protein or peptide of interest such as antibodies, antagonists,
agonists or the like particularly to the extent that the protein is
expressed on the membrane of an adherent cell type. Still further
membrane anchored forms of proteins may be suitable for
administration to a subject for example, for generating monoclonal
antibodies to the protein. This may be due to host cells providing
a convenient source of the protein that is likely to be correctly
folded and have appropriate post-translational modifications, for
example, glycosylation and disulphide bond formation. In addition,
a host cell may provide adjuvant properties, for example, antigenic
differences from a recipient subject, notably in major
histocompatibility complexes (MHC).
[0093] Alternatively, secreted proteins can be suitable where a
protein is to be harvested and purified. A nucleic acid molecule
encoding a signal sequence may be positioned in the construct at
any suitable location which can be determined as a matter of
routine procedure by a person of skill in the art. In some
embodiments, a signal sequence may be positioned immediately 5' to
the nucleic acid sequence encoding a peptide, protein or construct
of interest (such that it can be expressed as an immediately
adjacent fusion with the protein of interest) but 3' to a promoter
such that expression of a signal sequence is placed under control
of the promoter. A nucleic acid sequence encoding a signal sequence
can form part of a first nucleic acid region of a construct.
[0094] It is contemplated herein that constructs and vaccine
compositions disclosed can be used as therapies for conditions such
as diabetes, Alzheimer's and cancer or other condition. Constructs
may be generated for use in vaccines that protect against or as
therapies for certain conditions (e.g. for cancer, diabetes,
Alzheimer's disease, etc.). In addition, vaccine compositions and
pre-boost compositions described herein can be used in subjects to
boost their immune system.
Tumor Markers
[0095] Tumor markers and associated tumor peptides are contemplated
for using in constructs described herein. Tumor markers and
peptides associated with tumors (e.g. non-poxvirus peptides) can be
used in combination with elements described herein in order to
develop vaccines to treat or prevent cancer in a subject. Some
tumor markers include, but are not limited to the following,
707-AP=707 alanine proline AFP=alpha (.alpha.)-fetoprotein,
ART-4=adenocarcinoma antigen recognized by T cells 4, BAGE=B
antigen; b-catenin/m, .beta.-catenin/mutated, Bcr-abl=breakpoint
cluster region-Abelson, CAMEL=CTL-recognized antigen on melanoma,
CAP-1=carcinoembryonic antigen peptide-1, CASP-8=caspase-8,
CDC27m=cell-divisioncycle, 27 mutated, CDK4/m=cycline-dependent
kinase 4 mutated, CEA=carcinoembryonic antigen, CT=cancer/testis
(antigen), Cyp-B=cyclophilin B, DAM=differentiation antigen
melanoma (the epitopes of DAM-6 and DAM-10 are equivalent, but the
gene sequences are different. DAM-6 and DAM-10, ELF2M=elongation
factor 2 mutated, ETV6-AML1=Ets, variant gene 6/acute myeloid
leukemia 1 gene ETS, G250=glycoprotein 250 GAGE=G antigen,
GnT-V=N-acetylglucosaminyltransferase V, Gp100=glycoprotein 100 kD,
HAGE=helicose antigen, HER-2/neu=human epidermal
receptor-2/neurological, HLA-A*0201-R1701=arginine (R) to
isoleucine (I) exchange at residue 170 of the .alpha.-helix of the
.alpha.2-domain in the HLA-A2 gene, HPV-E7=human papilloma virus
E7, HSP70-2M=heat shock protein 70-2 mutated, HST-2=human signet
ring tumor-2, hTERT or hTRT=human telomerase reverse transcriptase,
iCE=intestinal carboxyl, sterase, KIAA0205=name of the gene as it
appears in databases, LAGE=L antigen, LDLR/FUT=low density lipid
receptor/GDP-L-fucose: .beta.-D-galactosidase
2-.alpha.-Lfucosyltransferase, MAGE=melanoma antigen,
MART-1/Melan-A=melanoma, antigen recognized by T cells-1/Melanoma
antigen A, MC1R=melanocortin 1 receptor, Myosin/m=myosin mutated,
MUC1=mucin, MUM-1, -2, -3=melanoma, ubiquitous mutated 1, 2, 3
NA88-A=NA cDNA clone of patient M88, NY-ESO-1=New York-esophageous
1, P15=protein 15, p190 minor bcr-abl=protein of 190, KD bcr-abl,
Pml/RARa=promyelocytic leukaemia/retinoic acid receptor .alpha.,
FRAME=preferentially expressed antigen of melanoma,
PSA=prostate-specific antigen, PSM=prostate-specific membrane
antigen, RAGE=renal antigen, RU1 or RU2=renal, ubiquitous 1 or 2,
SAGE=sarcoma antigen, SART-1 or SART-3=squamous antigen, rejecting
tumor 1 or 3, TEL/AML1=translocation Ets-family leukemia/acute
myeloid, leukemia 1, TPI/m=triosephosphate isomerase mutated,
TRP-1=tyrosinase related, protein 1, or gp75, TRP-2=tyrosinase
related protein 2, TRP-2/INT2=TRP-2/intron, WT1=Wilms' tumor gene
and any other tumor antigen known in the art. In certain
embodiments, a pre-boost having an MVA construct can be used alone
or prior to administering a vaccine having a tumor antigen derived
peptide to a subject in need thereof.
[0096] Anti-microbial peptides are contemplated of use in
constructs disclosed herein. Anti-microbial peptides can be
expressed in constructs described and used alone or after a subject
is administered a pre-immune boost to treat or prevent an
infection.
Selection Markers
[0097] In certain embodiments, additional selection markers may be
used, for example, one may insert any number of selection markers
which may be designed, for example, to facilitate the use of the
vectors in a variety of ways, such as purification of a molecule of
interest. For example, glutathione S-transferase (GST) gene fusion
system provides a convenient means of harvesting a construct,
protein or peptide of interest, Without limiting to any one theory
or mode of action, a GST-fusion protein can be purified, by virtue
of the GST tag, using glutathione agarose beads. Embodiments of the
present invention should be understood to extend to constructs
encoding a secretable GST-molecule fusion. This could be achieved,
for example, by designing the sequence of a first nucleic acid
region such that it encodes a cleavable signal sequence fused to a
cleavable GST which is, in turn, fused to the molecule of interest.
In another example, a fusion tag could be used. In accordance with
these embodiments, a fusion tag can be between 360 bp of protein A
(allowing purification of the secreted product) and beta lactamase
(a bacterial enzyme which allows testing of supernatants by a
simple colour reaction). Beta lactamase facilitates selection of an
assay for a molecule of interest in the absence of an assay for
molecule of interest. The protein A/beta lactamase fusion can be
separated from the molecule of interest by a cleavage site to
facilitate cleavage, so that after the molecule is purified, the
tug can be easily removed.
[0098] Other fusion tags that could be included to facilitate
purification of a molecule or construct of interest of use for
embodiments disclosed herein can include, but are not limited to,
staphylococcal protein A, streptococcal protein G, hexahistidine,
calmodulin-binding peptides and maltose-binding protein (e.g. the
latter is also useful to help ensure correct folding of a molecule
of interest). Yet another selectable marker may include an
antibiotic resistance gene. Other embodiments may include an
antibiotic resistance gene. These genes have previously been
utilized in the context of bicistronic vectors as the selection
marker or HAT-based selectable bicistronic vector may be used.
Electrophoresis
[0099] Electrophoresis may be used to separate molecules (e.g.
large molecules such as proteins or nucleic acids) based on their
size and electrical charge. There are many variations of
electrophoresis known in the art. A solution through which the
molecules move may be free, usually in capillary tubes, or it may
be embedded in a matrix. Common matrices include polyacrylamide
gels, agarose gels, and filter paper.
[0100] Proteins, peptides and/or antibodies or antibody fragments
thereof may be detected partially or wholly purified, or analyzed
by any means known in the art. In certain embodiments, methods for
separating and analyzing molecules may be used such as gel
electrophoresis and elution or column chromatography or other
separation/purification methods.
[0101] Any method known in the art for detecting, analyzing and/or
measuring levels of antibodies or antibody fragments may be used in
embodiments reported herein. For example, assays for antibodies or
antibody fragments may include, but are not limited to, ELISA
assays, chemiluminescence assays, flow cytometry, electroelution
and other techniques known in the art.
Imaging Agents and Radioisotopes
[0102] In certain embodiments, the claimed proteins or peptides may
be linked to a secondary binding ligand or to an enzyme (an enzyme
tag) that will generate a fluorescent, a luminescent, or a colored
product upon contact with a substrate. Examples of suitable enzymes
include luciferase, green fluorescent protein, urease, alkaline
phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase.
The use and identification of such labels is well known to those of
skill in the art.
[0103] In other embodiments, labels or molecules capable of
detecting peptides, antigens, constructs, antibodies or antibody
fragments may include using aptamers, Methods for making and using
aptamers are well known in the art and these methods and uses are
contemplated herein. In addition, aptamers may be generated against
construct elements disclosed herein and used for any purpose (e.g.
purification, detection, modifying effects of the construct
etc).
[0104] Some embodiments can include methods for detecting and/or
making polyclonal or monoclonal antibodies produced by a subject
exposed to vaccine compositions disclosed in some embodiments of
the present invention. For example, antibodies produced capable of
inducing passive immunity to a subject may be isolated, analyzed
and/or produced as a whole antibody or fragment thereof, or a
polyclonal or a monoclonal antibody. Any means for producing or
analyzing these antibodies or antibody fragments known in the art
are contemplated.
Nucleic Acid Amplification
[0105] Nucleic acid sequences used as a template for amplification
can be isolated from viruses, bacteria, cells or cellular
components contained in the biological sample, according to
standard methodologies, A nucleic acid sequence may be genomic DNA
or fractionated or Whole cell RNA. Where RNA is used, it may be
desired to convert the to a complementary cDNA. In one embodiment,
the RNA is whole cell RNA and is used directly as the template for
amplification. Any method known in the art for amplifying nucleic
acid molecules are contemplated (e.g. PCR, LCR, Qbeta Replicase
etc).
Expressed Proteins or Peptides
[0106] Genes can be expressed in any number of different
recombinant DNA expression systems to generate large amounts of the
polypeptide product, which can then be purified and used in methods
and compositions reported herein. Any method known in the art for
generating and using constructs is contemplated. In certain
embodiments, genes or gene fragments encoding one or more
polypeptide mays be inserted into an expression vector by standard
cloning or subcloning techniques known in the art.
[0107] Some embodiments, using a gene or gene fragment encoding a
polypeptide may be inserted into an expression vector by standard
subcloning techniques. An expression vector may be used which
produces the recombinant polypeptide as a fusion protein, allowing
rapid affinity purification of a peptide or protein. Examples of
such fusion protein expression systems are the glutathione
S-transferase system (Pharmacia. Piscataway, N.J.), the maltose
binding protein system (NEB, Beverley, Mass.), the FLAG system
(IBI, New Haven, Conn.), and the 6.times.His system (Qiagen,
Chatsworth, Calif.).
