U.S. patent application number 14/489213 was filed with the patent office on 2015-01-08 for compositions and methods for vaccinating against hsv-2.
This patent application is currently assigned to Vical Incorporated. The applicant listed for this patent is Lichun Dong, David M. Koelle, Michal Margalith, Adrian VILALTA. Invention is credited to Lichun Dong, David M. Koelle, Michal Margalith, Adrian VILALTA.
Application Number | 20150010597 14/489213 |
Document ID | / |
Family ID | 38851640 |
Filed Date | 2015-01-08 |
United States Patent
Application |
20150010597 |
Kind Code |
A1 |
VILALTA; Adrian ; et
al. |
January 8, 2015 |
COMPOSITIONS AND METHODS FOR VACCINATING AGAINST HSV-2
Abstract
This invention relates to therapeutic polynucleotide
compositions and methods for systemic immune activation which are
effective for eliciting both a systemic, non-antigen specific
immune response and a strong antigen-specific immune response in
mammals. The polynucleotide compositions are particularly effective
for protecting mammals from herpes simplex virus (HSV), such as HSV
gD2, VP11/12, and VP13/14 polypeptides.
Inventors: |
VILALTA; Adrian; (San Diego,
CA) ; Margalith; Michal; (Solana Beach, CA) ;
Dong; Lichun; (Seattle, WA) ; Koelle; David M.;
(Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VILALTA; Adrian
Margalith; Michal
Dong; Lichun
Koelle; David M. |
San Diego
Solana Beach
Seattle
Seattle |
CA
CA
WA
WA |
US
US
US
US |
|
|
Assignee: |
Vical Incorporated
San Diego
CA
The University Of Washington
Seattle
WA
|
Family ID: |
38851640 |
Appl. No.: |
14/489213 |
Filed: |
September 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13571247 |
Aug 9, 2012 |
8840903 |
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14489213 |
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|
12604694 |
Oct 23, 2009 |
8263087 |
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13571247 |
|
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11781153 |
Jul 20, 2007 |
7628993 |
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12604694 |
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60807911 |
Jul 20, 2006 |
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Current U.S.
Class: |
424/231.1 |
Current CPC
Class: |
A61K 2039/572 20130101;
A61K 2039/54 20130101; C12N 2320/31 20130101; A61K 2039/55511
20130101; A61K 2039/545 20130101; C12N 2710/16622 20130101; C07K
14/005 20130101; A61K 39/39 20130101; C12N 7/00 20130101; A61K
39/12 20130101; A61K 2039/55572 20130101; C12N 15/117 20130101;
A61K 2039/57 20130101; A61K 39/245 20130101; C12N 2310/17 20130101;
C12N 2710/16634 20130101; A61K 2039/53 20130101; A61K 2039/575
20130101; A61P 31/22 20180101; A61P 37/04 20180101 |
Class at
Publication: |
424/231.1 |
International
Class: |
A61K 39/245 20060101
A61K039/245; C07K 14/005 20060101 C07K014/005; C12N 7/00 20060101
C12N007/00 |
Goverment Interests
GRANT INFORMATION
[0002] This invention was made with government support under Grant
No. 1 R41 AI065015-01 awarded by National Institutes of Health,
National Institute of Allergy and Infectious Diseases and Grant No.
AI50132 awarded by National Institutes of Health. The government
has certain rights in the invention.
Claims
1. A method for raising an immune response to a herpes simplex
virus infection in a vertebrate comprising administering to the
vertebrate, an isolated polynucleotide comprising SEQ ID NOS:9, 11,
and 12, wherein the polynucleotide is administered in an amount
sufficient to elicit an immune response to the infection.
2. The method of claim 1 further comprising a pharmaceutically
acceptable carrier.
3. The method of claim 2 wherein the carrier is an adjuvant.
4. The method of claim 3 wherein the adjuvant comprises
(+/-)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-pr-
opanaminium bromide (GAP-DMORIE) and
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE).
5. The method of claim 1 further comprising a heterologous nucleic
acid.
6. The method of claim 5 wherein the heterologous nucleic acid
encodes a self-assembly polypeptide, and the polypeptide self
assembles into multimers.
7. The method of claim 5 wherein the heterologous nucleic acid
encodes a secretory signal peptide.
8. The method of claim 5 wherein the heterologous nucleic acid is a
promoter.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 13/571,247, filed Aug. 9, 2012, now pending,
which is a divisional application of U.S. application Ser. No.
12/604,694 filed Oct. 23, 2009, now issued as U.S. Pat. No.
8,263,087, issued Sep. 11, 2012, which is a divisional application
of U.S. application Ser. No. 11/781,153 filed Jul. 20, 2007, now
issued as U.S. Pat. No. 7,628,993, issued Dec. 8, 2009; which
claims the benefit under 35 USC .sctn.119(e) to U.S. Application
Ser. No. 60/807,911 filed Jul. 20, 2006, now abandoned. The
disclosure of each of the prior applications is considered part of
and is incorporated by reference in the disclosure of this
application.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to prophylactic and
therapeutic compositions and methods for inducing an immune
response to herpes simplex virus type 2 (HSV-2). More particularly,
the invention pertains to prophylactic and therapeutic compositions
and methods for inducing an immune response in a vertebrate by
introducing and expressing a DNA vaccine encoding at least one of
the HSV-2 proteins such as: gD, VP11/12, VP13/14 and/or VP22.
[0005] 2. Background Information
[0006] Vaccination with immunogenic proteins has eliminated or
reduced the incidence of many diseases; however there are major
difficulties in using proteins associated with certain pathogens
and disease states as immunogens. Many protein antigens are not
intrinsically immunogenic. More often, they are not effective as
vaccines because of the manner in which the immune system
operates.
[0007] The immune system of vertebrates consists of several
interacting components. The best characterized and most important
parts are the humoral and cellular (cytolytic) branches. Humoral
immunity involves antibodies, proteins which are secreted into the
body fluids and which directly recognize an antigen. The cellular
system, in contrast, relies on special cells which recognize and
kill other cells which are producing foreign antigens. This basic
functional division reflects two different strategies of immune
defense. Humoral immunity is mainly directed at antigens which are
exogenous to the animal whereas the cellular system responds to
antigens which are actively synthesized within the animal.
[0008] Antibody molecules, the effectors of humoral immunity, are
secreted by special B lymphoid cells, B cells, in response to
antigen. Antibodies can bind to and inactivate antigen directly
(neutralizing antibodies) or activate other cells of the immune
system to destroy the antigen.
[0009] Cellular immune recognition is mediated by a special class
of lymphoid cells, the cytotoxic T cells. These cells do not
recognize whole antigens but instead they respond to degraded
peptide fragments thereof which appear on the surface of the target
cell bound to proteins called class I major histocompatibility
complex (MHC) molecules. Essentially all nucleated cells have class
I molecules. It is believed that proteins produced within the cell
are continually degraded to peptides as part of normal cellular
metabolism. These fragments are bound to the MHC molecules and are
transported to the cell surface. Thus the cellular immune system is
constantly monitoring the spectra of proteins produced in all cells
in the body and is poised to eliminate any cells producing foreign
antigens.
[0010] Vaccination is the process of preparing an animal to respond
to an antigen. Vaccination is more complex than immune recognition
and involves not only B cells and cytotoxic T cells, but other
types of lymphoid cells as well. During vaccination, cells which
recognize the antigen (B cells or cytotoxic T cells) are clonally
expanded. In addition, the population of ancillary cells (helper T
cells) specific for the antigen also increase. Vaccination also
involves specialized antigen presenting cells which can process the
antigen and display it in a form which can stimulate one of the two
pathways.
[0011] Vaccination has changed little since the time of Louis
Pasteur. A foreign antigen is introduced into an animal where it
activates specific B cells by binding to surface immunoglobulins.
It is also taken up by antigen processing cells, wherein it is
degraded, and appears in fragments on the surface of these cells
bound to Class II MHC molecules. Peptides bound to class II
molecules are capable of stimulating the helper class of T cells.
Both helper T cells and activated B cells are required to produce
active humoral immunization. Cellular immunity is thought to be
stimulated by a similar but less understood mechanism.
[0012] Thus two different and distinct pathways of antigen
processing produce exogenous antigens bound to class II MHC
molecules where they can stimulate T helper cells, as well as
endogenous proteins degraded and bound to class I MHC molecules and
recognized by the cytotoxic class of T cells.
[0013] There is little or no difference in the distribution of MHC
molecules. Essentially all nucleated cells express class I
molecules whereas class II MHC proteins are restricted to some few
types of lymphoid cells.
[0014] Normal vaccination schemes will produce a humoral immune
response. They may also provide cytotoxic immunity. The humoral
system protects a vaccinated individual from subsequent challenge
from a pathogen and can prevent the spread of an intracellular
infection if the pathogen goes through an extracellular phase
during its life cycle; however, it can do relatively little to
eliminate intracellular pathogens. Cytotoxic immunity complements
the humoral system by eliminating the infected cells. Thus
effective vaccination should activate both types of immunity.
[0015] A cytotoxic T cell response is necessary to remove
intracellular pathogens, such as viruses, as well as malignant
cells. It has proven difficult to present an exogenously
administered antigen in adequate concentrations in conjunction with
Class I molecules to assure an adequate response. This has severely
hindered the development of vaccines against tumor-specific
antigens (e.g., on breast or colon cancer cells), and against
weakly immunogenic viral proteins (e.g., HIV, Herpes, non-A, non-B
hepatitis, CMV and EBV).
[0016] It would be desirable to provide a cellular immune response
alone in immunizing against agents, such as viruses, for which
antibodies have been shown to enhance infectivity. It would also be
useful to provide such a response against both chronic and latent
viral infections and against malignant cells.
[0017] The use of synthetic peptide vaccines does not necessarily
solve these problems because either the peptides do not readily
associate with histocompatibility molecules, have a short serum
half-life, are rapidly proteolyzed, or do not specifically localize
to antigen-presenting monocytes and macrophages. At best, all
exogenously administered antigens must compete with the universe of
self-proteins for binding to antigen-presenting macrophages.
[0018] Major efforts have been mounted to elicit immune responses
to poorly immunogenic viral proteins from the herpes viruses,
non-A, non-B hepatitis, HIV, and the like. These pathogens are
difficult and hazardous to propagate in vitro. Genital herpes is a
highly prevalent sexually transmitted disease worldwide, and is
considered to be a major health burden. The causative agent is
usually herpes simplex virus type 2 (HSV-2). Cellular immune
responses to HSV-2 are believed to be important for both the
prevention of disease and the control of recurrent disease. The
HSV-2 tegument proteins VP11/12, VP13/14, VP22, and gD are
respectively known as, or encoded by genes, UL46, UL47, UL49, and
US6. These proteins contain human CD8+ T-cell epitopes restricted
by HLA A*0101, A*0201 (.times.2), and B*0702, respectively.
[0019] As mentioned above, synthetic peptide vaccines corresponding
to viral-encoded proteins have been made, but have severe pitfalls.
Attempts have also been made to use vaccinia virus vectors to
express proteins from other viruses. However, the results have been
disappointing, since (a) recombinant vaccinia viruses may be
rapidly eliminated from the circulation in already immune
individuals, and (b) the administration of complex viral antigens
may induce a phenomenon known as "antigenic competition," in which
weakly immunogenic portions of the virus fail to elicit an immune
response.
[0020] Another major problem with protein or peptide vaccines is
anaphylactic reaction which can occur when injections of antigen
are repeated in efforts to produce a potent immune response. In
this phenomenon, IgE antibodies formed in response to the antigen
cause severe and sometimes fatal allergic reactions.
[0021] Accordingly, there is a need for a method for invoking a
safe and effective immune response to a protein or polypeptide
associated with herpes simplex virus type 2 (HSV-2). Moreover,
there is a great need for a method that will associate these
antigens with Class I histocompatibility antigens on the cell
surface to elicit a cytotoxic T cell response, avoid anaphylaxis
and proteolysis of the material in the serum, and facilitate
localization of the material to monocytes and macrophages.
SUMMARY OF THE INVENTION
[0022] The present invention provides for DNA vaccines, some of
which comprise the HSV-2 tegument genes UL46, UL47 and UL49 and
another alternative comprises the HSV-2 gD gene all of which were
individually cloned into expression plasmids (VR1012) and used to
immunize a vertebrate. Each animal received three 100 .mu.g doses
of formulated DNA vaccine by intramuscular (IM) injection.
Formulations based on VAXFECTIN adjuvant (an adjuvant composition
comprising GAP-DMORIE and DPyPE) and poloxamer were evaluated for
their ability to boost the immune responses to the HSV DNA
vaccines. Plasmid DNA formulated with PBS was used as a control.
Each tegument protein DNA vaccine induced strong humoral responses.
Regardless of vaccine formulation, UL49 and UL47 elicited stronger
cellular responses than did UL46. Poloxamer significantly boosted
the cellular immune responses to the UL47 DNA vaccine, relative to
the other vaccine formulations. VAXFECTIN adjuvant boosted by about
two-fold the antibody responses to the UL46 and UL49 DNA
vaccines.
[0023] The present invention provides a method for immunizing a
vertebrate against herpes simplex virus, comprising obtaining a
formulated polynucleotide, that is, a positively charged liposome
containing an expressible polynucleotide coding for an immunogenic
peptide, and introducing the formulated polynucleotide into a
vertebrate, whereby the liposome is incorporated into a monocyte, a
macrophage, or another cell, where an immunogenic translation
product of the polynucleotide is formed, and the product is
processed and presented by the cell in the context of the major
histocompatibility complex, thereby eliciting an immune response
against the immunogen. Again, the polynucleotide is DNA, although
mRNA may also be used.
[0024] In another embodiment, there is provided a method for
delivering a pharmaceutical or immunogenic polypeptide to the
interior of a cell of a vertebrate in vivo comprising introducing
an unformulated polynucleotide, that is, a preparation comprising a
pharmaceutically acceptable injectable carrier and a polynucleotide
operatively coding for the herpes simplex virus polypeptide, into
the interstitial space of a tissue comprising the cell, whereby the
polynucleotide is taken up into the interior of the cell and has an
immunogenic or pharmacological effect on the vertebrate. Also
provided is a method for introducing a polynucleotide into muscle
cells in vivo, comprising providing a composition comprising a
polynucleotide in a pharmaceutically acceptable carrier, and
contacting the composition with muscle tissue of a vertebrate in
vivo, whereby the polynucleotide is introduced into muscle cells of
the tissue. In this embodiment, the carrier preferably is isotonic,
hypotonic, or weakly hypertonic, and has a relatively low ionic
strength, such as provided by a sucrose solution.
[0025] One particularly attractive aspect of the invention is a
method for obtaining long term administration of a herpes simplex
polypeptide to a vertebrate, comprising introducing an unformulated
or formulated DNA sequence operatively coding for the polypeptide
interstitially into tissue of the vertebrate, whereby cells of the
tissue produce the polypeptide for at least one month or at least 3
months, more preferably at least 6 months. In this embodiment of
the invention, the cells producing the polypeptide are
nonproliferating cells, such as muscle cells.
[0026] Another method according to the invention is a method for
obtaining transitory expression of a herpes simplex polypeptide in
a vertebrate, comprising introducing unformulated or formulated
mRNA sequence operatively coding for the polypeptide interstitially
into tissue of the vertebrate, whereby cells of the tissue produce
the polypeptide for less than about 20 days, usually less than
about 10 days, and often less than 3 or 5 days. For many of the
methods of the invention, administration into solid tissue is
preferred.
[0027] One important aspect of the present invention is a method
for treatment of genital herpes, comprising introducing a
therapeutic amount of a composition comprising at least one
polynucleotide operatively coding for gD, VP11/12, VP13/14 and/or
VP22 in a pharmaceutically acceptable injectable carrier in vivo
into muscle tissue of an animal suffering from genital herpes,
whereby the polynucleotide is taken up into the cells and gD,
VP11/12, VP13/14 and/or VP22 is produced in vivo. Preferably, the
polynucleotide is a formulated polynucleotide and the composition
is introduced interstitially into the muscle tissue; however, an
unformulated polynucleotide is also contemplated.
[0028] The present invention also includes pharmaceutical products
for all of the uses contemplated in the methods described herein.
For example, there is a pharmaceutical product, comprising
unformulated or formulated polynucleotide, operatively coding for a
herpes simplex polypeptide, in physiologically acceptable
administrate form, in a container.
[0029] In another embodiment, the invention provides a
pharmaceutical product, comprising unformulated or formulated
polynucleotide, operatively coding for a herpes simplex peptide, in
solution in a physiologically acceptable injectable carrier and
suitable for introduction interstitially into a tissue to cause
cells of the tissue to express the polypeptide, and a container
enclosing the solution. The peptide may be immunogenic and
administration of the solution to a human may serve to vaccinate
the human, or an animal. Similarly, the peptide may be therapeutic
and administration of the solution to a vertebrate in need of
therapy relating to the polypeptide will have a therapeutic
effect.
[0030] Also provided by the present invention is a pharmaceutical
product, comprising unformulated or formulated antisense
polynucleotide, in solution in a physiologically acceptable
injectable carrier and suitable for introduction interstitially
into a tissue to cause cells of the tissue to take up the
polynucleotide and provide a therapeutic effect, and a container
enclosing the solution.
[0031] One particularly important aspect of the invention relates
to a pharmaceutical product for treatment of genital herpes,
comprising a sterile, pharmaceutically acceptable carrier, a
pharmaceutically effective amount of a unformulated or formulated
polynucleotide operatively coding for at least one gD, VP11/12,
VP13/14 and/or VP22 protein in the carrier, and a container
enclosing the carrier and the polynucleotide in sterile fashion.
Preferably, the polynucleotide is DNA.
[0032] From yet another perspective, the invention includes a
pharmaceutical product for use in supplying a herpes simplex
polypeptide to a vertebrate, comprising a pharmaceutically
effective amount of a unformulated or formulated polynucleotide
operatively coding for either gD, VP11/12, VP13/14, VP22 or a
combination thereof, a container enclosing the carrier and the
polynucleotide in a sterile fashion, and means associated with the
container for permitting transfer of the polynucleotide from the
container to the interstitial space of a tissue, whereby cells of
the tissue can take up and express the polynucleotide. The means
for permitting such transfer can include a conventional septum that
can be penetrated, e.g., by a needle. Alternatively, when the
container is a syringe, the means may be considered to comprise the
plunger of the syringe or a needle attached to the syringe.
Containers used in the present invention will usually have at least
1, preferably at least 5 or 10, and more preferably at least 50 or
100 micrograms of polynucleotide, to provide one or more unit
dosages. For many applications, the container will have at least
500 micrograms or 1 milligram, and often will contain at least 50
or 100 milligrams of polynucleotide.
[0033] Another aspect of the invention provides a pharmaceutical
product for use in immunizing a vertebrate, comprising a
pharmaceutically effective amount of an unformulated or formulated
polynucleotide operatively coding for either gD, VP11/12, VP13/14,
VP22 or a combination thereof, a sealed container enclosing the
polynucleotide in a sterile fashion, and means associated with the
container for permitting transfer of the polynucleotide from the
container to the interstitial space of a tissue, whereby cells of
the tissue can take up and express the polynucleotide.
[0034] Still another aspect of the present invention is the use of
unformulated or formulated polynucleotide operatively coding for a
physiologically active form of either gD, VP11/12, VP13/14, VP22 or
a combination thereof, in the preparation of a pharmaceutical for
introduction interstitially into tissue to cause cells comprising
the tissue to produce the either gD, VP11/12, VP13/14, VP22 or a
combination thereof for treatment of genital herpes.
[0035] The tissue into which the polynucleotide is introduced can
be a persistent, non-dividing cell. The polynucleotide may be
either a DNA or RNA sequence. When the polynucleotide is DNA, it
can also be a DNA sequence which is itself non-replicating, but is
inserted into a plasmid, and the plasmid further comprises a
replicator. The DNA may be a sequence engineered so as not to
integrate into the host cell genome. The polynucleotide sequences
may code for a herpes simplex virus polypeptide which is either
contained within the cells or secreted therefrom, or may comprise a
sequence which directs the secretion of the peptide.
[0036] The DNA sequence may also include a promoter sequence. In
one preferred embodiment, the DNA sequence includes a cell-specific
promoter that permits substantial transcription of the DNA only in
predetermined cells. The DNA may also code for a polymerase for
transcribing the DNA, and may comprise recognition sites for the
polymerase and the injectable preparation may include an initial
quantity of the polymerase.
[0037] In many instances, it is preferred that the polynucleotide
is translated for a limited period of time so that the polypeptide
delivery is transitory. The polypeptide may advantageously be a
therapeutic polypeptide, and may comprise an enzyme, a hormone, a
lymphokine, a receptor, particularly a cell surface receptor, a
regulatory protein, such as a growth factor or other regulatory
agent, or any other protein or peptide that one desires to deliver
to a cell in a living vertebrate and for which corresponding DNA or
mRNA can be obtained.
[0038] In preferred embodiments, the polynucleotide is introduced
into muscle tissue; in other embodiments the polynucleotide is
incorporated into tissues of skin, brain, lung, liver, spleen or
blood. The preparation is injected into the vertebrate by a variety
of routes, which may be intradermally, subdermally, intrathecally,
or intravenously, or it may be placed within cavities of the body.
In a preferred embodiment, the polynucleotide is injected
intramuscularly. In still other embodiments, the preparation
comprising the polynucleotide is impressed into the skin.
Transdermal administration is also contemplated, as is
inhalation.
[0039] In one preferred embodiment, the polynucleotide is DNA
coding for both a polypeptide and a polymerase for transcribing the
DNA, and the DNA includes recognition sites for the polymerase and
the injectable preparation further includes a means for providing
an initial quantity of the polymerase in the cell. The initial
quantity of polymerase may be physically present together with the
DNA. Alternatively, it may be provided by including mRNA coding
therefore, which mRNA is translated by the cell. In this embodiment
of the invention, the DNA is preferably a plasmid. Preferably, the
polymerase is phage T7 polymerase and the recognition site is a T7
origin of replication sequence.
[0040] In accordance with another aspect of the present invention,
there is provided a method for immunizing a vertebrate, comprising
the steps of obtaining a preparation comprising an expressible
polynucleotide coding for an immunogenic translation product (that
is, either gD, VP11/12, VP13/14, VP22 or a combination thereof),
and introducing the preparation into a vertebrate wherein the
translation product of the polynucleotide is formed by a cell of
the vertebrate, which elicits an immune response against the herpes
simplex virus immunogen. In one embodiment of the method, the
injectable preparation comprises a pharmaceutically acceptable
carrier containing an expressible polynucleotide coding for an
immunogenic peptide, and on the introduction of the preparation
into the vertebrate, the polynucleotide is incorporated into a cell
of the vertebrate wherein an immunogenic translation product of the
polynucleotide is formed, which elicits an immune response against
the immunogen.