Pharmaceutical Compositions and Routes of Administration
[0108] Aqueous compositions of some embodiments herein can include
an effective amount of a therapeutic protein, peptide, construct,
epitopic core region, stimulator, inhibitor, and the like,
dissolved or dispersed in a pharmaceutically acceptable carrier or
aqueous medium. Aqueous compositions of vectors expressing any of
the foregoing are also contemplated. The phrases "pharmaceutically
or pharmacologically acceptable" refer to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, or a human, as
appropriate.
[0109] Aqueous compositions of some embodiments herein can include
an effective amount of a therapeutic protein, peptide, construct,
an effective amount of the compound, dissolved or dispersed in a
pharmaceutically acceptable carrier or aqueous medium. Such
compositions can also be referred to as inocula. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutical active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated. Supplementary active
ingredients can also be incorporated into the compositions. For
human administration, preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biologics standards.
[0110] The biological material should be extensively dialyzed to
remove undesired small molecular weight molecules and/or
lyophilized for more ready formulation into a desired vehicle,
where appropriate. The active compounds or constructs will then
generally be formulated for parenteral administration, e.g.,
formulated for injection via the intravenous, intramuscular,
sub-cutaneous, intralesional, intranasal or even intraperitoneal
routes. Any route used for vaccination or boost of a subject can be
used. The preparation of an aqueous composition that contains an
active component or ingredient will be known to those of skill in
the art in light of the present disclosure. Typically, such
compositions can be prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for use in preparing
solutions or suspensions upon the addition of a liquid prior to
injection can also be prepared; and the preparations can also be
emulsified.
[0111] Pharmaceutical forms suitable for injectable use can include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms, such as bacteria and
fungi.
[0112] Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
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.
[0113] If formulations or constructs disclosed herein are used as a
therapeutic to boost an immune response in a subject, a therapeutic
agent can be formulated into a composition in a neutral or salt
form. Pharmaceutically acceptable salts, include the acid addition
salts (formed with the free amino groups of the protein) and which
are formed with inorganic acids such as, for example, hydrochloric
or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
[0114] A carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. 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. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0115] Sterile injectable solutions can be prepared by
incorporating the active compounds or constructs in the required
amount in the appropriate solvent with various of the other
ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the various sterilized active ingredients into a sterile vehicle
which contains the basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum-drying and
freeze-drying techniques which yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The preparation of more, or
highly, concentrated solutions for direct injection is also
contemplated, where the use of DMSO as solvent is envisioned to
result in extremely rapid penetration, delivering high
concentrations of the active agents to a small area.
[0116] 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, but slow release capsules or
microparticles and microspheres and the like can also be
employed.
[0117] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, intradermal, intranasal,
subcutaneous and intraperitoneal administration. In this
connection, sterile aqueous media that can be employed will be
known to those of skill in the art in light of the present
disclosure. For example, one dosage could be dissolved in 1 ml of
isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580).
[0118] The term "unit dose" refers to physically discrete units
suitable for use in a subject, each unit containing a predetermined
quantity of the construct composition or boost compositions
calculated to produce desired responses, discussed above, in
association with its administration, e.g., the appropriate route
and treatment regimen. The quantity to be administered, both
according to number of treatments or vaccinations and unit dose,
depends on the subject to be treated, the state of the subject and
the protection desired. The person responsible for administration
will, in any event, determine the appropriate dose for the
individual subject. For example, a subject may be administered a
construct composition disclosed herein on a daily or weekly basis
for a time period or on a monthly, bi-yearly or yearly basis
depending on need or exposure to a pathogenic organism or to a
condition in the subject (e.g. cancer).
[0119] The active therapeutic agents may be formulated within a
mixture to comprise 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 10 milligrams
per dose or so. Alternatively active agents (e.g. constructs) may
be formulated to comprise a certain number of constructs per dose
known to produce a desired effect in a subject. Multiple doses can
also be administered.
[0120] In addition to the compounds formulated for parenteral
administration, such as intravenous, intradermal or intramuscular
injection, other pharmaceutically acceptable forms include, e.g.,
tablets or other solids for oral administration; liposomal
formulations; time release capsules; biodegradable and any other
form currently used.
[0121] One may also use intranasal or inhalable solutions or
sprays, aerosols or inhalants. Nasal solutions can be aqueous
solutions designed to be administered to the nasal passages in
drops or sprays. Nasal solutions can be prepared so that they are
similar in many respects to nasal secretions. Thus, the aqueous
nasal solutions usually are isotonic and slightly buffered to
maintain a pH of 5.5 to 6.5. In addition, antimicrobial
preservatives, similar to those used in ophthalmic preparations,
and appropriate drug stabilizers, if required, may be included in
the formulation. Various commercial nasal preparations are known
and can include, for example, antibiotics and antihistamines and
are used for asthma prophylaxis.
[0122] Additional formulations which are suitable for other modes
of administration can 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%.
[0123] Oral formulations can include excipients as, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate and the
like. These compositions take the form of solutions, suspensions,
tablets, pills, capsules, sustained release formulations or
powders. In certain defined embodiments, oral pharmaceutical
compositions will comprise an inert diluent or assimilable edible
carrier, or they may be enclosed in hard or soft shell gelatin
capsule, or they may be compressed into tablets, or they 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 tablets, 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 compositions is such that a suitable dosage can be
obtained.
[0124] The tablets, troches, pills, capsules and the like may also
contain the following: a binder, as gum tragacanth, acacia,
cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin may be
added or a flavoring agent, such as peppermint, oil of wintergreen,
or cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup of elixir may contain the active compounds
sucrose as a sweetening agent methyl and propylparabens as
preservatives, a dye and flavoring, such as cherry or orange
flavor.
Kits
[0125] Further embodiments concerns kits for use with the methods
and compositions described herein. Some embodiments concern kits
having one or more vaccine or boost compositions of use to prevent
or treat subjects having or exposed to a pathogen or have a
condition. In certain embodiments, a pathogen can include a viral,
bacterial, fungal, or protozoan derived pathogen. A condition can
include a chronic condition or a condition like cancer. Other
embodiments concern kits having vaccine compositions of use to
prevent or treat subjects having or exposed to influenza or
poxvirus. Kits can be portable, for example, able to be transported
and used in remote areas. Other kits may be of use in a health
facility to treat a subject having been exposed to a virus or
suspected of being at risk of exposure to a pathogen (e.g. viral
pathogen). Kits can include one or more construct compositions that
can be administered before, during and/or after exposure to a
pathogen. Other kits can include dehydrated formulations of
constructs contemplated herein in order to prolong the half-life of
the constructs (e.g. for stockpiling the vaccinations in the event
of an outbreak or providing treatments to remote areas).
[0126] Other embodiments can concern kits for making and using
molecular constructs described herein. In certain embodiments,
compositions can include constructs having one or more of,
attenuated or modified MVA and poxvirus secretory signals. Other
constructs can also include at least one secretory signal sequence.
Yet other embodiments can have a construct that includes
translation control sequences (e.g. IRES). Other reagents for
making and using constructs are contemplated.
[0127] Kits can also include a suitable container, for example,
vials, tubes, mini- or microfuge tubes, test tube, flask, bottle,
syringe or other container. Where an additional component or agent
is provided, the kit can 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. Optionally, one or more additional agents such as other
anti-viral agents, anti-fungal or anti-bacterial agents may be
needed for compositions described, for example, for compositions of
use as a vaccine.
[0128] Dose ranges used during vaccination can vary depending on
the nature of the live attenuated vaccine and viral vector used.
For recombinant poxviruses these doses can range between
10.sup.5-10.sup.7 PFUs. In certain embodiments of the present
invention, immunogenic doses can be as low as 10.sup.2 pfu.
Frequency of vaccination can vary depending on the nature of the
vaccine, the condition of the subject and also the route of
administration used. One regimen can include a primary immunization
(prime) followed up by a boost administration four to six weeks
post-prime immunization. In certain embodiments of the present
invention, improvements in antigen translation and expression can
permit fewer and/or lower doses to be administered to a subject.
Some embodiments concern intramuscular administration and/or
intradermal vaccination of a subject.
[0129] Any method known to one skilled in the art may be used for
large scale production of recombinant peptides or proteins. In
accordance with these embodiments, large-scale production of MVA
can be used. For example, master and working seed stocks may be
prepared under GMP conditions in qualified primary CEFs. Cells may
be plated on large surface area flasks, grown to near confluence
and infected at selected MOI and vaccine virus purified. Cells may
be harvested and intracellular virus released by mechanical
disruption, cell debris removed by large-pore depth filtration and
host cell DNA digested with endonuclease. Virus particles may be
subsequently purified and concentrated by tangential-flow
filtration, followed by diafiltration. The resulting concentrated
bulk vaccine may be formulated by dilution with a buffer containing
stabilizers, filled into vials, and lyophilized. For use, the
lyophilized vaccine may be reconstituted by addition of
diluent.
[0130] Poxviruses are known for their stability. The ability to
lyophilize vaccinia for long term, room temperature storage and
distribution was one of the key attributes that permitted
widespread use of the vaccine and eradication of smallpox.
Recently, it was demonstrated that Dryvax vaccinia virus stockpiled
in the 60's was still potent after several decades. Procedures for
lyophilization and storage of poxviruses are well know in the art
and could be applied to the recombinant poxvirus vaccines for some
embodiments disclosed herein.
[0131] 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 that
follow represent techniques discovered to function well in the
practices disclosed herein. However, those of skill in the art
should, in light of the present disclosure, appreciate that many
changes can be made in the certain embodiments which are disclosed
and still obtain a like or similar result without departing from
the spirit and scope herein.
EXAMPLES
[0132] Many constructs described herein were generated, separated
and purified by methods disclosed herein (data not shown) for use
in various studies. Some of these constructs are detailed m the
descriptions below. In certain methods, constructs with and without
influenza gene segments and peptides were generated and used in
mouse models exposed to influenza challenges.
Example 1
[0133] In one exemplary method, a construct composition including
an influenza segment and a vaccinia secretory segment was tested
for induction of immune protection against influenza challenge.
FIGS. 1A and 1 B illustrate a mouse model vaccinated and challenged
with a virus. Here, Balb/C mice were vaccinated with
MVA/IRES/tPA/HA (107 pfu) and challenged with VN/1203 63
(A/Vietnam/1203/04 (H5N1)-10.sup.4 TCID.sub.50) days
post-vaccination. A) Weight loss, and B) Lungs titers, day 4
post-challenge. An MVA construct expressing an influenza segment
elicited protection against the viral challenge. All the MVA
vectored plague vaccines tested in this study were shown to be
completely safe in severe combined immuno-deficient (SCID) mice.
MVA has been stockpiled for use as a second-generation smallpox
vaccine, with superior safety to the original live, attenuated
vaccinia strains. Thus, a recombinant MVA/IRES/tPA/influenza
segment vaccine has the potential to simultaneously provide
protection against smallpox and influenza.