[0041] In an alternative embodiment, the preparation comprises one
or more cells obtained from the vertebrate and transfected in vitro
with the polynucleotide (that is, either, UL46, UL47, UL49, or US6
or a combination thereof), whereby the polynucleotide is
incorporated into said cells, where an immunogenic translation
product of the polynucleotide is formed, and whereby on the
introduction of the preparation into the vertebrate, an immune
response against the immunogen is elicited. In any of the
embodiments of the invention, the immunogenic product may be
secreted by the cells, or it may be presented by a cell of the
vertebrate in the context of the major histocompatibility antigens,
thereby eliciting an immune response against the immunogen. The
method may be practiced using non-dividing, differentiated cells
from the vertebrates, which cells may be lymphocytes, obtained from
a blood sample; alternatively, it may be practiced using partially
differentiated skin fibroblasts which are capable of dividing. In a
preferred embodiment, the method is practiced by incorporating the
polynucleotide coding for an immunogenic translation product into
muscle tissue.
[0042] The method may be used to selectively elicit a humoral
immune response, a cellular immune response, or a mixture of these.
In embodiments wherein the cell expresses major histocompatibility
complex of Class I, and the immunogenic peptide is presented in the
context of the Class I complex, the immune response is cellular and
comprises the production of cytotoxic T-cells.
[0043] In one such embodiment, the immunogenic peptide is
associated with the HSV-2 virus and is presented in the context of
Class I antigens, and stimulates cytotoxic T-cells which are
capable of destroying cells infected with the virus. A cytotoxic
T-cell response may also be produced according the method where the
polynucleotide codes for either a truncated gD, VP11/12, VP13/14,
VP22 or a combination thereof antigen lacking humoral epitopes.
[0044] In another embodiment, there is provided a method of
immunizing a vertebrate, comprising obtaining a positively charged
liposome containing an expressible polynucleotide coding for either
gD, VP11/12, VP13/14, VP22 or a combination thereof, and
introducing the liposome into a vertebrate, whereby the liposome is
incorporated into a monocyte, a macrophage, or another cell, where
an immunogenic translation product of the polynucleotide is formed,
and the product is processed and presented by the cell in the
context of the major histocompatibility complex, thereby eliciting
an immune response against the immunogen. Again, the polynucleotide
is preferably DNA, although mRNA may also be used. And as before,
the method may be practiced without the liposome, utilizing just
the polynucleotide in an injectable carrier.
[0045] The present invention is directed to enhancing the immune
response of a vertebrate or mammal in need of protection against
herpes simplex virus infection by administering in vivo, into a
tissue of the vertebrate, at least one polynucleotide, wherein the
polynucleotide comprises one or more nucleic acid fragments, where
the one or more nucleic acid fragments are optionally fragments of
codon-optimized coding regions operably encoding one or more herpes
simplex virus polypeptides, or fragments, variants, or derivatives
thereof. The present invention is further directed to enhancing the
immune response of a vertebrate in need of protection against
herpes simplex virus infection by administering, in vivo, into a
tissue of the vertebrate, a polynucleotide described above plus at
least one isolated herpes simplex virus polypeptide or a fragment,
a variant, or derivative thereof. The isolated herpes simplex virus
polypeptide can be, for example, a purified subunit, a recombinant
protein, a viral vector expressing an isolated herpes simplex virus
polypeptide, or can be an inactivated or attentuated herpes simplex
virus, such as those present in conventional herpes simplex virus
vaccines. According to either method, the polynucleotide is
incorporated into the cells of the vertebrate in vivo, and an
immunologically effective amount of an immunogenic epitope of the
encoded herpes simplex virus polypeptide, or a fragment, variant,
or derivative thereof, is produced in vivo. When utilized, an
isolated herpes simplex virus polypeptide or a fragment, variant,
or derivative thereof is also administered in an immunologically
effective amount
[0046] According to the present invention, the polynucleotide can
be administered either prior to, at the same time (simultaneously),
or subsequent to the administration of the isolated herpes simplex
virus polypeptide. The herpes simplex virus polypeptide or
fragment, variant, or derivative thereof encoded by the
polynucleotide comprises at least one immunogenic epitope capable
of eliciting an immune response to herpes simplex virus in a
vertebrate. In addition, an isolated herpes simplex virus
polypeptide or fragment, variant, or derivative thereof, when used,
comprises at least one immunogenic epitope capable of eliciting an
immune response in a vertebrate. The herpes simplex virus
polypeptide or fragment, variant, or derivative thereof encoded by
the polynucleotide can, but need not, be the same protein or
fragment, variant, or derivative thereof as the isolated herpes
simplex virus polypeptide which can be administered according to
the method.
[0047] The polynucleotide of the invention can comprise a nucleic
acid fragment, where the nucleic acid fragment is a fragment of a
codon-optimized coding region operably encoding any herpes simplex
virus polypeptide or fragment, variant, or derivative thereof,
including, but not limited to, gD, VP 11/12, VP13/14 and/or VP22
proteins or fragments, variants or derivatives thereof. A
polynucleotide of the invention can also encode a derivative fusion
protein, wherein two or more nucleic acid fragments, at least one
of which encodes a herpes simplex virus polypeptide or fragment,
variant, or derivative thereof, are joined in frame to encode a
single polypeptide, such as, but not limited to, gD, VP 11/12,
VP13/14 and/or VP22. Additionally, a polynucleotide of the
invention can further comprise a heterologous nucleic acid or
nucleic acid fragment. Such heterologous nucleic acid or nucleic
acid fragment may encode a heterologous polypeptide fused in frame
with the polynucleotide encoding the herpes simplex virus
polypeptide, e.g., a hepatitis B core protein or a secretory signal
peptide. Preferably, the polynucleotide encodes a herpes simplex
virus polypeptide or fragment, variant, or derivative thereof
comprising at least one immunogenic epitope of herpes simplex
virus, wherein the epitope elicits a B-cell (antibody) response, a
T-cell (e.g., CTL) response, or both.
[0048] Similarly, the isolated herpes simplex virus polypeptide or
fragment, variant, or derivative thereof to be delivered (either a
recombinant protein, a purified subunit, or viral vector expressing
an isolated herpes simplex virus polypeptide, or in the form of an
inactivated herpes simplex virus vaccine) can be any isolated
herpes simplex virus polypeptide or fragment, variant, or
derivative thereof, including but not limited to the gD, VP 11/12,
VP13/14 and/or VP22 proteins or fragments, variants or derivatives
thereof. In certain embodiments, a derivative protein can be a
fusion protein. In other embodiments, the isolated herpes simplex
virus polypeptide or fragment, variant, or derivative thereof can
be fused to a heterologous protein, e.g., a secretory signal
peptide or the hepatitis B virus core protein.
[0049] Nucleic acids and fragments thereof of the present invention
can be altered from their native state in one or more of the
following ways. First, a nucleic acid or fragment thereof which
encodes a herpes simplex virus polypeptide or fragment, variant, or
derivative thereof can be part or all of a codon-optimized coding
region, optimized according to codon usage in the animal in which
the vaccine is to be delivered. In addition, a nucleic acid or
fragment thereof which encodes a herpes simplex virus polypeptide
can be a fragment which encodes only a portion of a full-length
polypeptide, and/or can be mutated so as to, for example, remove
from the encoded polypeptide non-desired protein motifs present in
the encoded polypeptide or virulence factors associated with the
encoded polypeptide. For example, the nucleic acid sequence could
be mutated so as not to encode a membrane anchoring region that
would prevent release of the polypeptide from the cell. Upon
delivery, the polynucleotide of the invention is incorporated into
the cells of the vertebrate in vivo, and a prophylactically or
therapeutically effective amount of an immunologic epitope of a
herpes simplex virus is produced in vivo.
[0050] The invention further provides immunogenic compositions
comprising at least one polynucleotide, wherein the polynucleotide
comprises one or more nucleic acid fragments, where each nucleic
acid fragment is a fragment of a codon-optimized coding region
encoding a herpes simplex virus polypeptide or a fragment, a
variant, or a derivative thereof; and immunogenic compositions
comprising a polynucleotide as described above and at least one
isolated herpes simplex virus polypeptide or a fragment, a variant,
or derivative thereof. Such compositions can further comprise, for
example, carriers, excipients, transfection facilitating agents,
and/or adjuvants as described herein.
[0051] The immunogenic compositions comprising a polynucleotide and
an isolated herpes simplex virus polypeptide or fragment, variant,
or derivative thereof as described above can be provided so that
the polynucleotide and protein formulation are administered
separately, for example, when the polynucleotide portion of the
composition is administered prior (or subsequent) to the isolated
herpes simplex virus polypeptide portion of the composition.
Alternatively, immunogenic compositions comprising the
polynucleotide and the isolated herpes simplex virus polypeptide or
fragment, variant, or derivative thereof can be provided as a
single formulation, comprising both the polynucleotide and the
protein, for example, when the polynucleotide and the protein are
administered simultaneously. In another alternative, the
polynucleotide portion of the composition and the isolated herpes
simplex virus polypeptide portion of the composition can be
provided simultaneously, but in separate formulations.
[0052] Compositions comprising at least one polynucleotide
comprising one or more nucleic acid fragments, where each nucleic
acid fragment is optionally a fragment of a codon-optimized coding
region operably encoding a herpes simplex virus polypeptide or
fragment, variant, or derivative thereof together with and one or
more isolated herpes simplex virus polypeptides or fragments,
variants or derivatives thereof (as either a recombinant protein, a
purified subunit, a viral vector expressing the protein, or in the
form of an inactivated or attenuated herpes simplex virus vaccine)
will be referred to herein as "combinatorial polynucleotide (e.g.,
DNA) vaccine compositions" or "single formulation heterologous
prime-boost vaccine compositions."
[0053] The compositions of the invention can be univalent,
bivalent, trivalent or multivalent. A univalent composition will
comprise only one polynucleotide comprising a nucleic acid
fragment, where the nucleic acid fragment is optionally a fragment
of a codon-optimized coding region encoding a herpes simplex virus
polypeptide or a fragment, variant, or derivative thereof, and
optionally the same herpes simplex virus polypeptide or a fragment,
variant, or derivative thereof in isolated form. In a single
formulation heterologous prime-boost vaccine composition, a
univalent composition can include a polynucleotide comprising a
nucleic acid fragment, where the nucleic acid fragment is
optionally a fragment of a codon-optimized coding region encoding a
herpes simplex virus polypeptide or a fragment, variant, or
derivative thereof and an isolated polypeptide having the same
antigenic region as the polynucleotide. A bivalent composition will
comprise, either in polynucleotide or protein form, two different
herpes simplex virus polypeptides or fragments, variants, or
derivatives thereof, each capable of eliciting an immune response.
The polynucleotide(s) of the composition can encode two herpes
simplex virus polypeptides or alternatively, the polynucleotide can
encode only one herpes simplex virus polypeptide and the second
herpes simplex virus polypeptide would be provided by an isolated
herpes simplex virus polypeptide of the invention as in, for
example, a single formulation heterologous prime-boost vaccine
composition. In the case where both herpes simplex virus
polypeptides of a bivalent composition are delivered in
polynucleotide form, the nucleic acid fragments operably encoding
those herpes simplex virus polypeptides need not be on the same
polynucleotide, but can be on two different polynucleotides. A
trivalent or further multivalent composition will comprise three
herpes simplex virus polypeptides or fragments, variants or
derivatives thereof, either in isolated form or encoded by one or
more polynucleotides of the invention.