Example 2
[0134] Dose Sparing: In another exemplary method, various
constructs were tested in a range of doses to analyze their
protective effects and to test some of the limitations in these
dose ranges. FIGS. 2A-2C represents Balb/C mice (n=10) vaccinated
ID with 105, 6 or 7 pfu and challenged with VN/1203 on day 63
post-vaccination. Weight loss curves are displayed for A)
MVA/IRES/tPA/HA, B) MVA/IRES/C13L/HA, and C) MVA/HA native.
Example 3
Long-Term and Cross-Clade Protection
[0135] FIG. 3 illustrates that certain vaccine constructs presented
herein provide long-Term Immunity. Balb/C mice (n=7) were
intradermally (ID) vaccinated with 105 (HA) and/or 107 (NP) pfu and
challenged with VN/1203 at 28 wks post-vaccination
Example 4
Heterologous Clade 2 Challenge
[0136] FIG. 4 illustrates cross-clade protection. Here, Balb/C mice
(n=7) ID were vaccinated with 10.sup.5 (HA) or 10.sup.7 (NP)
pfu/mouse, and challenged with VN/UT 28 wks post-vaccination.
Safety
[0137] In another example, safeties of some of the vaccine
constructs were assessed. FIG. 5 represents mice tested with
certain constructs described herein. In this example, SCID/Balb/C
mice (n=6) were IP inoculated with 10.sup.8 pfu/animal
MVA-influenza constructs or 10.sup.6 pfu/animal Wild Type Vaccinia
and monitored for morbidity and pox lesions for 6 weeks.
[0138] Experiments conducted in herein demonstrate that recombinant
MVA influenza vaccines are safe & efficacious. It was
demonstrated that single dose intradermal injection was able to
provides 100% protection from lethal challenge. In addition, dose
sparing introductions at about 5.times.10.sup.5 offers 100%
protection. In certain examples, protection was demonstrated to
last up to 28 weeks. Other examples demonstrate that including NP
in the constructs may provide cross-clade protection. Using a mouse
model, it was demonstrated that recombinant MVA influenza vaccines
are safe in SCID mice. These experiments demonstrate that MVA
construct vaccinations may provide viable alternatives to
traditional influenza vaccination, particularly for emerging virus
subtypes.
[0139] B8R was used as a Vaccinia IFN-gamma soluble receptor. C13L
is associated with a non-expressed protein in Vaccinia that may be
a serpin homologue. As indicated these sequences are not present in
MVA. The signal scores are equivalent or better than those for tPA.
The scores are similar and not significantly different in the
context of other antigens.
[0140] Putative Vaccinia signal sequences were analyzed and C13L
signal was identified as a potent element for constructs generated
and used herein. B8R signal could be more obvious as it is part of
a known secreted Vaccinia protein.
Poxvirus Alternative Secretory Signals.
[0141] Alternative signal sequences from orthopox virus have been
identified to replace tPA in certain constructs for example, for
secretion enhancement from MVA. In this example, tPA cleavage site
is correctly identified in F1 construct according to program signal
P 3.0. Predicted cleavage after AG of NgoMIV site. Hidden Markov
model (HMM) score of 98.8%.
Example 5
Exemplary Secretory Signal Sequences and Constructs
[0142] Some Options for constructs are outlined below.
[0143] C13L, exemplary secretory signal sequence [0144] i. VV-cop:
12510-12313 (complete DNA sequence: 12510-11971). [0145] ii.
Unknown protein function. Located near serpin homologues. [0146]
iii. VV-cop version has a deletion following the signal peptide
that causes a frame shift and unrelated protein sequence prior to
termination 44 aa later. The DNA sequence is present in comparison
to orthopox orthologs. The last 100 bp are present at 179670-179767
as an inverted repeat. Full coding sequence equivalent to VV-WR,
loci 206. [0147] iv. Secretory signal: [0148] 1. 1
MMIYGLIACLIFVTSSIAASP 20 (SEQ ID NO:1) [0149] 2. Signal peptide
score=10.3, probability=6.1.times.10.sup.-5, VV-WR
1.1.times.10.sup.-3. [0150] 3. Cleavage in F1 either AGA-DL (neural
network) or SIA-SPAGAD (HMM) with 99.8% signal probability.
[0151] B8R exemplary secretory signal sequence [0152] i. VV-cop:
[0153] ii. IFN-gamma soluble receptor gene: [0154] 1. B8R is
secreted from the cell to bind host IFN-gamma [0155] 2. Secretory
signal: [0156] a. 1 MRYIIILAVLFINSIHAAKI (SEQ ID NO:2) [0157] b.
Signal peptide score=10.5, probability=4.1.times.10.sup.-4 [0158]
3. Cleavage with F1 either KAG-ADL (neural network) or HA-KAGAD
(HMM) with 99.1% signal probability.
[0159] Signal sequence design with and without IRES.
[0160] a. tPA without IRES.
[0161] b. With IRES, insert into XmaI site, not SalI site: [0162]
i. C13L: [0163] 1) For, 5' IRES, Xma, tm=64.7:
TABLE-US-00001 [0163] (SEQ ID NO: 3) a) 5'
TCGTCCCGGGTTATTTTCCACCATATTGCCGT 3'
[0164] 2) Rev, 3' C13L-Ngom, tm=64.7 with IRES sequence:
TABLE-US-00002 [0164] (SEQ ID NO: 4) a)
5'TCGTGCCGGCTGGACTAGCGATGGATGAAGTC
ACGAATATAAGACACGCTATTAATCCGTATATCAT CATATTATCATCGTGTTTTTCAAAGGA
3'
[0165] 3) pI41(pI4,C13L) created and annotated in CLC. [0166] ii.
B8R: [0167] 1) For, 5' IRES, Xma, tm=64.7:
TABLE-US-00003 [0167] (SEQ ID NO: 5) a) 5'
TCGTCCCGGGTTATTTTCCACCATATTGCCGT 3'
[0168] 2) Rev, 3' B8R-Ngom, tm=64.7 with IRES sequence:
TABLE-US-00004 [0168] (SEQ ID NO: 6) a)
5'TCGTGCCGGCTTTAGCGTGTATACTATTAATGA
ACAAAACTGCGAGAATTATAATATATCTCATATTAT CATCGTGTTTTTCAAAGGA 3'
[0169] 3) pI142(pI14,C13L) created and annotated in CLC.
[0170] c. Without IRES: [0171] i. C13L [0172] 1) For: 5'
C13L-Xma,Ngom,Nhe
TABLE-US-00005 [0172] (SEQ ID NO: 7) a)
5'CCGGGATGATGATATACGGATTAATAGCGTGTCT
TATATTCGTGACTTCATCCATCGCTAGTCCA 3'
[0173] 2) Rev: 3' C13L Xma, Ngom,Nhe
TABLE-US-00006 [0173] (SEQ ID NO: 8) a) 5' CTAG
TGGACTAGCGATGGATGAAGTC ACGAATATAAGACACGCTATTAATCCGTATATCATCA TC
3'
[0174] 3) pI44(sel,C13L) created and annotated in CLC. [0175] ii.
B8R [0176] 1) For: 5' B8R-Xma,Ngom,Nhe
TABLE-US-00007 [0176] (SEQ ID NO: 9) a)
5'CCGGGATGAGATATATTATAATTCTCGCAGTTTT GTTCATTAATAGTATACACGCTAAA G
3'
[0177] 2) Rev: 3' B8R-Xma, Ngom,Nhe
TABLE-US-00008 [0177] (SEQ ID NO: 10) a) 5'CTAG
TTTAGCGTGTATACTATTAATGA ACAAAACTGCGAGAATTATAATATATCTCATC 3'
[0178] 3) pI45(sel,B8R) created and annotated in CLC.
Materials and Methods
Construction of MVA Recombinant Vaccines
[0179] The transfer plasmid was used to generate recombinant MVA
expressing influenza gene segments. Any method known in the art can
be used to generate these constructs.
[0180] Some Construct Test Groups include the following in the
presence or absence of various native and non-IRES constructs (e.g.
IRES, tPA, C13L and B8R).
[0181] 1. MVA/HA (IM) prime
[0182] 2. MVA/HA (IM) prime/boost
[0183] 3. MVA/HA (IM) prime+MVA/flagellin (Adjuvant)
[0184] 4. MVA/HA (ID) prime
[0185] 5. MVA/HA (ID) prime/boost
[0186] 6. MVA/HA (ID) prime+MVA/flagellin (Adjuvant)
[0187] 7. MVA/GFP prime/boost (IM)
[0188] 8. MVA/GFP prime/boost (IM) flagellin
[0189] 9. Formalin Inactivated VN/1203 5 .mu.g (IM) prime/boost
[0190] Some of these constructs have been generated in E. coli.
Some constructs were expressed in CEF (chicken embryo fibroblasts,
data not shown). Some constructs include one or more influenza gene
segment(s) (e.g. HA, NA, NP, Hat, Some constructs include native or
IRES or non-IRES constructs. Other constructs include native, C13L
and IRES/C13L constructs with and without an pathogen associated
gene segment.
Immunization and Challenge
[0191] Groups of mice (e.g Harlan Sprague Dawley, Indianapolis,
Ind.) received primary and booster immunizations with each vaccine
candidate via intramuscular injections into hind legs. Then the
mice were challenges with various viruses disclosed herein for
protection.
Serology
[0192] Serum samples were collected post-primary vaccination and
post-boost (pre-challenge) by means known in the art to assess
antibody titers against influenza or poxvirus.
Statistical Analysis
[0193] The Student's t-test and the Log rank test were used to
compare groups of data. Probability values <0.05 were considered
significant using the GraphPad Prism 5 software (La Jolla, Calif.)
for all statistical analyses.
[0194] Possible Secretory Signal Sequences of Use for Constructs
Disclosed Herein
TABLE-US-00009 VV-cop have SSP IN MVA A13L A14L A39R A41L A56R yes
B19R no B25R B5R B7R B8R no B9R C11R yes C13L no C19L no C3L F5L
G3L K2L M2L no
[0195] In certain experiments it was noted that IM vaccinations
such as Prime/Boost scheme were very effective, that there was
increased morbidity with prime only. An adjuvant may not be
effective and that in certain experiments it was observed that
there was an increase in morbidity & mortality with the
flagellin. In other experiments, ID Vaccinations using all tested
schemes provide complete protection with the least morbidity
occurring with prime/boost. An adjuvant was not contributing and
adjuvant alone does not provide protection.
[0196] Some dose ranges were tested in a mouse model for certain
constructs disclosed herein. Some of the doses ranges were about
5.times.10.sup.5 to about 5.times.10.sup.7. Weight loss of test
animals was one way to monitor effectiveness of vaccination
formulations and constructs tested.
[0197] FIGS. 6A and 6B represent exemplary plots of parameters in
mice after intramuscular (IM) introduction of various constructs of
some embodiments described herein to the mice followed by challenge
with influenza, A) Weight loss and B) Survival post-challenge.
[0198] FIGS. 7A and 7B represent exemplary plots of parameters in
mice after intradermal (ID) introduction of various constructs of
some embodiments described herein to the mice followed by challenge
with influenza, A) Weight loss and B) Survival
post-challenge/infection.