[0054] The present invention further provides plasmids and other
polynucleotide constructs for delivery of nucleic acid fragments of
the invention to a vertebrate, e.g., a human, which provide
expression of herpes simplex virus polypeptides, or fragments,
variants, or derivatives thereof. The present invention further
provides carriers, excipients, transfection-facilitating agents,
immunogenicity-enhancing agents, e.g., adjuvants, or other agent or
agents to enhance the transfection, expression or efficacy of the
administered gene and its gene product.
[0055] In one embodiment, a multivalent composition comprises a
single polynucleotide, e.g., plasmid, comprising one or more
nucleic acid regions operably encoding herpes simplex virus
polypeptides or fragments, variants, or derivatives thereof.
Reducing the number of polynucleotides, e.g., plasmids in the
compositions of the invention can have significant impacts on the
manufacture and release of product, thereby reducing the costs
associated with manufacturing the compositions. There are a number
of approaches to include more than one expressed antigen coding
sequence on a single plasmid. These include, for example, the use
of Internal Ribosome Entry Site (IRES) sequences, dual
promoters/expression cassettes, and fusion proteins.
[0056] The invention also provides methods for enhancing the immune
response of a vertebrate to herpes simplex virus infection by
administering to the tissues of a vertebrate one or more
polynucleotides each comprising one or more nucleic acid fragments,
where each nucleic acid fragment is optionally a fragment of a
codon-optimized coding region encoding a herpes simplex virus
polypeptide or fragment, variant, or derivative thereof; and
optionally administering to the tissues of the vertebrate one or
more isolated herpes simplex virus polypeptides, or fragments,
variants, or derivatives thereof. The isolated herpes simplex virus
polypeptide can be administered prior to, at the same time
(simultaneously), or subsequent to administration of the
polynucleotides encoding herpes simplex virus polypeptides.
[0057] In addition, the invention provides consensus amino acid
sequences for herpes simplex virus polypeptides, or fragments,
variants or derivatives thereof, including, but not limited to, the
gD, VP 11/12, VP13/14 and/or VP22 proteins or fragments, variants
or derivatives thereof. Polynucleotides which encode the consensus
polypeptides or fragments, variants or derivatives thereof, are
also embodied in this invention. Such polynucleotides can be
obtained by known methods, for example by backtranslation of the
amino acid sequence and PCR synthesis of the corresponding
polynucleotide as described below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] The accompanying drawings, which are incorporated in and
form a part of the specifications, illustrate the preferred
embodiments of the present invention, and together with the
description serve to explain the principles of the invention.
[0059] FIG. 1 is a schematic representation of the VR1012 DNA
vaccine backbone or plasmid.
[0060] FIGS. 2A through 2C demonstrate sample data for CD4 and CD8
enrichment by negative selection. Fractions were stained with
labeled monoclonal antibody (mAb) and analyzed by flow
cytometry.
[0061] FIGS. 3A through 3C illustrate comparison of codon-optimized
and wild-type plasmids encoding full-length HSV-2 genes for
activation of cloned CD8+ T-cells specific for HSV-2 epitopes.
Cos-7 cells were transfected with either ("codon optimized")
vaccines, or wild-type (strain HG52) plasmids, and 50 ng/well
relevant human class I HLA cDNA. These APC were incubated with
CD8.sup.+ T cell clones known to respond to the relevant proteins.
Supematants were assayed for IFN-.gamma. by ELISA.
[0062] FIG. 4 is a graph illustrating the reactivity of human serum
pools with recombinant HSV-2 tegument proteins, each plated at a
1:5 dilution for use as capture antigen. Binding of human IgG from
pooled sera of donors with HSV-2, or without either HSV-1 or HSV-2
infection was detected by routine ELISA.
[0063] FIGS. 5A through 5I contain graphs illustrating the IgG
responses induced by HSV-2 tegument DNA vaccines in BALB/c mice
detected by ELISA. Serum was collected before each immunization and
at terminal sacrifice (X axis at days 0, 14, 28 and 42). Bars are
geometric means. Antibody titers (Y axis) were determined from
OD.sub.450 values.
[0064] FIGS. 6A through 6C graphically demonstrate the cellular
responses to HSV-2 tegument DNA vaccines at day 42. Mice (10/group)
were immunized on Days 0, 14, and 28, and splenocytes tested on Day
42. Each stacked bar represents a single animal. Between 4 and 8
peptide pools (18-24 peptides/pool) were tested per ORF. The
heights of single bars indicate IFN-.gamma. spot forming units
(SFU)/10.sup.6 splenocytes. Note the differing Y-axes. If the SFU
were too numerous to count (TNTC), they were arbitrarily shown as
1,000.
[0065] FIG. 7 is an example of peptide truncation for minimal
epitopes and CD4.sup.+ vs. CD8.sup.+ responses. The epitopes begin
at amino acid position 183 or 181 of UL46 peptide and are 9- or
11-mers.
[0066] FIG. 8 is an example of peptide truncation for minimal
epitopes and CD4.sup.+ vs. CD8.sup.+ responses. The epitopes begin
at amino acid position 388, 391, 389 or 399 and are 11- or
13-mers.
[0067] FIG. 9 graphically demonstrates the peptide dose curve for
selected UL49 peptides; this region of the protein forms a
CD8.sup.+ epitope. The peptides are 9-, 11- or 13-mers beginning at
amino acid positions 199, 200 or 201.
[0068] FIG. 10 graphically demonstrates the peptide dose curve for
selected UL46 peptides; this region of the protein forms a
CD4.sup.+ epitope. The peptides are 11- or 13-mers beginning at
amino acid positions 388, 389, 391 or 393.
[0069] FIG. 11 schematically demonstrates the identified human and
H-2.sup.d CD4.sup.+ and CD8.sup.+ epitopes in UL46, UL47, and UL49.
For H-2.sup.d epitopes, bar height is proportional to EC.sub.50.
Footnotes 1-5 as marked are:
[0070] 1. Verjans et al, J Infect Dis 2000; 182: 923-927
[0071] 2. Koelle et al Proc Nat Acad Sci USA 2003; 100:
12899-12904
[0072] 3. Posavad et al, J Immunol 2003; 170: 4380-4388
[0073] 4. Koelle et al, J Immunol 2001; 166: 4049-4058 and
[0074] 5. Koelle et al, J Virol 1998; 72: 7476-7483
[0075] FIG. 12 is the plasmid details of the present invention
encoding gD, including the VR2139 plasmid construct, and the amino
acid sequence (SEQ ID NO:1) and codon-optimized nucleic acid
sequence (SEQ ID NO:2) for gD.
[0076] FIG. 13 is the plasmid details of the present invention
encoding UL49, including the VR 2143 plasmid construct, and the
amino acid sequence (SEQ ID NO:3) and codon-optimized nucleic acid
sequence (SEQ ID NO:4) for UL 49.
[0077] FIG. 14 is the plasmid details of the present invention
encoding UL47, including the VR 2144 plasmid construct, and the
amino acid sequence (SEQ ID NO:5) and codon-optimized nucleic acid
sequence (SEQ ID NO:6) for UL 47.
[0078] FIG. 15 is the plasmid details of the present invention
encoding UL46, including the VR 2145 plasmid construct, and the
amino acid sequence (SEQ ID NO:7) and codon-optimized nucleic acid
sequence (SEQ ID NO:8) for UL 46.
[0079] FIG. 16 provides the codon-optimized nucleic acid sequence
for truncated HSV-2 gD (SEQ ID NO:9).
[0080] FIG. 17 provides the codon-optimized nucleic acid sequence
for UL49 (SEQ ID NO:10).
[0081] FIG. 18 provides the codon-optimized nucleic acid sequence
for UL47 (SEQ ID NO:11).
[0082] FIG. 19 provides the codon-optimized nucleic acid sequence
for UL 46 (SEQ ID NO:12).
[0083] FIGS. 20A through 20D show the immunogenicity of HSV-2
tegument DNA vaccines in BALB/c mice. Serum was collected before
each immunization and at terminal sacrifice. Top three panels show
antibody titers (Y axes) determined by ELISA against proteins made
from transfected VM92 cells (Kumar, S., et al., A DNA vaccine
encoding the 42 kDa C-terminus of merozoite surface protein 1 of
Plasmodium falciparum induces antibody, interferon-gamma and
cytotoxic T cell responses in rhesus monkeys: immuno-stimulatory
effects of granulocyte macrophage-colony stimulating factor.
Immunol Lett, 2002. 81(1): p. 13-24). Peroxidase-conjugated goat
anti-mouse IgG and colorimetric detection was used to measure mouse
IgG. Each symbol represents an individual animal; solid bars are
the geometric means from 10 mice per group. Titers<1:100 are
plotted as 100; every naive mouse had titers<1:100 at all time
points (not shown). Antibody titers are significantly different
between each sequential vaccination time points (*p<0.05,
**p<0.005, paired two-tailed t-test). FIG. 20D shows that the
antibody response is against crude mixed native HSV-2 proteins at
day 42.
[0084] FIGS. 21A through 21J show that T cells specific for
tegument proteins have high avidity. Splenocytes were pooled from
2-3 immunized mice and tested by IFN-.gamma. ELISPOT with 13-amino
acid and shorter peptides. Peptides were titrated in 10-fold
dilutions from 10 .mu.M to 10.sup.-6 .mu.M. In general, responder
cells reacting to CD8.sup.+ epitopes showed higher avidity than
cells reacting to CD4.sup.+ epitopes. In some cases, strong ELISPOT
responses were at 10.sup.-12 M. The amino acid positions are
designated for each peptide.
[0085] FIGS. 22A through 22C show the detection of
tegument-specific CD8+ T-cells by intracellular cytokine cytometry.
Splenocytes from a mouse vaccinated three times with UL47 pDNA and
then surviving challenge with virulent HSV-2 were harvested 8 weeks
later and stimulated with a pool of five optimal UL47 CD8 peptides
at 1 .mu.M each (FIG. 22A). FIG. 22C is same mouse, DMSO control.
FIG. 22B is naive mouse splenocytes stimulated with UL47 CD8
epitope peptide pool.
[0086] FIG. 23 provides that splenocytes from
tk.sup.--HSV-2-infected BALB/c mice recognize tegument protein
epitopes. Mice were challenged with 4.times.10.sup.7 pfu
tk.sup.--HSV-2 five days after Depo-provera. Cells at day 14 were
testing in IFN-.gamma. ELISPOT with CD8 peptide epitopes. A
previously described ICP27 CD8.sup.+ epitope is the positive
control (Haynes, J., Arlington J, Dong L, Braun R P, Payne L G,
Potent protective cellular immune responses generated by a DNA
vaccine encoding HSV-2 ICP27 and the E. coli heat labile
enterotoxin. Vaccine, 2006. 24(23): p. 5016-26)
[0087] FIGS. 24A through 24C establish that tegument vaccines are
beneficial in an HSV-2 intravaginal challenge model. Groups of 10
mice were challenged with 50.times.LD.sub.50 of HSV-2 strain 186
and observed for 14 days. FIG. 24A: mortality. FIG. 24B: mean
intravaginal HSV-2 DNA copy numbers. FIG. 24C: Clinical scores in
surviving animals.
[0088] FIGS. 25A and 25B show the immune responses to pDNA vaccine
VR2139 encoding gD.sub.2 amino acid positions 1-340 administered IM
to BALB/c mice with VAXFECTIN adjuvant. IgG titers by ELISA before
each vaccine and at day 42 (FIG. 25A). Raw IFN-.gamma. sfu/million
splenocytes on day 42 (FIG. 25B) using pooled overlapping gD.sub.2
peptides as antigen. Each dot is an individual mouse (n=9) and bars
are mean.
DETAILED DESCRIPTION OF THE INVENTION
[0089] The practice of the present invention requires obtaining a
formulated or unformulated polynucleotide operatively coding for a
polypeptide for incorporation into vertebrate cells. A
polynucleotide operatively codes for a polypeptide when it has all
the genetic information necessary for expression by a target cell,
such as promoters and the like. These polynucleotides can be
administered to the vertebrate by any method that delivers
injectable materials to cells of the vertebrate, such as by
injection into the interstitial space of tissues such as muscles or
skin, introduction into the circulation or into body cavities or by
inhalation or insufflation. A formulated polynucleotide is injected
or otherwise delivered to a vertebrate with a pharmaceutically
acceptable lipid or liposome, for example, when the polynucleotide
is to be associated with a liposome, it requires a material for
forming liposomes, preferably cationic or positively charged
liposomes, and requires that liposomal preparations be made from
these materials. With the liposomal material in hand, the
polynucleotide may advantageously be used to transfect cells in
vitro for use as immunizing agents, or to administer
polynucleotides into bodily sites where liposomes may be taken up
by phagocytic cells.
[0090] Alternatively an unformulated polynucleotide is injected or
otherwise delivered to the animal with a pharmaceutically
acceptable liquid carrier. For all applications, the liquid carrier
is aqueous or partly aqueous, comprising sterile, pyrogen-free
water. The pH of the preparation is suitably adjusted and
buffered.
Polynucleotide Materials
[0091] The formulated or unformulated polynucleotide materials used
according to the methods of the invention comprise DNA and RNA
sequences or DNA and RNA sequences coding for either gD, VP11/12,
VP13/14, VP22 or a combination thereof. (See U.S. Pat. Nos.
6,413,518; 6,855,317; and 7,037,509; and U.S. Patent Publication
US2006/0216304). These polynucleotide sequences are unformulated in
the sense that they are free from any delivery vehicle that can act
to facilitate entry into the cell, for example, the polynucleotide
sequences are free of viral sequences, particularly any viral
particles which may carry genetic information. Alternatively, these
polynucleotide sequences are formulated with a material which
promotes transfection, such as liposomal formulations, charged
lipids such as, but not limited to, LIPOFECTIN reagent (cationic
lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride
(DOTMA) and dioleoyl phophotidylethanolamine (DOPE)), or VAXFECTIN
adjuvant disclosed in U.S. Pat. No. 7,105,574.
[0092] The DNA sequences used in these methods can be those
sequences which do not integrate into the genome of the host cell.
These may be non-replicating DNA sequences, or specific replicating
sequences genetically engineered to lack the genome-integration
ability.
[0093] The polynucleotide sequences of the invention are DNA or RNA
sequences of either HSV-2 proteins gD, VP11/12, VP13/14, VP22 or a
combination thereof. The polynucleotides of the invention also can
code for therapeutic polypeptides. A polypeptide is understood to
be any translation product of a polynucleotide regardless of size,
and whether glycosylated or not. Therapeutic polypeptides include
as a primary example, those polypeptides that can compensate for
defective or deficient species in an animal, or those that act
through toxic effects to limit or remove harmful cells from the
body. Polynucleotide sequences of the invention preferably code for
either gD, VP11/12, VP13/14, VP22 or a combination thereof, and
these sequences may be used in association with other
polynucleotide sequences coding for regulatory proteins that
control the expression of these polypeptides. The regulatory
protein can act by binding to genomic DNA so as to regulate its
transcription; alternatively, it can act by binding to messenger
RNA to increase or decrease its stability or translation
efficiency.
[0094] Where the polynucleotide is DNA, promoters suitable for use
in various vertebrate systems are well known. For example, for use
in murine systems, suitable strong promoters include RSV LTR, MPSV
LTR, SV40 IEP, and metallothionein promoter. In humans, on the
other hand, promoters such as CMV IEP may advantageously be used.
All forms of DNA, whether replicating or non-replicating, which do
not become integrated into the genome, and which are expressible,
are within the methods contemplated by the invention.
[0095] With the availability of automated nucleic acid synthesis
equipment, both DNA and RNA can be synthesized directly when the
nucleotide sequence is known or by a combination of PCR cloning and
fermentation. Moreover, when the sequence of the desired
polypeptide is known, a suitable coding sequence for the
polynucleotide can be inferred.
[0096] When the polynucleotide is mRNA, it can be readily prepared
from the corresponding DNA in vitro. For example, conventional
techniques utilize phage RNA polymerases SP6, T3, or T7 to prepare
mRNA from DNA templates in the presence of the individual
ribonucleoside triphosphates. An appropriate phage promoter, such
as a T7 origin of replication site is placed in the template DNA
immediately upstream of the gene to be transcribed. Systems
utilizing T7 in this manner are well known, and are described in
the literature, e.g., in Current Protocols in Molecular Biology,
.sctn.3.8 (vol. 1 1988).
DNA and mRNA Vaccines
[0097] According to the methods of the invention, both expressible
DNA and mRNA can be delivered to cells to form therein a
polypeptide translation product. If the nucleic acids contain the
proper control sequences, they will direct the synthesis of
relatively large amounts of either gD, VP11/12, VP13/14, VP22 or a
combination thereof. When the DNA and mRNA delivered to the cells
code either gD, VP11/12, VP13/14, VP22 or a combination thereof,
the methods can be applied to achieve improved and more effective
immunity. Since the immune systems of all vertebrates operate
similarly, the applications described can be implemented in all
vertebrate systems, comprising mammalian and avian species, as well
as fish.
[0098] The methods of the invention may be applied by direct
injection of the polynucleotide into cells of the animal in vivo,
or by in vitro transfection of some of the animal cells which are
then re-introduced into the animal body.
[0099] The polynucleotides may be delivered to various cells of the
animal body, including muscle, skin, brain, lung, liver, spleen, or
to the cells of the blood. Delivery of the polynucleotides directly
in vivo is preferably to the cells of muscle or skin. The
polynucleotides may be injected into muscle or skin using an
injection syringe. They may also be delivered into muscle or skin
using a vaccine gun.
[0100] It has recently been shown that cationic lipids can be used
to facilitate the transfection of cells in certain applications,
particularly in vitro transfection. Cationic lipid based
transfection technology is preferred over other methods; it is more
efficient and convenient than calcium phosphate, DEAE dextran or
electroporation methods, and retrovirus mediated transfection, as
discussed previously, can lead to integration events in the host
cell genome that result in oncogene activation or other undesirable
consequences. The knowledge that cationic lipid technology works
with messenger RNA is a further advantage to this approach, because
RNA is turned over rapidly by intracellular nucleases and is not
integrated into the host genome. A transfection system that results
in high levels of reversible expression is preferred to alternative
methodology requiring selection and expansion of stably transformed
clones because many of the desired primary target cells do not
rapidly divide in culture.
[0101] The ability to transfect cells at high efficiency with
cationic liposomes provides an alternative method for immunization.
The gene for an antigen is introduced into cells which have been
removed from an animal. The transfected cells, now expressing the
antigen, are reinjected into the animal where the immune system can
respond to the (now) endogenous antigen. The process can possibly
be enhanced by coinjection of either an adjuvant or lymphokines to
further stimulate the lymphoid cells.
[0102] Vaccination with nucleic acids containing either gD,
VP11/12, VP13/14, VP22 or a combination thereof provides a way to
specifically target the cellular immune response. Cells expressing
at least one gD, VP11/12, VP13/14, and/or VP22 proteins which are
secreted will enter the normal antigen processing pathways and
produce both a humoral and cytotoxic response. The response to
proteins which are not secreted is more selective. Non-secreted
proteins synthesized in cells expressing only class I MHC molecules
are expected to produce only a cytotoxic vaccination. Expression of
the same antigen in cells bearing both class I and class II
molecules may produce a more vigorous response by stimulating both
cytotoxic and helper T cells. Enhancement of the immune response
may also be possible by injecting the gene for either gD, VP11/12,
VP13/14, VP22 or a combination thereof along with a peptide
fragment of the protein. The antigen is presented via class I MHC
molecules to the cellular immune system while the peptide is
presented via class II MHC molecules to stimulate helper T cells.
In any case, this method provides a way to stimulate and modulate
the immune response in a way which has not previously been
possible.
Liposome-Forming Materials
[0103] Liposomes are unilamellar or multilamellar vesicles, having
a membrane portion formed of lipophilic material and an interior
aqueous portion. The aqueous portion is used in the present
invention to contain the polynucleotide material to be delivered to
the target cell. It is preferred that the liposome forming
materials used herein have a cationic group, such as a quaternary
ammonium group, and one or more lipophilic groups, such as
saturated or unsaturated alkyl groups having from about 6 to about
30 carbon atoms. One group of suitable materials is described in
European Patent Publication No. 0187702 and is incorporated herein
by reference. These compounds may be prepared as detailed in the
above-identified patent application; alternatively, at least one of
these compounds,
N-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammonium
chloride (DOTMA), is commercially available from Bethesda Research
Laboratories (BRL), Gaithersburg, Md. 20877, USA.
[0104] Moreover, many suitable liposome-forming cationic lipid
compounds are described in the literature. See, e.g., L.
Stamatatos, et al., Biochemistry, 27:3917-3925 (1988); H. Eibl, et
al, Biophysical Chemistry, 10:261-271 (1979).
Liposome Preparation
[0105] Suitable liposomes for use in the present invention are
commercially available. DOTMA liposomes, for example, are available
under the trademark Lipofectin from Bethesda Research Labs,
Gaithersburg, Md.
[0106] Alternatively, liposomes can be prepared from
readily-available or freshly synthesized starting materials of the
type previously described. Preparation of DOTMA liposomes is
explained in the literature, see, e.g., p. Felgner, et al., Proc.
Nat'l Acad. Sci. USA, 84:7413-7417. Similar methods can be used to
prepare liposomes from other cationic lipid materials. Moreover,
conventional liposome forming materials can be used to prepare
liposomes having negative charge or neutral charge. Such materials
include phosphatidylcholine, cholesterol,
phosphatidyl-ethanolamine, and the like. These materials can also
advantageously be mixed with the DOTAP or DOTMA starting materials
in ratios from 0% to about 75%.
[0107] Conventional methods can be used to prepare other,
noncationic liposomes. These liposomes do not fuse with cell walls
as readily as cationic liposomes. However, they are taken up by
macrophages in vivo, and are thus particularly effective for
delivery of polynucleotide to these cells. For example,
commercially dioleoyl-phosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl
ethanolamine (DOPE) can be used in various combinations to make
conventional liposomes, with or without the addition of
cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared
by drying 50 mg each of DOPG and DOPC under a stream of nitrogen
gas into a sonication vial. The sample is placed under a vacuum
pump overnight and is hydrated the following day with deionized
water. The sample is then sonicated for 2 hours in a capped vial,
using a Heat Systems model 350 sonicator equipped with an inverted
cup (bath type) probe at the maximum setting while the bath is
circulated at 15.degree. C. Alternatively, negatively charged
vesicles can be prepared without sonication to produce
multilamellar vesicles or by extrusion through nucleopore membranes
to produce unilamellar vesicles of discrete size. Other methods are
known and available to those of skill in the art.