[0199] FIGS. 8A and 8B represent exemplary plots of parameters in
mice after (A) intramuscular (IM) or (B) intradermal (ID)
introduction of various constructs of some embodiments described
herein to the mice followed by challenge with influenza. These
exemplary experiments assess several clinical indications. The
indications were graded in the mouse model on a scale of 0 to 4.
0=no signs of illness, 1=ruffled fur; 2=pitted coat, hunched
posture, shivering and slow movement; 3=labored breathing,
anorexia, little/no movement and 4=paralysis, moribund.
[0200] FIG. 9 represents an exemplary plot that illustrates viral
titers in lung after introduction of various constructs of some
embodiments described herein to the mice followed by challenge with
influenza. Mice were sacrificed in each group on day 4 (post
challenge/infection, 3 mice per group) and lungs were homogenized
and tittered on MDCKs. Log virus titer is presented.
[0201] Table 1 represents some of the MVA influenza transfer
vectors and constructs generated and tested.
[0202] FIGS. 10A and 10B represent exemplary plots of percent
weight change in mice after introduction of 2 different constructs
of some embodiments described herein having the HA gene segment in
each construct followed by challenge with influenza. These
construct were administered at different doses (5.times.10.sup.5 to
5.times.10.sup.7)
[0203] FIGS. 11A and 11B represent exemplary plots of percent
weight change in mice after introduction of 2 different constructs
of some embodiments described herein followed by challenge with
influenza. In A), these construct were administered at different
doses (5.times.105 to 5.times.107). In B. a traceable compound was
linked to an MVA construct.
[0204] FIGS. 12A and 12B represent exemplary plots of percent
weight change in mice after introduction of 2 different constructs
at various concentrations of some embodiments described herein
followed by challenge with influenza. These construct were
administered at different doses (5.times.105 to 5.times.107).
[0205] FIGS. 13A and 13B represent exemplary plots of percent
survival in mice after introduction of 2 different constructs at
various concentrations of some embodiments described herein
followed by challenge with influenza. These construct were
administered at different doses (5.times.105 to 5.times.107). Some
of the constructs included additional elements, tPA and IRES. It
was observed at day 8 that mice having constructs with an IRES and
tPA element had decreased survival than MVA/HA alone in a
construct.
[0206] FIGS. 14A and 14B represent exemplary plots of survival in
mice after introduction of 2 different constructs at various
concentrations of some embodiments described herein followed by
challenge with influenza. These construct were administered at
different doses (5.times.105 to 5.times.107). Some of the
constructs included additional elements, tPA and IRES sequences
(A). It was observed at day 8 that mice having constructs with an
IRES and tpa element had decreased survival than MVA/HA alone in a
construct. When the tPA element was replaced with another secretory
signal C13L, survival was 100 percent for the time period
tested.
[0207] FIGS. 15A and 15B represent exemplary plots of survival in
mice after introduction of 2 different constructs at various
concentrations of some embodiments described herein followed by
challenge with influenza (dpi represents days post infection).
These construct were administered at different doses (5.times.105
to 5.times.107).
[0208] FIGS. 16A and 16B represent exemplary plots of clinical
scores (e.g. physical and physiological parameters, see above
scores from 0 to 4) in mice after introduction of 2 different
constructs at various concentrations of some embodiments described
herein followed by challenge with influenza. These construct were
administered at different doses (5.times.105 to 5.times.107). In
addition an MVA construct linked to a detectible marker was also
introduced and followed in the mice.
[0209] FIGS. 17A and 17B represent exemplary plots of clinical
scores (e.g. physical and physiological parameters) in mice after
introduction of 2 different constructs at various concentrations of
some embodiments described herein followed by challenge with
influenza. These construct were administered at different doses
(5.times.105 to 5.times.107). In addition an MVA construct linked
to a detectible marker (GFP) was also introduced and followed in
the mice.
[0210] FIG. 18 represents an exemplary plot of clinical scores
(e.g. physical and physiological parameters) in mice after
introduction of a construct at various concentrations of some
embodiments described herein followed by challenge with influenza.
These construct were administered at different doses (5.times.105
to 5.times.107). In addition an MVA construct linked to a
detectible marker (GFP) was also introduced and followed in the
mice.
Example 6
[0211] Groups of mice (n=8) were inoculated intradermally with
modified vaccinia Ankara (MVA) three month prior to intradermal
vaccination with MVA/flu vaccines expressing hemagglutinin and/or
nucleoprotein in with or without secretory signal (C13L).
TABLE-US-00010 TABLE 2 represents Antibody titers (Geometric mean
titer - GMT) of serum samples following prime and booster
(intradermal) vaccination with MVA/influenza vaccines in mice with
pre-existing immunity to vaccinia: Vaccine Sampling Construct
Pre-Boost Post-Boost MVA/HA 3.61.sup.c 697.92.sup.a MVA/C13L/HA
1.00.sup.c 65.42.sup.b MVA/C13L/NP 1.00.sup.c 1.00.sup.c
MVA/HA/C13L/NP 2.11.sup.c 697.92.sup.a MVA/GFP 1.00.sup.c
1.00.sup.c .sup.a-cgroup different letters differ significantly (P
< 0.05) by ANOVA
[0212] FIGS. 19A and 19B represent (A) mean weigh changes in
immunized mice challenged with Influenza A/Vietnam/1203-H5N1 virus
(10.sup.4 TCID.sub.50) 4 wks post-booster vaccination with MVA/Flu
vaccines. Mice had pre-existing immunity to vaccinia. Mice
immunized with MVA/Flu containing the hemagglutinin antigen did not
lose weight; and (B) represents Clinical score of mice challenged
with Influenza A/Vietnam/1203-H5N1 virus (10.sup.4 TCID.sub.50) 4
wks post-booster vaccination with MVA/Flu vaccines. Mice had
pre-existing immunity to vaccinia prior to immunization of MVA/Flu
vaccines. Clinical scores 0-4 are detailed above FIG. 20 represents
survival rates of immunized mice (using the same constructs as in
FIGS. 19A and B above) challenged with Influenza
A/Vietnam/1203-H5N1 virus (104 TCID50) 4 wks post-booster
vaccination with MVA/Flu vaccines. Mice had pre-existing immunity
to vaccinia prior to immunization of MVA/Flu vaccines. All mice
immunized with MVA/Flu containing the hemagglutinin antigen
survived challenge with lethal dose of Influenza
A/Vietnam/1203-H5N1 virus.
Example 7
In Vitro Expression of Influenza Virus Proteins by MVA/Flu
Viruses
[0213] FIG. 21 represents a schematic construct of having two
influenza gene segments from different subtypes each having a
secretory signal associated with the gene segment. In certain
examples, the secretory signal can be naturally occurring and in
other examples it can be inserted next to or fused to the influenza
gene segment, or a combination thereof.
[0214] In one exemplary method, expression levels of the HAL HA5
and NP antigens of influenza virus were assessed by immunoblot
analyses of proteins from CEF cells infected with the MVA/Flu
recombinant viruses. Expression levels were evaluated in whole cell
extracts and in cell culture supernatants 48 hr post-infection
(FIG. 22). Both HA1 and HA5 proteins were detected (based on their
predicted sizes) in whole cell extract (FIGS. 22Ai and 22Aii, FIGS.
22Bi and 22Bii) as well as in the culture supernatant (FIGS. 22Bi
and 22Bii). To facilitate secretion of NP into the culture
supernatant the C13L secretory signal sequence was fused to the 5'
end of the NP protein. As illustrated in FIG. 22C NP expression was
detected in both cellular and supernatant fractions from cells
infected with the MVA/C13L/NP virus. The native NP protein had the
expected molecular weight of 56 kD (FIG. 22C). In contrast, the
C13L/NP fusion protein appears slightly larger than the native NP
suggesting that the signal peptide is not cleaved upon expression
or secretion (FIG. 22C). Results indicate that the C13L secretory
signal increases NP secretion into the culture supernatant as
compared to the native NP construct. As with the single antigen
constructs, recombinant viruses expressing both HA and NP proteins
(MVA/HA1/C13L/NP and MVA/HA5/C13L/NP) demonstrated increased levels
of NP protein in cellular fractions than the supernatant fraction
(data not shown).
Example 8
Efficacy of a Single Dose of MVA/Flu Vaccine Against Homologous
Challenge
[0215] In another exemplary methods, groups of BALB/c mice (n=10)
were vaccinated ID (intradermally) with a single dose of MVA/HA1 or
MVA/HA5 recombinant vaccine and challenged by the IN route eight
weeks later. Antibody titers were measured by hemagglutination
Inhibition (HI) assays against A/Norway/3487-2/09(NW/09) or
A/Vietnam/1203/04 (VN/1203), respectively and survival rates
following challenge recorded. The vaccines elicited significantly
higher (P.ltoreq.0.05) HI titers (FIGS. 23A and 23B) compared to
the control virus (MVA/GFP) and conferred complete protection
against homologous challenge with NW/09 or VN/1203, respectively
(FIGS. 23C and 23D). Although one out of three mice vaccinated with
a single dose of MVA/HA1 or MVA/HA5 showed high titers of virus in
their lungs on day 3 post challenge, the overall viral loads in the
lungs of immunized mice were significantly lower (p<0.05)
compared to those from MVA/GFP immunized mice (FIGS. 23E and 23F).
Furthermore, mice receiving a prime and boost immunization with
MVA/Flu vaccine in subsequent studies, cleared the virus from their
lungs three days post-challenge (FIG. 24). Mice in the control
group lost weight and were humanely euthanized when they reached
20% weight loss. No significant (P>0.05) weight losses were
recorded in animals that received any of the MVA/Flu vaccines
(MVA/HA1 or MVA/HA5) upon challenge with NW/09 or VN/1203,
respectively (FIGS. 23G and 23H). In an ancillary long-term
immunity study, mice were immunized with a single dose of MVA/HA5
and seven months later they were challenged with a lethal dose of
VN/1203. All vaccinated animals were protected while control
animals succumbed to challenge with a median survival day of
seven.
Example 9
Cross-Protective Immunity of MVA/Flu Vaccines
[0216] In yet another exemplary method, cross-protective efficacy
of MVA/Flu vaccines expressing for example, both HA and NP
influenza proteins (MVA/HA1/C13L/NP and MVA/HA5/C13L/NP) were
tested against IN challenge with 100 LD50 of the H5N1 strain
A/Vietnam/1203/04 (VN/1203) (1.times.104 pfu), the H1N1 pandemic
strain A/Norway/3487-2/09 (NW/09) (1.0.times.106 pfu), the H1N1
seasonal strain A/Puerto Rico/8/34 (PR8) (3.99.times.105 pfu) or
the H3N2 seasonal strain A/Aichi/2/68 (Aichi/68) (2.5.times.104
pfu) virus. Following prime and booster vaccinations, strong
antibody titers were induced against the homologous virus strains;
however no cross-reactive antibodies were detected against
heterologous viruses (Table 3).