Therapeutic Formulations Polynucleotide Salts
[0108] Administration of pharmaceutically acceptable salts of the
polynucleotides described herein is included within the scope of
the invention. Such salts may be prepared from pharmaceutically
acceptable non-toxic bases including organic bases and inorganic
bases. Salts derived from inorganic bases include sodium,
potassium, lithium, ammonium, calcium, magnesium, and the like.
Salts derived from pharmaceutically acceptable organic non-toxic
bases include salts of primary, secondary, and tertiary amines,
basic amino acids, and the like. For a helpful discussion of
pharmaceutical salts, see S. M. Berge et al., Journal of
Pharmaceutical Sciences 66:1-19 (1977).
[0109] Polynucleotides for injection, a preferred route of
delivery, may be prepared in unit dosage form in ampules, or in
multidose containers. The polynucleotides may be present in such
forms as suspensions, solutions, or emulsions in oily or preferably
aqueous vehicles. Alternatively, the polynucleotide salt may be in
lyophilized form for reconstitution, at the time of delivery, with
a suitable vehicle, such as sterile pyrogen-free water. Both liquid
as well as lyophilized forms that are to be reconstituted will
comprise agents, preferably buffers, in amounts necessary to
suitably adjust the pH of the injected solution. For any parenteral
use, particularly if the formulation is to be administered
intravenously, the total concentration of solutes should be
controlled to make the preparation isotonic, hypotonic, or weakly
hypertonic. Nonionic materials, such as sugars, are preferred for
adjusting tonicity, and sucrose is particularly preferred. Any of
these forms may further comprise suitable formulatory agents, such
as starch or sugar, glycerol or saline. The compositions per unit
dosage, whether liquid or solid, may contain from 0.1% to 99% of
polynucleotide material.
[0110] The units dosage ampules or multidose containers, in which
the polynucleotides are packaged prior to use, may comprise an
hermetically sealed container enclosing an amount of polynucleotide
or solution containing a polynucleotide suitable for a
pharmaceutically effective dose thereof, or multiples of an
effective dose. The polynucleotide is packaged as a sterile
formulation, and the hermetically sealed container is designed to
preserve sterility of the formulation until use.
[0111] The container in which the polynucleotide is packaged is
labeled, and the label bears a notice in the form prescribed by a
governmental agency, for example the Food and Drug Administration,
which notice is reflective of approval by the agency under Federal
law, of the manufacture, use, or sale of the polynucleotide
material therein for human administration.
[0112] Federal law requires that the use of pharmaceutical agents
in the therapy of humans be approved by an agency of the Federal
government. Responsibility for enforcement is the responsibility of
the Food and Drug Administration, which issues appropriate
regulations for securing such approval, detailed in 21 U.S.C.
.sctn..sctn.301-392. Regulation for biologic material, comprising
products made from the tissues of animals is provided under 42
U.S.C. .sctn.262. Similar approval is required by most foreign
countries. Regulations vary from country to country, but the
individual procedures are well known to those in the art.
Dosage and Route of Administration
[0113] The dosage to be administered depends to a large extent on
the condition and size of the subject being treated as well as the
frequency of treatment and the route of administration. Regimens
for continuing therapy, including dose and frequency may be guided
by the initial response and clinical judgment. The parenteral route
of injection into the interstitial space of tissues is preferred,
although other parenteral routes, such as inhalation of an aerosol
formulation, may be required in specific administration, as for
example to the mucous membranes of the nose, throat, bronchial
tissues or lungs.
[0114] In preferred protocols, a formulation comprising the naked
polynucleotide in an aqueous carrier is injected into tissue in
amounts of from 10 .mu.l per site to about 1 ml per site. The
concentration of polynucleotide in the formulation is from about
0.1 .mu.g/ml to about 20 mg/ml.
[0115] The present invention is directed to compositions and
methods for enhancing the immune response of a vertebrate in need
of protection against herpes simplex virus infection by
administering in vivo, into a tissue of a vertebrate, at least one
polynucleotide comprising one or more nucleic acid fragments, where
each nucleic acid fragment is optionally a fragment of a
codon-optimized coding region operably encoding a herpes simplex
virus polypeptide, or a fragment, variant, or derivative thereof in
cells of the vertebrate in need of protection. The present
invention is also directed to administering in vivo, into a tissue
of the vertebrate the above described polynucleotide and at least
one isolated herpes simplex virus polypeptide, or a fragment,
variant, or derivative thereof. The isolated herpes simplex virus
polypeptide or fragment, variant, or derivative thereof can be, for
example, a recombinant protein, a purified subunit protein, a
protein expressed and carried by a heterologous live or inactivated
or attentuated viral vector expressing the protein, or can be an
inactivated herpes simplex virus, such as those present in
conventional, commercially available, inactivated herpes simplex
virus vaccines. According to either method, the polynucleotide is
incorporated into the cells of the vertebrate in vivo, and an
immunologically effective amount of the herpes simplex protein, or
fragment or variant encoded by the polynucleotide is produced in
vivo. The isolated protein or fragment, variant, or derivative
thereof is also administered in an immunologically effective
amount. The polynucleotide can be administered to the vertebrate in
need thereof either prior to, at the same time (simultaneously), or
subsequent to the administration of the isolated herpes simplex
virus polypeptide or fragment, variant, or derivative thereof.
[0116] Non-limiting examples of herpes simplex virus polypeptides
within the scope of the invention include, but are not limited to,
gD, VP 11/12, VP13/14 and/or VP22 polypeptides, and fragments,
derivatives, and variants thereof. Nucleotide and amino acid
sequences of herpes simplex virus polypeptides from a wide variety
of herpes simplex virus types and subtypes are known in the
art.
[0117] The present invention also provides vaccine compositions and
methods for delivery of herpes simplex virus coding sequences to a
vertebrate with optimal expression and safety conferred through
codon optimization and/or other manipulations. These vaccine
compositions are prepared and administered in such a manner that
the encoded gene products are optimally expressed in the vertebrate
of interest. As a result, these compositions and methods are useful
in stimulating an immune response against herpes simplex virus
infection. Also included in the invention are expression systems,
delivery systems, and codon-optimized herpes simplex virus coding
regions.
[0118] In a specific embodiment, the invention provides
combinatorial polynucleotide (e.g., DNA) vaccines which combine
both a polynucleotide vaccine and polypeptide (e.g., either a
recombinant protein, a purified subunit protein, a viral vector
expressing an isolated herpes simplex virus polypeptide, or in the
form of an inactivated or attenuated herpes simplex virus vaccine)
vaccine in a single formulation. The single formulation comprises a
herpes simplex virus polypeptide-encoding polynucleotide vaccine as
described herein, and optionally, an effective amount of a desired
isolated herpes simplex virus polypeptide or fragment, variant, or
derivative thereof. The polypeptide may exist in any form, for
example, a recombinant protein, a purified subunit protein, a viral
vector expressing an isolated herpes simplex virus polypeptide, or
in the form of an inactivated or attenuated herpes simplex virus
vaccine. The herpes simplex virus polypeptide or fragment, variant,
or derivative thereof encoded by the polynucleotide vaccine may be
identical to the isolated herpes simplex virus polypeptide or
fragment, variant, or derivative thereof. Alternatively, the herpes
simplex virus polypeptide or fragment, variant, or derivative
thereof encoded by the polynucleotide may be different from the
isolated herpes simplex virus polypeptide or fragment, variant, or
derivative thereof.
[0119] It is to be noted that the term "a" or "an" entity refers to
one or more of that entity, for example, "a polynucleotide," is
understood to represent one or more polynucleotides. As such, the
terms "a" (or "an"), "one or more," and "at least one" can be used
interchangeably herein.
[0120] The term "polynucleotide" is intended to encompass a
singular nucleic acid or nucleic acid fragment as well as plural
nucleic acids or nucleic acid fragments, and refers to an isolated
molecule or construct, e.g., a virus genome (e.g., a non-infectious
viral genome), messenger RNA (mRNA), plasmid DNA (pDNA), or
derivatives of pDNA (e.g., minicircles as described in (Darquet,
A-M et al., Gene Therapy 4:1341-1349 (1997)) comprising a
polynucleotide. A polynucleotide may comprise a conventional
phosphodiester bond or a non-conventional bond (e.g., an amide
bond, such as found in peptide nucleic acids (PNA)).
[0121] The terms "nucleic acid" or "nucleic acid fragment" refer to
any one or more nucleic acid segments, e.g., DNA or RNA fragments,
present in a polynucleotide or construct. A nucleic acid or
fragment thereof may be provided in linear (e.g., mRNA) or circular
(e.g., plasmid) form as well as double-stranded or single-stranded
forms. By "isolated" nucleic acid or polynucleotide is intended a
nucleic acid molecule, DNA or RNA, which has been removed from its
native environment. For example, a recombinant polynucleotide
contained in a vector is considered isolated for the purposes of
the present invention. Further examples of an isolated
polynucleotide include recombinant polynucleotides maintained in
heterologous host cells or purified (partially or substantially)
polynucleotides in solution. Isolated RNA molecules include in vivo
or in vitro RNA transcripts of the polynucleotides of the present
invention. Isolated polynucleotides or nucleic acids according to
the present invention further include such molecules produced
synthetically.
[0122] As used herein, a "coding region" is a portion of nucleic
acid which consists of codons translated into amino acids. Although
a "stop codon" (TAG, TGA, or TAA) is not translated into an amino
acid, it may be considered to be part of a coding region, but any
flanking sequences, for example promoters, ribosome binding sites,
transcriptional terminators, and the like, are not part of a coding
region. Two or more nucleic acids or nucleic acid fragments of the
present invention can be present in a single polynucleotide
construct, e.g., on a single plasmid, or in separate polynucleotide
constructs, e.g., on separate (different) plasmids. Furthermore,
any nucleic acid or nucleic acid fragment may encode a single
herpes simplex virus polypeptide or fragment, derivative, or
variant thereof, e.g., or may encode more than one polypeptide,
e.g., a nucleic acid may encode two or more polypeptides. In
addition, a nucleic acid may include a regulatory element such as a
promoter, ribosome binding site, or a transcription terminator, or
may encode heterologous coding regions fused to the herpes simplex
virus coding region, e.g., specialized elements or motifs, such as
a secretory signal peptide or a heterologous functional domain.
[0123] The terms "fragment," "variant," "derivative" and "analog"
when referring to herpes simplex virus polypeptides of the present
invention include any polypeptides which retain at least some of
the immunogenicity or antigenicity of the corresponding native
polypeptide. Fragments of herpes simplex virus polypeptides of the
present invention include proteolytic fragments, deletion fragments
and in particular, fragments of herpes simplex virus polypeptides
which exhibit increased secretion from the cell or higher
immunogenicity or reduced pathogenicity when delivered to an
animal. Polypeptide fragments further include any portion of the
polypeptide which comprises an antigenic or immunogenic epitope of
the native polypeptide, including linear as well as
three-dimensional epitopes. Variants of herpes simplex virus
polypeptides of the present invention include fragments as
described above, and also polypeptides with altered amino acid
sequences due to amino acid substitutions, deletions, or
insertions. Variants may occur naturally, such as an allelic
variant. By an "allelic variant" is intended alternate forms of a
gene occupying a given locus on a chromosome or genome of an
organism or virus. Genes II, Lewin, B., ed., John Wiley & Sons,
New York (1985). For example, as used herein, variations in a given
gene product. When referring to herpes simplex virus gD, VP 11/12,
VP13/14 and/or VP22 proteins, each such protein is a "variant," in
that native herpes simplex virus strains are distinguished by the
type of proteins encoded by the virus. However, within a single gD,
VP 11/12, VP13/14 and/or VP22 variant type, further naturally or
non-naturally occurring variations such as amino acid deletions,
insertions or substitutions may occur. Non-naturally occurring
variants may be produced using art-known mutagenesis techniques.