[0217] In one example, the MVA/HA1/C13L/NP vaccine elicited strong
HI antibody titers to NW/09 virus but failed to induce any antibody
responses to the heterologous strains PR8 \, VN/1203 or Aichi/68
viruses (Table 3). Despite the lack of cross-reactivity in HA
titers, complete protection against challenge with PR8 or VN/1203
was observed (Table 3). In addition, the MVA/HA1/C13L/NP vaccine
conferred partial protection (57.1%) against challenge with
Aichi/68 virus (Table 3). In the case of the MVA/HA5/C13L/NP virus,
it did not induce any detectable antibody responses to any of the
H1N1 viruses (NW/09 and PR8) or H3N2 (Aichi/68) virus. However, it
did protect immunized mice against challenge with the H1N1 PR8
strain (Table 3). Analysis of lung viral loads following challenge
with the H1N1 pandemic NW/09 strain showed significantly higher
(P.ltoreq.0.05) virus titers in mice vaccinated with
MVA/HA5/C13L/NP or MVA/GFP virus as compared to mice immunized with
MVA/HA1/C13L/NP virus (FIG. 24).
[0218] Mice immunized with MVA/HA1/C13L/NP vaccine had no
significant weight loss following challenge with the H5N1 VN/1203
strain whereas those immunized with the MVA/HA5/C13L/NP virus had
significant weight loss upon challenge with H1N1 pandemic NW/09
strain (FIGS. 25A and 25B). Mice in the control group consistently
lost weight upon challenge with VN/1203, NW/09, PR8 or Aichi/68
virus (FIGS. 25A, 25B, 25C and 25D). In conjunction with the
cross-protection survival data, mice immunized with MVA/HA5/C13L/NP
demonstrated significant weight loss (P.ltoreq.0.05) compared to
mice immunized with MVA/HA1/C13L/NP upon challenge with the H3N2
Aichi/68 virus (FIG. 25D); however no significant differences in
weight were recorded in mice immunized with MVA/HA1/C13L/NP or
MVA/HA5/C13L/NP vaccines following challenge with seasonal H1N1 PR8
virus (FIG. 25C). Taken together, these data indicate that certain
constructs can provide broader cross-protective immunity that
others, for example, the MVA/HA1/C13L/NP vaccine that conferred
complete protection from all three group 1 A strains and partial
protection against the group 2 H3N2 strain. Surprisingly, the
MVA/HA5/C13L/NP strain completely protected against heterologous
challenge with the seasonal H1N1 strain, but not pandemic H1N1
strain, nor the H3N2 strain.
Example 10
Vector Immunity
[0219] In another exemplary method, MVA was used to pre-expose a
group of mice to an influenza vaccine having proteins from multiple
subtypes. In these experiments, pre-exposure of mice to MVA
significantly (P<0.05) affected the neutralizing antibody titers
elicited post-priming with MVA/HA5 vaccine. This effect could be
reversed following a booster vaccination with the same virus (FIG.
26A). Moreover, pre-existing immunity to MVA did not have any
significant impact on the protective efficacy of the vaccine after
prime and booster immunizations (FIG. 26B).
Example 11
Safety of the MVA/Flu Vaccine
[0220] In another method, SCID mice were injected intraperitoneally
with the MVA/HA5 or MVA/C13L/NP viruses or the control MVA/GFP
virus. These mice did not develop any pox lesions or any other
signs of morbidity. In contrast, animals that were infected with
vaccinia-Wyeth, developed pox lesions on their tails, feet and
mouth region, and demonstrated gradual weight loss. By week four,
the vaccinia-Wyeth group displayed average weight loss of
approximately 11% in addition to ruffled fur, hunched posture and
pox lesions (data not shown). Weight losses in the vaccinia-Wyeth
group was significantly greater (P<0.0001) than in groups that
were infected with MVA/Flu vaccines (FIG. 27).
TABLE-US-00011 TABLE 3 Cross-protective immunity of MVA/Flu
vaccines Challenge virus H5N1 H1N1pdm H1N1 seasonal H3N2 seasonal
(VN/1203) (NW/09pdm) (PR8) (Aichi/68) Post- Post- Post- Post- Boost
Boost Boost Boost Titer % Titer % Titer % Titer % Vaccines (GMT)
Survival (GMT) Survival (GMT) Survival (GMT) Survival
MVA/HA.sub.1/C13L/NP <20.sup.a 100 373 100 <20 100 <20
57.1 MVA/HA.sub.5/C13L/NP 149 100 <20 28.6 <20 100 <20
14.3 MVA/GFP <20 0 <20 28.6 <20 14.3 <20 28.6
.sup.aBelow limit of detection. Hemagglutination inhibition
antibody titers are presented as geometric mean
Materials and Methods
Cells and Viruses
[0221] Mardin-Darby canine kidney (MDCK) cells obtained from the
American Type Culture Collection (ATCC, Manassas, Va.) were
propagated in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics.
Stocks of chicken embryo fibroblasts (CEF) were produced as
previously described. The CEF were used for propagating modified
vaccinia Ankara (MVA) virus. Highly pathogenic avian influenza
(H5N1) virus (A/Vietnam/1203/04), pandemic H1N1 virus
(A/Norway/3487-2/09), seasonal H1N1 virus (A/Puerto Rico/8/34) and
H3N2 virus (A/Aichi/2/68) were obtained. The influenza viruses were
propagated and titrated by TCID50 in MDCK cells with DMEM that
contained 1% bovine serum albumin and 20 mM HEPES and were, stored
as infectious stocks at -80.degree. C. Viral stock titers were
determined by endpoint dilution and recorded as 50% tissue culture
infectious dose (TCID50) as previously described. Culture media for
H1N1 and H3N2 viruses also included 1 .mu.g/ml of trypsin treated
with tosyl phenylalanyl chloromethyl ketone (TPCK). Work with H5N1
influenza virus was conducted in a BSL3+ facility in compliance
with the UW Madison Office of Biological Safety.
[0222] Construction of plasmids and production of MVA recombinant
vaccines
[0223] In one exemplary embodiment, transfer plasmid pdIIIGFP
encoding green fluorescent protein were used to generate
recombinant MVA viruses expressing influenza virus antigens as
previously described but other methods can be used. Hemagglutinin
(HA1) gene from H1N1pdm (A/California/04/09) and HA5 gene from H5N1
(A/VN1203/04) virus were synthesized after codon optimization for
mammalian expression by GeneScript (Piscataway, N.J.). Coding
regions of HA1 and HA5 proteins were amplified by PCR from the
GeneScript constructs using 5'HA1 ssp/3'HA1 or 5' HA5 ssp/3' HA5,
respectively, (Table 3) to generate appropriate restriction sites
and their native secretory signals and then sub-cloned into plasmid
pdIIIGFP, generating plasmids pdIIIGFP/HA1 and pdIIIGFP/HA5
respectively. An alternative transfer plasmid with a secretory
signal, pdIIIGFP/C13L, was generated by inserting a linker, 5'/3'
C13L-ssp, (Table 3) fused with a secretory signal from vaccinia
virus (from the N terminus of the C13L vaccinia gene) at the 5' end
of the multiple cloning site (MCS) such that antigens could be
inserted in frame with the secretory signal. The entire coding
region of nucleoprotein (NP) of H5N1 influenza virus
(A/Vietnam/1203/04) was amplified by PCR from cDNA clone and then
inserted into plasmid pdIIIGFP/C13L to generate the plasmid
pdIIIGFP/C13L/NP. A dual transfer vector pdIIIGFP-d was constructed
by inverting the GFP cassette in pdIIIGFP and then inserting a
second promoter/MCS cassette in an inverted orientation to the
primary promoter cassette (FIG. 21). The coding regions of HAL HA5
and C13L/NP were sub-cloned from the single expression constructs
to generate dual expression constructs pdIIIGFP/HA1/C13L/NP and
pdIIIGFP/HA5/C13L/NP, respectively (FIG. 21).
[0224] The recombinant MVA/Flu viruses were generated as described
previously for each HA and NP constructs. Briefly, CEF cells grown
in 35 mm 6-well plates were infected with wild-type MVA at a
multiplicity of infection (MOI) of 0.05 pfu/cell for one hour (h)
and then transfected with each of the expression plasmids using
Lipofectamine.TM. (Invitrogen, Carlsbad, Calif.). At 48-72 h
post-transfection, cell monolayers were harvested, centrifuged at
500.times.g for 5 min at 4.degree. C. and disrupted by freeze-thaw
and sonication (2 times for 15 seconds using a Virtis600 at setting
3). The disrupted cell extracts containing possible recombinant
viruses expressing GFP were plated onto fresh CEF and overlaid with
0.8% agarose. After 48-72 h of incubation, recombinant
virus-generated plaques were detected by fluorescence using an
inverted microscope and picked into media with a glass pipette. The
cell/virus samples were sonicated and plated as described above.
After three consecutive rounds of plaque purification, high titer
virus stocks were prepared in CEF for subsequent in vitro and in
vivo characterization.
TABLE-US-00012 TABLE 4 Sequence of primers used for amplification
of influenza virus proteins Restriction Primer PCR Primer
Sequence.sup.a Enzyme 5' HA.sub.5
5'-tctcCCCGGGatggagaaaatagtgcttctttttgcaa XmaI ssp
tagtcagtcttgttaaaagtgatcagatttgcattggttaccat-3' (SEQ ID NO:11) 3'
HA.sub.5 5'-ggcGGATCCttaaatgcaaattctgcattgt-3' NgomIV (SEQ ID NO:
12) 5'HA.sub.1 5'-gtgaCCCGGGatgaaggccatcctggtcgtgctgctgtacaccttcg
XmaI ssp ccaccgccaacgccgacacactgtgtatcgggtat-3' (SEQ ID NO: 13)
3'HA.sub.1 5'-gatccGCTAGCtcagatacagat-3' NheI (SEQ ID NO: 14) 5' NP
5'-gtgaGCCGGCgcgtctcaaggcaccaaa-3' NgomIV (SEQ ID NO: 15) 3'NP
5'-gcGAATTCttaattgtcatactcctctgcattgt-3' EcoRI (SEQ ID NO: 16) 5'
5'-CCGGGatgatgatatacggattaatagcgtgtcttatattcgtgacttc XmaI, NgomIV,
NheI C13L-ssp atccatcgctagtccaGCCGGCG-3' (SEQ ID NO: 17) 3'
5'-CTAGCGCCGGCtggactagcgatggatgaagtcacgaatata XmaI, NgomIV, NheI
C13L-ssp agacacgctattaatccgtatatcatcatC-3' (SEQ ID NO: 18)
.sup.aRestriction enzyme sites are capitalized.