Variant polypeptides may comprise conservative or non-conservative
amino acid substitutions, deletions or additions. Derivatives of
herpes simplex virus polypeptides of the present invention, are
polypeptides which have been altered so as to exhibit additional
features not found on the native polypeptide. Examples include
fusion proteins. An analog is another form of a herpes simplex
virus polypeptide of the present invention. An example is a
proprotein which can be activated by cleavage of the proprotein to
produce an active mature polypeptide.
[0124] The terms "infectious polynucleotide" or "infectious nucleic
acid" are intended to encompass isolated viral polynucleotides
and/or nucleic acids which are solely sufficient to mediate the
synthesis of complete infectious virus particles upon uptake by
permissive cells. Thus, "infectious nucleic acids" do not require
pre-synthesized copies of any of the polypeptides it encodes, e.g.,
viral replicases, in order to initiate its replication cycle in a
permissive host cell.
[0125] The terms "non-infectious polynucleotide" or "non-infectious
nucleic acid" as defined herein are polynucleotides or nucleic
acids which cannot, without additional added materials, e.g.,
polypeptides, mediate the synthesis of complete infectious virus
particles upon uptake by permissive cells. An infectious
polynucleotide or nucleic acid is not made "non-infectious" simply
because it is taken up by a non-permissive cell. For example, an
infectious viral polynucleotide from a virus with limited host
range is infectious if it is capable of mediating the synthesis of
complete infectious virus particles when taken up by cells derived
from a permissive host (i.e., a host permissive for the virus
itself). The fact that uptake by cells derived from a
non-permissive host does not result in the synthesis of complete
infectious virus particles does not make the nucleic acid
"non-infectious." In other words, the term is not qualified by the
nature of the host cell, the tissue type, or the species taking up
the polynucleotide or nucleic acid fragment.
[0126] In some cases, an isolated infectious polynucleotide or
nucleic acid may produce fully-infectious virus particles in a host
cell population which lacks receptors for the virus particles,
i.e., is non-permissive for virus entry. Thus viruses produced will
not infect surrounding cells. However, if the supernatant
containing the virus particles is transferred to cells which are
permissive for the virus, infection will take place.
[0127] The terms "replicating polynucleotide" or "replicating
nucleic acid" are meant to encompass those polynucleotides and/or
nucleic acids which, upon being taken up by a permissive host cell,
are capable of producing multiple, e.g., one or more copies of the
same polynucleotide or nucleic acid. Infectious polynucleotides and
nucleic acids are a subset of replicating polynucleotides and
nucleic acids; the terms are not synonymous. For example, a
defective virus genome lacking the genes for virus coat proteins
may replicate, e.g., produce multiple copies of itself, but is NOT
infectious because it is incapable of mediating the synthesis of
complete infectious virus particles unless the coat proteins, or
another nucleic acid encoding the coat proteins, are exogenously
provided.
[0128] In certain embodiments, the polynucleotide, nucleic acid, or
nucleic acid fragment is DNA. In the case of DNA, a polynucleotide
comprising a nucleic acid which encodes a polypeptide normally also
comprises a promoter and/or other transcription or translation
control elements operably associated with the polypeptide-encoding
nucleic acid fragment. An operable association is when a nucleic
acid fragment encoding a gene product, e.g., a polypeptide, is
associated with one or more regulatory sequences in such a way as
to place expression of the gene product under the influence or
control of the regulatory sequence(s). Two DNA fragments (such as a
polypeptide-encoding nucleic acid fragment and a promoter
associated with the 5' end of the nucleic acid fragment) are
"operably associated" if induction of promoter function results in
the transcription of mRNA encoding the desired gene product and if
the nature of the linkage between the two DNA fragments does not
(1) result in the introduction of a frame-shift mutation, (2)
interfere with the ability of the expression regulatory sequences
to direct the expression of the gene product, or (3) interfere with
the ability of the DNA template to be transcribed. Thus, a promoter
region would be operably associated with a nucleic acid fragment
encoding a polypeptide if the promoter was capable of effecting
transcription of that nucleic acid fragment. The promoter may be a
cell-specific promoter that directs substantial transcription of
the DNA only in predetermined cells. Other transcription control
elements, besides a promoter, for example enhancers, operators,
repressors, and transcription termination signals, can be operably
associated with the polynucleotide to direct cell-specific
transcription. Suitable promoters and other transcription control
regions are disclosed herein.
[0129] A variety of transcription control regions are known to
those skilled in the art. These include, without limitation,
transcription control regions which function in vertebrate cells,
such as, but not limited to, promoter and enhancer segments from
cytomegaloviruses (the immediate early promoter, in conjunction
with intron-A), simian virus 40 (the early promoter), and
retroviruses (such as Rous sarcoma virus). Other transcription
control regions include those derived from vertebrate genes such as
actin, heat shock protein, bovine growth hormone and rabbit
.beta.-globin, as well as other sequences capable of controlling
gene expression in eukaryotic cells. Additional suitable
transcription control regions include tissue-specific promoters and
enhancers as well as lymphokine-inducible promoters (e.g.,
promoters inducible by interferons or interleukins).
[0130] Similarly, a variety of translation control elements are
known to those of ordinary skill in the art. These include, but are
not limited to ribosome binding sites, translation initiation and
termination codons, elements from picornaviruses (particularly an
internal ribosome entry site, or IRES, also referred to as a CITE
sequence).
[0131] A DNA polynucleotide of the present invention may be a
circular or linearized plasmid or vector, or other linear DNA which
may also be non-infectious and nonintegrating (i.e., does not
integrate into the genome of vertebrate cells). A linearized
plasmid is a plasmid that was previously circular but has been
linearized, for example, by digestion with a restriction
endonuclease. Linear DNA may be advantageous in certain situations
as discussed, e.g., in Cherng, J. Y., et al., J. Control. Release
60:343-53 (1999), and Chen, Z. Y., et al. Mol. Ther. 3:403-10
(2001). As used herein, the terms plasmid and vector can be used
interchangeably.
[0132] Alternatively, DNA virus genomes may be used to administer
DNA polynucleotides into vertebrate cells. In certain embodiments,
a DNA virus genome of the present invention is nonreplicative,
noninfectious, and/or nonintegrating. Suitable DNA virus genomes
include without limitation, herpes simplex virus genomes,
adenovirus genomes, adeno-associated virus genomes, and poxvirus
genomes. References citing methods for the in vivo introduction of
non-infectious virus genomes to vertebrate tissues are well known
to those of ordinary skill in the art.
[0133] In other embodiments, a polynucleotide of the present
invention is RNA, for example, in the form of messenger RNA (mRNA).
Methods for introducing RNA sequences into vertebrate cells are
described in U.S. Pat. No. 5,580,859.
[0134] Polynucleotides, nucleic acids, and nucleic acid fragments
of the present invention may be associated with additional nucleic
acids which encode secretory or signal peptides, which direct the
secretion of a polypeptide encoded by a nucleic acid fragment or
polynucleotide of the present invention. According to the signal
hypothesis, proteins secreted by mammalian cells have a signal
peptide or secretory leader sequence which is cleaved from the
mature protein once export of the growing protein chain across the
rough endoplasmic reticulum has been initiated. Those of ordinary
skill in the art are aware that polypeptides secreted by vertebrate
cells generally have a signal peptide fused to the N-terminus of
the polypeptide, which is cleaved from the complete or "full
length" polypeptide to produce a secreted or "mature" form of the
polypeptide. In certain embodiments, the native leader sequence is
used, or a functional derivative of that sequence that retains the
ability to direct the secretion of the polypeptide that is operably
associated with it. Alternatively, a heterologous mammalian leader
sequence, or a functional derivative thereof, may be used. For
example, the wild-type leader sequence may be substituted with the
leader sequence of human tissue plasminogen activator (TPA) or
mouse .beta.-glucuronidase.
[0135] In accordance with one aspect of the present invention,
there is provided a polynucleotide construct, for example, a
plasmid, comprising a nucleic acid fragment, where the nucleic acid
fragment is a fragment of a codon-optimized coding region operably
encoding a herpes simplex virus-derived polypeptide, where the
coding region is optimized for expression in vertebrate cells, of a
desired vertebrate species, e.g., humans, to be delivered to a
vertebrate to be treated or immunized. Suitable herpes simplex
virus polypeptides, or fragments, variants, or derivatives thereof
may be derived from, but are not limited to, the herpes simplex
virus gD, VP 11/12, VP13/14 and/or VP22 proteins. Additional herpes
simplex virus-derived coding sequences, may also be included on the
plasmid, or on a separate plasmid, and expressed, either using
native herpes simplex virus codons or codons optimized for
expression in the vertebrate to be treated or immunized. When such
a plasmid encoding one or more optimized herpes simplex sequences
is delivered, in vivo to a tissue of the vertebrate to be treated
or immunized, one or more of the encoded gene products will be
expressed, i.e., transcribed and translated. The level of
expression of the gene product(s) will depend to a significant
extent on the strength of the associated promoter and the presence
and activation of an associated enhancer element, as well as the
degree of optimization of the coding region.
[0136] As used herein, the term "plasmid" refers to a construct
made up of genetic material (i.e., nucleic acids). Typically a
plasmid contains an origin of replication which is functional in
bacterial host cells, e.g., Escherichia coli, and selectable
markers for detecting bacterial host cells comprising the plasmid.
Plasmids of the present invention may include genetic elements as
described herein arranged such that an inserted coding sequence can
be transcribed and translated in eukaryotic cells. Also, the
plasmid may include a sequence from a viral nucleic acid. However,
such viral sequences normally are not sufficient to direct or allow
the incorporation of the plasmid into a viral particle, and the
plasmid is therefore a non-viral vector. In certain embodiments
described herein, a plasmid is a closed circular DNA molecule.
[0137] The term "expression" refers to the biological production of
a product encoded by a coding sequence. In most cases a DNA
sequence, including the coding sequence, is transcribed to form a
messenger-RNA (mRNA). The messenger-RNA is then translated to form
a polypeptide product which has a relevant biological activity.
Also, the process of expression may involve further processing
steps to the RNA product of transcription, such as splicing to
remove introns, and/or post-translational processing of a
polypeptide product.
[0138] As used herein, the term "polypeptide" is intended to
encompass a singular "polypeptide" as well as plural
"polypeptides," and comprises any chain or chains of two or more
amino acids. Thus, as used herein, terms including, but not limited
to "peptide," "dipeptide," "tripeptide," "protein," "amino acid
chain," or any other term used to refer to a chain or chains of two
or more amino acids, are included in the definition of a
"polypeptide," and the term "polypeptide" can be used instead of,
or interchangeably with any of these terms. The term further
includes polypeptides which have undergone post-translational
modifications, for example, glycosylation, acetylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, or modification
by non-naturally occurring amino acids.
[0139] Also included as polypeptides of the present invention are
fragments, derivatives, analogs, or variants of the foregoing
polypeptides, and any combination thereof. Polypeptides, and
fragments, derivatives, analogs, or variants thereof of the present
invention can be antigenic and immunogenic polypeptides related to
herpes simplex virus polypeptides, which are used to prevent or
treat, i.e., cure, ameliorate, lessen the severity of, or prevent
or reduce contagion of infectious disease caused by the herpes
simplex virus.