In Vitro Expression of Influenza Virus Antigens
[0225] The in vitro expression of HA or NP antigens by recombinant
MVA/Flu viruses was determined by immuno-blot analyses. CEF cells
were seeded into 6-well plates and then infected with either of the
recombinant viruses at a multiplicity of infection (MOI) of 0.5
pfu/cell under serum free conditions. At 48 h post-infection, the
infected cells were harvested in the presence of a protease
inhibitor cocktail (Mini Protease tabs, Roche Diagnostics,
Indianapolis, Ind.), washed, resuspended in 1.times. loading buffer
and heated to 95.degree. C. for 5 min. The supernatants from the
infected cells were centrifuged and concentrated by ultrafiltration
with a 3 kDa cutoff membrane (Nanosep 3K Omega, Pall, Inc., East
Hills, N.Y.). The concentrated supernatants were then combined with
an equal volume of 2.times. gel loading buffer and heated to
95.degree. C. for 5 min. Supernatant and cell samples were resolved
by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to a nitrocellulose membrane for immuno-blot analysis
using anti-H1 (NR-15429, ferret polyclonal), anti-H5 (NR-2730,
mouse monoclonal) or anti-NP (NR-4282, mouse monoclonal) antibodies
(ATCC, BEI Resources, Manassas, Va.).
Animal Studies
[0226] All mouse studies were conducted at University of
Wisconsin-Madison animal facilities and were approved by its
Interinstitutional Animal Care and Use Committee (IACUC). Challenge
experiments involving H5N1 virus (A/Vietnam/1203/04) were conducted
in an ABSL3+ facility in compliance with the University of
Wisconsin-Madison Office of Biological Safety.
Vaccine Efficacy
[0227] A series of experiments were performed to assess the
immunogenicity and protective efficacy of MVA/Flu viruses (MVA/HA1,
MVA/HA5, MVA/C13L/NP, MVA/HA1/C13L/NP and MVA/HA5/C13L/NP). Groups
(n=10) of five week old BALB/c mice (Harlan, Indianapolis, Ind.)
were vaccinated with 1.times.107 plaque forming unit (pfu) of
recombinant MVA/Flu virus contained in 50 .mu.l of PBS via the
intradermal (ID) route. The animals received a single dose or two
vaccine doses (28 days apart) and then blood samples were collected
28 and 56 days post-primary vaccination, respectively for
serological analysis. At 56 days post-primary vaccination, mice
were challenged by intranasal (IN) instillation under isoflurane
anesthesia with 100 LD50 of either A/Vietnam/1203/04 (VN/1203)
(1.times.104 pfu), A/Norway/3487-2/09 (NW/09) (1.0.times.106 pfu),
A/Puerto Rico/8/34 (PR8) (3.99.times.105 pfu) or A/Aichi/2/68
(Aichi/68) (2.5.times.104 pfu) virus contained in 50 .mu.l of PBS.
At day three post-challenge, three mice from each group were
euthanized and lung tissues were collected and homogenized using a
mechanical homogenizer (MP Biochemicals, Solon, Ohio). Viral titers
in the homogenates were quantified by plaque assay on MDCK cells.
Body weight was measured on alternative days and survival rates
were recorded daily for 14 days. Mice showing 20% or more of body
weight loss were considered to have reached the experimental end
point and were humanely euthanized.
Vector Immunity
[0228] To assess the effect of pre-existing immunity to the vector
virus on MVA/Flu vaccines, groups of 4 week old BALB/c mice (n=7)
were first injected ID with MVA/GFP (1.times.107 pfu) virus and
then vaccinated with the recombinant MVA/FLU vaccine (same route
and dose) three months later. Following the initial MVA/GFP
inoculation, mice were vaccinated with 2 doses (28 days apart) of
either 1.times.107 pfu of MVA/HA5 or MVA/GFP virus followed (28
days post-boost) by a challenge with 100 LD50 of VN/1203 virus.
Mice were monitored for morbidity and survival rates recorded over
14 days.
Vaccine Safety
[0229] Safety of MVA/Flu viruses was evaluated in severe combined
immunodeficiency (SCID) BALB/c mice. Briefly, 4-6 week old SCID
mice (n=6) were injected intraperitoneally with 1.times.108 pfu of
the MVA/HA5, MVA/C13L/NP, MVA/GFP virus, or 1.times.106 pfu of
wild-type (WT) vaccinia virus (Wyeth strain). Mice were monitored
for 9 weeks for morbidity including weight loss and appearance of
pox lesions.
Serology
[0230] Blood samples were collected from all animals via the
maxillary vein at different time-points post-vaccination and prior
to challenge to determine antibody titers by hemagglutination
inhibition (HI) or by microneutralization assays. Briefly, aliquots
of serum samples were treated with receptor destroying enzyme (RDE,
Denka Seiken, TKY). One volume of pooled serum was added to three
volumes of RDE, incubated at 37.degree. C. for 16 hours, then
heat-inactivated at 56.degree. C. for 1 h. Hemagglutination
inhibition (HI) assay was conducted by mixing 25 .mu.l of two-fold
serial dilutions of sera in with 8 HA units of virus re-suspended
in the same volume of PBS and incubated in 96-well plates for 45
min at room temperature (RT). Following this, 50 .mu.l of turkey
red blood cells (0.5%) were added and plates were incubated at RT
for 45 min. HI titers of sera were determined as the highest
dilution that displayed hemagglutinin inhibition activity. For
microneutralization assay, 50 .mu.l of serially (two-fold) treated
serum samples were added to 50 .mu.l of virus containing 200 TCID50
units and then incubated at 37.degree. C. for 1 hour. The
virus-serum mixture from each dilution was added to duplicate wells
of MDCK cells in 96-well plates, incubated at 37.degree. C. for 72
hours, fixed and stained with 10% crystal violet in 10% formalin to
determine the TCID. The titer was defined as the serum dilution
resulting in complete neutralization of the virus.
Statistical Analysis
[0231] One way ANOVA was used to evaluate the vaccine group effects
on pre-boost and pre-challenge antibody titers. If the vaccine
group effect was statistically significant (P<0.05 by
Kruskal-Wallis test), an all pair-wise comparison among groups was
performed using an unadjusted P-value of 0.05. Survival analyses
were performed to assess vaccine effectiveness against challenge
viruses; reported P-values are from Fisher's exact test.
Probability values <0.05 were considered significant using the
GraphPad Prism 5 software (La Jolla, Calif.) for all statistical
analyses.
[0232] FIG. 21: Represents a schematic of a recombinant plasmid
construction. Expression cassettes were generated by PCR for each
of the influenza virus hemagglutinin antigens, HA1 and HA5 as
described in the materials and methods. The cassettes were cloned
into the pdIIIGFP vector and the resulting plasmids were designated
as pdIIIGFP/HA1 and pdIIIGFP/HA5. The coding regions of HAL HA5 and
C13L/NP were sub-cloned from the single expression constructs to
generate dual expression constructs pdIIIGFP/HA1/C13L/NP and
pdIIIGFP/HA5/C13L/NP. Homologous recombination into MVA was
successfully completed and recombinant MVA/Flu viruses were
identified by GFP expression. HA, NP and C13L are represented with
orange, green and pink color codings. MCS1 and MCS2-multiple
cloning sites; Flanks1 and Flanks2-open box, GFP-striped box;
dSP--orthopoxvirus secretory signals, either p11 (gfp) or synthetic
early/late promoter (MCS1/2) and delIII--MVA deletion region
III.
[0233] FIG. 22 illustrates expression of influenza virus proteins
by recombinant MVA/flu viruses in infected cells. Monolayers of CEF
cells were infected with exemplary recombinant MVA/flu constructs
including, MVA/C13L/NP, MVA/HA1, MVA/HA5, MVA/HA1/C13L/NP or
MVA/HA5/C13L/NP virus at MOI of 0.05 pfu/cell. At 48 h
post-infection cells were harvested and subjected to SDS-PAGE
followed by Western blot analysis. (A) HA expression in CEF cell
(c) and supernatant (s) fractions detected with anti-H1 antibody in
(Ai) MVA/HA1 and (Aii) MVA/HA1/C13L/NP. (B) HA expression in CEF
cell (c) and supernatant (s) fractions detected with anti-H5
antibody in (Bi) MVA/HA5 and (Bii) MVA/HA5/C13L/NP. 50 ng of HA5
protein was loaded as a positive control for size (C) NP expression
in CEF cell (c) and supernatant (s) fractions detected with anti-NP
antibody in MVA/NP and MVA/C13L/NP. M indicates molecular weight
markers.
[0234] FIG. 23 represents an example of efficacy of MVA/Flu vaccine
against homologous challenge: Mice were immunized with the (n=10)
were challenged against pandemic H1N1 (NW/09) (A, C, E, G) or H5N1
(VN/1203) (B, D, F, H). Fifty-six days following primary
immunization antibody titers were determined by HI assay (A, B).
Mice were challenged 56 days post primary immunization and survival
rates were recorded (C, D). Three days post-challenge lung viral
load were determined (E, F) and clinical scores (weight) measured
over 14 days post-challenge (G, H).
[0235] FIG. 24 represents an example of lung viral titers in
vaccinated and control animals following challenge with NW/09pdm:
Mice (n=3) immunized with two doses (28 days apart) of
MVA/HA1/C13L/NP or MVA/HA5/C13L/NP vaccine were challenged with
A/H1N1pdm (NW/09) virus 28 days post-boost. Three days
post-challenge lungs from mice were harvested and analyzed for
viral load by plaque assay. Data points represent antibody titers
per gram of tissue (PFU/g) in individual mice. Horizontal bars
indicate the mean.
[0236] FIG. 25 represents and example of cross protective efficacy
of MVA/Flu vaccines: Mean weight of immunized mice (n=7) following
challenge with A (VN/1203), B (NW/09), C (PR8) or D (Aichi/68).
[0237] FIG. 26 represents an example of effects of vector immunity
on vaccine efficacy: Cohorts of mice (n=7) were first injected with
MVA vector and then received two doses of MVA/Flu vaccine three
months later. Following prime and booster vaccinations, antibody
titers were measured by microneutralization (A) and twenty eight
days following the booster vaccination, mice were challenged with
VN/1203 and survival rates were recorded (B).
[0238] FIG. 27 represents an example of safety of MVA-vectored
vaccines in SCID mice: Following inoculation of mice (n=6) with
MVA/Flu vaccine (1.times.108 pfu) or with Wyeth strain of vaccinia
virus (1.times.106 pfu), mice were monitored 9 weeks for (A)
ruffleness, hunched posture, pox lesions and (B) for weight loss.
Mean body weights are presented.
[0239] FIG. 28 illustrates an exemplary plasmid construct of some
embodiments disclosed herein. FIG. 29 illustrates a histogram plot
of cross-protective activity using certain constructs described
herein.
[0240] 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.
Sequence CWU 1
1
20118PRTArtificial Sequencevaccinia virus secretory signal 1Met Met
Ile Tyr Gly Leu Ile Ala Cys Leu Ile Phe Val Thr Ser Ser 1 5 10 15
Ile Ala 217PRTArtificial Sequencevaccinia virus secretory signal
2Met Arg Tyr Ile Ile Ile Leu Ala Val Leu Phe Ile Asn Ser Ile His 1
5 10 15 Ala 332DNAArtificial Sequenceprimer 3tcgtcccggg ttattttcca
ccatattgcc gt 32494DNAArtificial Sequenceprimer 4tcgtgccggc
tggactagcg atggatgaag tcacgaatat aagacacgct attaatccgt 60atatcatcat
attatcatcg tgtttttcaa agga 94532DNAArtificial Sequenceprimer
5tcgtcccggg ttattttcca ccatattgcc gt 32688DNAArtificial
Sequenceprimer 6tcgtgccggc tttagcgtgt atactattaa tgaacaaaac
tgcgagaatt ataatatatc 60tcatattatc atcgtgtttt tcaaagga
88772DNAArtificial Sequenceprimer 7ccgggatgat gatatacgga ttaatagcgt
gtcttatatt cgtgacttca tccatcgcta 60gtccagccgg cg 72872DNAArtificial
Sequenceprimer 8ctagcgccgg ctggactagc gatggatgaa gtcacgaata
taagacacgc tattaatccg 60tatatcatca tc 72966DNAArtificial
Sequenceprimer 9ccgggatgag atatattata attctcgcag ttttgttcat
taatagtata cacgctaaag 60ccggcg 661066DNAArtificial Sequenceprimer
10ctagcgccgg ctttagcgtg tatactatta atgaacaaaa ctgcgagaat tataatatat
60ctcatc 661182DNAArtificial Sequenceprimer sequence 11tctccccggg
atggagaaaa tagtgcttct ttttgcaata gtcagtcttg ttaaaagtga 60tcagatttgc
attggttacc at 821231DNAArtificial Sequenceprimer sequence
12ggcggatcct taaatgcaaa ttctgcattg t 311382DNAArtificial
Sequenceprimer sequence 13gtgacccggg atgaaggcca tcctggtcgt
gctgctgtac accttcgcca ccgccaacgc 60cgacacactg tgtatcgggt at
821423DNAArtificial Sequenceprimer sequence 14gatccgctag ctcagataca
gat 231528DNAArtificial Sequenceprimer sequence 15gtgagccggc
gcgtctcaag gcaccaaa 281634DNAArtificial Sequenceprimer sequence
16gcgaattctt aattgtcata ctcctctgca ttgt 341772DNAArtificial
Sequenceprimer sequence 17ccgggatgat gatatacgga ttaatagcgt
gtcttatatt cgtgacttca tccatcgcta 60gtccagccgg cg
721872DNAArtificial Sequenceprimer sequence 18ctagcgccgg ctggactagc
gatggatgaa gtcacgaata taagacacgc tattaatccg 60tatatcatca tc
72198385DNAArtificial SequenceMVA construct 19gaattaattc gttggtggtc
gccatggatg gtgttattgt atactgtcta aacgcgttag 60taaaacatgg cgaggaaata
aatcatataa aaaatgattt catgattaaa ccatgttgtg 120aaaaagtcaa
gaacgttcac attggcggac aatctaaaaa caatacagtg attgcagatt
180tgccatatat ggataatgcg gtatccgatg tatgcaattc actgtataaa
aagaatgtat 240caagaatatc cagatttgct aatttgataa agatagatga
cgatgacaag actcctactg 300gtgtatataa ttattttaaa cctaaagatg
ccattcctgt tattatatcc ataggaaagg 360atagagatgt ttgtgaacta
ttaatctcat ctgataaagc gtgtgcgtgt atagagttaa 420attcatataa
agtagccatt cttcccatgg atgtttcctt ttttaccaaa ggaaatgcat
480cattgattat tctcctgttt gatttctcta tcgatgcggc acctctctta
agaagtgtaa 540ccgataataa tgttattata tctagacacc agcgtctaca
tgacgagctt ccgagttcca 600attaattggt tcaagtttta cataagtata
aagtccgact attgttctat attatatatg 660gttgttgatg gatctgtgat
gcatgcaata gctgataata gaacttacgc aaatattagc 720aaaaatatat
tagacaatac tacaattaac gatgagtgta gatgctgtta ttttgaacca
780cagattagga ttcttgatag agatgagatg ctcaatggat catcgtgtga
tatgaacaga 840cattgtatta tgatgaattt acctgatgta ggcgaatttg
gatctagtat gttggggaaa 900tatgaacctg acatgattaa gattgctctt
tcggtggctg ggtaccttgc ggccgcttac 960ttgtacagct cgtgcatgcc
gagagtgatc ccggcggcgg tcacgaactc cagcaggacc 1020atgtgatcgc
gcttctcgtt ggggtctttg ctcagggcgg actgggtgct caggtagtgg
1080ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta
gtggtcggcg 1140agctgcacgc tgccgtcctc gatgttgtgg cggatcttga
agttcacctt gatgccgttc 1200ttctgcttgt cggccatgat atagacgttg
tggctgttgt agttgtactc cagcttgtgc 1260cccaggatgt tgccgtcctc
cttgaagtcg atgcccttca gctcgatgcg gttcaccagg 1320gtgtcgccct
cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag
1380atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc
gtgctgcttc 1440atgtggtcgg ggtagcggct gaagcactgc acgccgtagg
tcagggtggt cacgagggtg 1500ggccagggca cgggcagctt gccggtggtg
cagatgaact tcagggtcag cttgccgtag 1560gtggcatcgc cctcgccctc
gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc 1620agctcgacca
ggatgggcac caccccggtg aacagctcct cgcccttgct caccatttat
1680agcatagaaa aaaacaaaat gaaaggcgcg cctggtaccg caataccaac
caggaagaca 1740cgaacattta ctgttctgtg ttccggtaca gaaaaacaat
tgataattct taattgtcat 1800actcctctgc attgtctccg aagaaataag
atccttcatt attcatgtca aaggaaggca 1860cgatcgggtt cgttgccttt
tcgtccgaga gctcgaagac tccccgcccc tggaatgaca 1920catcttctgg
tctggcactt tccatcattc ttatgatttc agtcctcatg tcagacgttc
1980tgccctcagt atttcctgta aatgctgcca taatggtcgc tctttcaaag
ggaaggtttc 2040tctggaccga gaaagtgggc tgaacgctga tctgtcctgc
agatgccctc tgctggttgg 2100tgtttcctcc gcttctggtt cttatagccc
aatatctgct tctcagttca agagtgttgg 2160agtccattgc ctccatgttc
tcatttgaag caatttgaac ccctctggtg gatagctgtc 2220ctcttgggac
cactcttgtc cctctgatga aacttgagac tctaaggtcc tcaaatgctg
2280cagagtggca tgccatccac actaattgac tcttatgtgc tggattctca
tttggtctaa 2340tgagactaaa gacctggctg ttttgaagca ggcggaaagg
atctattcca accagagagt 2400acccttctct ctcaaagtca tatccactgg
ccactgcaag tccgtacaca caagcaggca 2460agcaggactt atgggccact
gatcctctca ggatgagtgc agaccgtgcc agaaaaatga 2520gatcttcaat
ttcagcattc ccaggatttc tgctctctcg cacttgatcc atcattgctc
2580tttgtgctgc tgtttggaat ttccctttga ggatgttgca cattctctca
tatgcaatcc 2640ttgttcttct tccattttcg cctctccaga aattccggtc
gttgatccct cgttttatca 2700tccgaatcag ctccatcacc attgtcccta
ccccctttac tgctgcaccg gcagctccag 2760atctcctcgg gagagttgac
ccttgcatca gagagcacat ccttgggtcc attccagtac 2820gcacgagagc
tctcgttctc tgatatgtgg catcatttag attggaatgc catatcatca
2880ggtgggtaag accagcagtt gcgtcctctc cattgttcgc ttgacgccaa
atcctcctga 2940tctcctcttt gtcgtacaga attagctctc tcacccattt
cccgtctctc ctccgataaa 3000ttggacctcc agtcttcttc gggtcctttc
ccgcactggg gtgttcttcc aggtatctgt 3060tccttctttc atcaaatgca
gagagtacca ttctctctat tgttatgctg ttctggatca 3120gcctcccttc
atagtcactg agtttgagtt ctgtgcacat ctgtatgtag aacctcccaa
3180tgccactaac cattcttcca acagatgccc tgatctcagt agcattctgg
cgttccccac 3240cagtttccat ctgttcataa gatcgtttgg tgccttgaga
cgcgccggct ggactagcga 3300tggatgaagt cacgaatata agacacgcta
ttaatccgta tatcatcatc ccggtcttat 3360ttatattcca aaaaaaaaaa
ataaaatttc aatttttccg gaaaaattga aattttattt 3420tttttttttg
gaatataaat aagcccggga tgaaggccat cctggtcgtg ctgctgtaca
3480ccttcgccac cgccaacgcc gacacactgt gtatcgggta tcacgcaaac
aatagtactg 3540ataccgtcga cacagtgctg gagaagaacg tcactgtgac
ccattcagtc aatctgctgg 3600aagataaaca caacggaaag ctgtgcaaac
tgcggggcgt ggcccctctg catctgggga 3660agtgtaatat tgctggatgg
atcctgggca acccagagtg cgaaagcctg tccacagcat 3720ctagttggtc
atacattgtc gagactccca gctccgacaa tgggacctgt tatcctggag
3780attttatcga ctacgaagag ctgagagaac agctgtctag tgtgtcaagc
ttcgagaggt 3840ttgaaatttt cccaaaaaca tcctcttggc ccaaccacga
tagtaataag ggcgtcactg 3900ccgcttgccc tcatgcaggg gccaaatcat
tttataagaa cctgatctgg ctggtgaaaa 3960agggaaatag ctacccaaaa
ctgtccaagt cttatattaa cgacaaaggc aaggaggtcc 4020tggtgctgtg
ggggatccac catcccagta cctcagctga tcagcagagc ctgtaccaga
4080atgcagacac atatgtcttc gtgggatcct ctcgctacag taaaaagttt
aaacctgaaa 4140ttgccatccg accaaaggtc cgggatcagg agggcagaat
gaactattac tggactctgg 4200tggaacctgg agacaaaatt accttcgagg
ctacaggaaa tctggtcgtg cctagatatg 4260catttgccat ggaacgcaac
gctggctcag ggatcattat cagcgatact ccagtccacg 4320actgtaatac
cacatgccag actcccaagg gagcaattaa cacctccctg cctttccaga
4380atatccatcc aattacaatc ggcaaatgtc ccaagtacgt gaaatctact
aagctgcgac 4440tggccaccgg gctgcggaac attcctagta tccagtcaag
aggactgttt ggcgctattg 4500cagggttcat cgagggaggc tggacaggga
tggtcgatgg atggtatggc taccaccatc 4560agaatgaaca ggggagcgga
tatgccgctg acctgaaatc cactcagaac gcaattgatg 4620agatcaccaa
taaggtgaac tctgtcattg aaaaaatgaa tacacagttt actgccgtgg
4680gcaaggagtt caaccacctg gaaaaaagga tcgagaatct gaacaagaaa
gtggacgatg 4740ggtttctgga catttggacc tacaatgctg aactgctggt
gctgctggag aacgaacgca 4800cactggatta tcatgacagt aatgtgaaga
acctgtacga gaaagtgcga agccagctga 4860agaataacgc caaagaaatc
ggaaatggct gcttcgagtt ttatcacaag tgtgataaca 4920cctgcatgga
aagcgtgaag aatgggacat acgactatcc caagtactcc gaggaggcta
4980agctgaaccg cgaggagatt gatggcgtga agctggagtc taccagaatc
taccagatcc 5040tggccatcta cagcaccgtg gccagctccc tggtgctggt
ggtgtctctg ggcgccatca 5100gcttctggat gtgtagcaac ggctccctgc
agtgccggat ctgtatctga gctagcggat 5160ccgaattcct gcagggaaag
ttttataggt agttgataga acaaaataca taattttgta 5220aaaataaatc
actttttata ctaatatgac acgattacca atacttttgt tactaatatc
5280attagtatac gctacacctt ttcctcagac atctaaaaaa ataggtgatg
atgcaacttt 5340atcatgtaat cgaaataata caaatgacta cgttgttatg
agtgcttggt ataaggagcc 5400caattccatt attcttttag ctgctaaaag
cgacgtcttg tattttgata attataccaa 5460ggataaaata tcttacgact
ctccatacga tgatctagtt acaactatca caattaaatc 5520attgactgct
agagatgccg gtacttatgt atgtgcattc tttatgacat cgcctacaaa
5580tgacactgat aaagtagatt atgaagaata ctccacagag ttgattgtaa
atacagatag 5640tgaatcgact atagacataa tactatctgg atctacacat
tcaccagaaa ctagttaagc 5700ttgtctccct atagtgagtc gtattagagc
ttggcgtaat catggtcata gctgtttcct 5760gtgtgaaatt gttatccgct
cacaattcca cacaacatac gagccggaag cataaagtgt 5820aaagcctggg
gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
5880gctttcgagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
acgcgcgggg 5940agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
tcactgactc gctgcgctcg 6000gtcgttcggc tgcggcgagc ggtatcagct
cactcaaagg cggtaatacg gttatccaca 6060gaatcagggg ataacgcagg
aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 6120cgtaaaaagg
ccgcgttgct ggcgtttttc gataggctcc gcccccctga cgagcatcac
6180aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
ataccaggcg 6240tttccccctg gaagctccct cgtgcgctct cctgttccga
ccctgccgct taccggatac 6300ctgtccgcct ttctcccttc gggaagcgtg
gcgctttctc atagctcacg ctgtaggtat 6360ctcagttcgg tgtaggtcgt
tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 6420cccgaccgct
gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
6480ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
tgtaggcggt 6540gctacagagt tcttgaagtg gtggcctaac tacggctaca
ctagaaggac agtatttggt 6600atctgcgctc tgctgaagcc agttaccttc
ggaaaaagag ttggtagctc ttgatccggc 6660aaacaaacca ccgctggtag
cggtggtttt tttgtttgca agcagcagat tacgcgcaga 6720aaaaaaggat
ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
6780gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
cacctagatc 6840cttttaaatt aaaaatgaag ttttaaatca atctaaagta
tatatgagta aacttggtct 6900gacagttacc aatgcttaat cagtgaggca
cctatctcag cgatctgtct atttcgttca 6960tccatagttg cctgactccc
cgtcgtgtag ataactacga tacgggaggg cttaccatct 7020ggccccagtg
ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
7080ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
atccgcctcc 7140atccagtcta ttaattgttg ccgggaagct agagtaagta
gttcgccagt taatagtttg 7200cgcaacgttg ttggcattgc tacaggcatc
gtggtgtcac gctcgtcgtt tggtatggct 7260tcattcagct ccggttccca
acgatcaagg cgagttacat gatcccccat gttgtgcaaa 7320aaagcggtta
gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
7380tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
cgtaagatgc 7440ttttctgtga ctggtgagta ctcaaccaag tcattctgag
aatagtgtat gcggcgaccg 7500agttgctctt gcccggcgtc aatacgggat
aataccgcgc cacatagcag aactttaaaa 7560gtgctcatca ttggaaaacg
ttcttcgggg cgaaaactct caaggatctt accgctgttg 7620agatccagtt
cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
7680accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
gggaataagg 7740gcgacacgga aatgttgaat actcatactc ttcctttttc
aatattattg aagcatttat 7800cagggttatt gtctcatgag cggatacata
tttgaatgta tttagaaaaa taaacaaata 7860ggggttccgc gcacatttcc
ccgaaaagtg ccacctgacg tctaagaaac cattattatc 7920atgacattaa
cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
7980gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
ttgtctgtaa 8040gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
cgggtgttgg cgggtgtcgg 8100ggctggctta actatgcggc atcagagcag
attgtactga gagtgcacca tatgcggtgt 8160gaaataccgc acagatgcgt
aaggagaaaa taccgcatca ggcgccattc gccattcagg 8220ctgcgcaact
gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
8280aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
ccagtcacga 8340cgttgtaaaa cgacggccag tgaattggat ttaggtgaca ctata
8385203361DNAArtificial SequenceMVA construct 20ttaattgtca
tactcctctg cattgtctcc gaagaaataa gatccttcat tattcatgtc 60aaaggaaggc
acgatcgggt tcgttgcctt ttcgtccgag agctcgaaga ctccccgccc
120ctggaatgac acatcttctg gtctggcact ttccatcatt cttatgattt
cagtcctcat 180gtcagacgtt ctgccctcag tatttcctgt aaatgctgcc
ataatggtcg ctctttcaaa 240gggaaggttt ctctggaccg agaaagtggg
ctgaacgctg atctgtcctg cagatgccct 300ctgctggttg gtgtttcctc
cgcttctggt tcttatagcc caatatctgc ttctcagttc 360aagagtgttg
gagtccattg cctccatgtt ctcatttgaa gcaatttgaa cccctctggt
420ggatagctgt cctcttggga ccactcttgt ccctctgatg aaacttgaga
ctctaaggtc 480ctcaaatgct gcagagtggc atgccatcca cactaattga
ctcttatgtg ctggattctc 540atttggtcta atgagactaa agacctggct
gttttgaagc aggcggaaag gatctattcc 600aaccagagag tacccttctc
tctcaaagtc atatccactg gccactgcaa gtccgtacac 660acaagcaggc
aagcaggact tatgggccac tgatcctctc aggatgagtg cagaccgtgc
720cagaaaaatg agatcttcaa tttcagcatt cccaggattt ctgctctctc
gcacttgatc 780catcattgct ctttgtgctg ctgtttggaa tttccctttg
aggatgttgc acattctctc 840atatgcaatc cttgttcttc ttccattttc
gcctctccag aaattccggt cgttgatccc 900tcgttttatc atccgaatca
gctccatcac cattgtccct acccccttta ctgctgcacc 960ggcagctcca
gatctcctcg ggagagttga cccttgcatc agagagcaca tccttgggtc
1020cattccagta cgcacgagag ctctcgttct ctgatatgtg gcatcattta
gattggaatg 1080ccatatcatc aggtgggtaa gaccagcagt tgcgtcctct
ccattgttcg cttgacgcca 1140aatcctcctg atctcctctt tgtcgtacag
aattagctct ctcacccatt tcccgtctct 1200cctccgataa attggacctc
cagtcttctt cgggtccttt cccgcactgg ggtgttcttc 1260caggtatctg
ttccttcttt catcaaatgc agagagtacc attctctcta ttgttatgct
1320gttctggatc agcctccctt catagtcact gagtttgagt tctgtgcaca
tctgtatgta 1380gaacctccca atgccactaa ccattcttcc aacagatgcc
ctgatctcag tagcattctg 1440gcgttcccca ccagtttcca tctgttcata
agatcgtttg gtgccttgag acgcgccggc 1500tggactagcg atggatgaag
tcacgaatat aagacacgct attaatccgt atatcatcat 1560cccggtctta
tttatattcc aaaaaaaaaa aataaaattt caatttttcc ggaaaaattg
1620aaattttatt tttttttttt ggaatataaa taagcccggg atgaaggcca
tcctggtcgt 1680gctgctgtac accttcgcca ccgccaacgc cgacacactg
tgtatcgggt atcacgcaaa 1740caatagtact gataccgtcg acacagtgct
ggagaagaac gtcactgtga cccattcagt 1800caatctgctg gaagataaac
acaacggaaa gctgtgcaaa ctgcggggcg tggcccctct 1860gcatctgggg
aagtgtaata ttgctggatg gatcctgggc aacccagagt gcgaaagcct
1920gtccacagca tctagttggt catacattgt cgagactccc agctccgaca
atgggacctg 1980ttatcctgga gattttatcg actacgaaga gctgagagaa
cagctgtcta gtgtgtcaag 2040cttcgagagg tttgaaattt tcccaaaaac
atcctcttgg cccaaccacg atagtaataa 2100gggcgtcact gccgcttgcc
ctcatgcagg ggccaaatca ttttataaga acctgatctg 2160gctggtgaaa
aagggaaata gctacccaaa actgtccaag tcttatatta acgacaaagg
2220caaggaggtc ctggtgctgt gggggatcca ccatcccagt acctcagctg
atcagcagag 2280cctgtaccag aatgcagaca catatgtctt cgtgggatcc
tctcgctaca gtaaaaagtt 2340taaacctgaa attgccatcc gaccaaaggt
ccgggatcag gagggcagaa tgaactatta 2400ctggactctg gtggaacctg
gagacaaaat taccttcgag gctacaggaa atctggtcgt 2460gcctagatat
gcatttgcca tggaacgcaa cgctggctca gggatcatta tcagcgatac
2520tccagtccac gactgtaata ccacatgcca gactcccaag ggagcaatta
acacctccct 2580gcctttccag aatatccatc caattacaat cggcaaatgt
cccaagtacg tgaaatctac 2640taagctgcga ctggccaccg ggctgcggaa
cattcctagt atccagtcaa gaggactgtt 2700tggcgctatt gcagggttca
tcgagggagg ctggacaggg atggtcgatg gatggtatgg 2760ctaccaccat
cagaatgaac aggggagcgg atatgccgct gacctgaaat ccactcagaa
2820cgcaattgat gagatcacca ataaggtgaa ctctgtcatt gaaaaaatga
atacacagtt 2880tactgccgtg ggcaaggagt tcaaccacct ggaaaaaagg
atcgagaatc tgaacaagaa 2940agtggacgat gggtttctgg acatttggac
ctacaatgct gaactgctgg tgctgctgga 3000gaacgaacgc acactggatt
atcatgacag taatgtgaag aacctgtacg agaaagtgcg 3060aagccagctg
aagaataacg ccaaagaaat cggaaatggc tgcttcgagt tttatcacaa
3120gtgtgataac acctgcatgg aaagcgtgaa gaatgggaca tacgactatc
ccaagtactc 3180cgaggaggct aagctgaacc gcgaggagat tgatggcgtg
aagctggagt ctaccagaat 3240ctaccagatc ctggccatct acagcaccgt
ggccagctcc ctggtgctgg tggtgtctct 3300gggcgccatc agcttctgga
tgtgtagcaa cggctccctg cagtgccgga tctgtatctg 3360a 3361
* * * * *