[0140] As used herein, an "antigenic polypeptide" or an
"immunogenic polypeptide" is a polypeptide which, when introduced
into a vertebrate, reacts with the vertebrate's immune system
molecules, i.e., is antigenic, and/or induces an immune response in
the vertebrate, i.e., is immunogenic. It is quite likely that an
immunogenic polypeptide will also be antigenic, but an antigenic
polypeptide, because of its size or conformation, may not
necessarily be immunogenic. Examples of antigenic and immunogenic
polypeptides of the present invention include, but are not limited
to, e.g., gD, VP 11/12, VP13/14 and/or VP22 or fragments or
variants thereof, or any of the foregoing polypeptides or fragments
fused to a heterologous polypeptide, for example, a hepatitis B
core antigen. Isolated antigenic and immunogenic polypeptides of
the present invention in addition to those encoded by
polynucleotides of the invention, may be provided as a recombinant
protein, a purified subunit, a viral vector expressing the protein,
or may be provided in the form of an inactivated herpes simplex
virus vaccine, e.g., a live-attenuated virus vaccine, a heat-killed
virus vaccine, etc.
[0141] Immunospecific binding excludes non-specific binding but
does not exclude cross-reactivity with other antigens. Where all
immunogenic epitopes are antigenic, antigenic epitopes need not be
immunogenic.
[0142] By an "isolated" herpes simplex virus polypeptide or a
fragment, variant, or derivative thereof is intended a herpes
simplex virus polypeptide or protein that is not in its natural
form. No particular level of purification is required. For example,
an isolated herpes simplex virus polypeptide can be removed from
its native or natural environment. Recombinantly produced herpes
simplex virus polypeptides and proteins expressed in host cells are
considered isolated for purposed of the invention, as are native or
recombinant herpes simplex virus polypeptides which have been
separated, fractionated, or partially or substantially purified by
any suitable technique, including the separation of herpes simplex
virus virions from eggs or culture cells in which they have been
propagated. In addition, an isolated herpes simplex virus
polypeptide or protein can be provided as a live or inactivated
viral vector expressing an isolated herpes simplex virus
polypeptide and can include those found in inactivated herpes
simplex virus vaccine compositions. Thus, isolated herpes simplex
virus polypeptides and proteins can be provided as, for example,
recombinant herpes simplex virus polypeptides, a purified subunit
of herpes simplex virus, a viral vector expressing an isolated
herpes simplex virus polypeptide, or in the form of an inactivated
or attenuated herpes simplex virus vaccine.
[0143] The term "epitopes," as used herein, refers to portions of a
polypeptide having antigenic or immunogenic activity in a
vertebrate, for example a human. An "immunogenic epitope," as used
herein, is defined as a portion of a protein that elicits an immune
response in an animal, as determined by any method known in the
art. The term "antigenic epitope," as used herein, is defined as a
portion of a protein to which an antibody or T-cell receptor can
immunospecifically bind as determined by any method well known in
the art.
[0144] The term "immunogenic carrier" as used herein refers to a
first polypeptide or fragment, variant, or derivative thereof which
enhances the immunogenicity of a second polypeptide or fragment,
variant, or derivative thereof. Typically, an "immunogenic carrier"
is fused to or conjugated to the desired polypeptide or fragment
thereof. An example of an "immunogenic carrier" is a recombinant
hepatitis B core antigen expressing, as a surface epitope, an
immunogenic epitope of interest. See, e.g., European Patent No. EP
0385610 B1.
[0145] In the present invention, antigenic epitopes preferably
contain a sequence of at least 4, at least 5, at least 6, at least
7, at least 8, at least 9, at least 10, at least 15, at least 20,
at least 25, or between about 8 to about 30 amino acids contained
within the amino acid sequence of a herpes simplex virus
polypeptide of the invention, e.g., an gD, VP 11/12, VP13/14 and/or
VP22 polypeptide. Certain polypeptides comprising immunogenic or
antigenic epitopes are at least 5, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues
in length. Antigenic as well as immunogenic epitopes may be linear,
i.e., be comprised of contiguous amino acids in a polypeptide, or
may be three dimensional, i.e., where an epitope is comprised of
non-contiguous amino acids which come together due to the secondary
or tertiary structure of the polypeptide, thereby forming an
epitope.
[0146] As to the selection of peptides or polypeptides bearing an
antigenic epitope (e.g., that contain a region of a protein
molecule to which an antibody or T cell receptor can bind), it is
well known in that art that relatively short synthetic peptides
that mimic part of a protein sequence are routinely capable of
eliciting an antiserum that reacts with the partially mimicked
protein. See, e.g., Sutcliffe, J. G., et al., Science 219:660-666
(1983).
[0147] Peptides capable of eliciting an immunogenic response are
frequently represented in the primary sequence of a protein, can be
characterized by a set of simple chemical rules, and are confined
neither to immunodominant regions of intact proteins nor to the
amino or carboxyl terminals. Peptides that are extremely
hydrophobic and those of six or fewer residues generally are
ineffective at inducing antibodies that bind to the mimicked
protein; longer peptides, especially those containing proline
residues, usually are effective. Sutcliffe et al., supra, at
661.
Codon Optimization
[0148] "Codon optimization" is defined as modifying a nucleic acid
sequence for enhanced expression in the cells of the vertebrate of
interest, e.g. human, by replacing at least one, more than one, or
a significant number, of codons of the native sequence with codons
that are more frequently or most frequently used in the genes of
that vertebrate. Various species exhibit particular bias for
certain codons of a particular amino acid.
[0149] In one aspect, the present invention relates to
polynucleotides comprising nucleic acid fragments of
codon-optimized coding regions which encode herpes simplex virus
polypeptides, or fragments, variants, or derivatives thereof, with
the codon usage adapted for optimized expression in the cells of a
given vertebrate, e.g., humans. These polynucleotides are prepared
by incorporating codons preferred for use in the genes of the
vertebrate of interest into the DNA sequence. Also provided are
polynucleotide expression constructs, vectors, and host cells
comprising nucleic acid fragments of codon-optimized coding regions
which encode herpes simplex virus polypeptides, and fragments,
variants, or derivatives thereof, and various methods of using the
polynucleotide expression constructs, vectors, host cells to treat
or prevent herpes simplex disease in a vertebrate.
[0150] As used herein the term "codon-optimized coding region"
means a nucleic acid coding region that has been adapted for
expression in the cells of a given vertebrate by replacing at least
one, or more than one, or a significant number, of codons with one
or more codons that are more frequently used in the genes of that
vertebrate.
[0151] Deviations in the nucleotide sequence that comprise the
codons encoding the amino acids of any polypeptide chain allow for
variations in the sequence coding for the gene. Since each codon
consists of three nucleotides, and the nucleotides comprising DNA
are restricted to four specific bases, there are 64 possible
combinations of nucleotides, 61 of which encode amino acids (the
remaining three codons encode signals ending translation (stop or
termination)). The "genetic code" which shows which codons encode
which amino acids is reproduced herein as Table 1. As a result,
many amino acids are designated by more than one codon. For
example, the amino acids alanine and proline are coded for by four
triplets, serine and arginine by six, whereas tryptophan and
methionine are coded by just one triplet. This degeneracy allows
for DNA base composition to vary over a wide range without altering
the amino acid sequence of the proteins encoded by the DNA.
TABLE-US-00001 TABLE 1 The Standard Genetic Code T(U) C A G T(U)
TTT Phe (F) TCT Ser (S) TAT Tyr (Y) TGT Cys (C) TTC Phe TCC Ser TAC
Tyr TGC Cys TTA Leu (L) TCA Ser TAA Ter TGA Ter TTG Leu TCG Ser TAG
Ter TGG Trp (W) C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R)
CTC Leu CCC Pro CAC His CGC Arg CTA Leu CCA Pro CAA Gln (Q) CGA Arg
CTG Leu CCG Pro CAG Gln CGG Arg A ATT Ile (I) ACT Thr (T) AAT Asn
(N) AGT Ser (S) ATC Ile ACC Thr AAC Asn AGC Ser ATA Ile ACA Thr AAA
Lys (K) AGA Arg (R) ATG Met (M) ACG Thr AAG Lys AGG Arg G GTT Val
(V) GCT Ala (A) GAT Asp (D) GGT Gly (G) GTC Val GCC Ala GAC Asp GGC
Gly GTA Val GCA Ala GAA Glu (E) GGA Gly GTG Val GCG Ala GAG Glu GGG
Gly
[0152] Many organisms display a bias for use of particular codons
to code for insertion of a particular amino acid in a growing
peptide chain. Codon preference or codon bias, differences in codon
usage between organisms, is afforded by degeneracy of the genetic
code, and is well documented among many organisms. Codon bias often
correlates with the efficiency of translation of messenger RNA
(mRNA), which is in turn believed to be dependent on, inter alia,
the properties of the codons being translated and the availability
of particular transfer RNA (tRNA) molecules. The predominance of
selected tRNAs in a cell is generally a reflection of the codons
used most frequently in peptide synthesis. Accordingly, genes can
be tailored for optimal gene expression in a given organism based
on codon optimization.
[0153] Given the large number of gene sequences available for a
wide variety of animal, plant and microbial species, it is possible
to calculate the relative frequencies of codon usage. Codon usage
tables are readily available, for example, at the "Codon Usage
Database" available on the world wide web at kazusa.or.jp/codon
(Jul. 9, 2002), and these tables can be adapted in a number of
ways. See Nakamura, Y., et al. "Codon usage tabulated from the
international DNA sequence databases: status for the year 2000"
Nucl. Acids Res. 28:292 (2000). As examples, the codon usage tables
for human, mouse, domestic cat, and cow, calculated from GenBank
Release 128.0 (15 Feb. 2002), are reproduced below as Tables 2-5.
These Tables use mRNA nomenclature, and so instead of thymine (T)
which is found in DNA, the Tables use uracil (U) which is found in
RNA. The Tables have been adapted so that frequencies are
calculated for each amino acid, rather than for all 64 codons.
TABLE-US-00002 TABLE 2 Codon Usage Table for Human Genes (Homo
sapiens) Amino Acid Codon Number Frequency Phe UUU 326146 0.4525
Phe UUC 394680 0.5475 Total 720826 Leu UUA 139249 0.0728 Leu UUG
242151 0.1266 Leu CUU 246206 0.1287 Leu CUC 374262 0.1956 Leu CUA
133980 0.0700 Leu CUG 777077 0.4062 Total 1912925 Ile AUU 303721
0.3554 Ile AUC 414483 0.4850 Ile AUA 136399 0.1596 Total 854603 Met
AUG 1430946 1.0000 Total 1430946 Val GUU 210423 0.1773 Val GUC
282445 0.2380 Val GUA 134991 0.1137 Val GUG 559044 0.4710 Total
1186903 Ser UCU 282407 0.1840 Ser UCC 336349 0.2191 Ser UCA 225963
0.1472 Ser UCG 86761 0.0565 Ser AGU 230047 0.1499 Ser AGC 373362
0.2433 Total 1534889 Pro CCU 333705 0.2834 Pro CCC 386462 0.3281
Pro CCA 322220 0.2736 Pro CCG 135317 0.1149 Total 1177704 Thr ACU
247913 0.2419 Thr ACC 371420 0.3624 Thr ACA 285655 0.2787 Thr ACG
120022 0.1171 Total 1025010 Ala GCU 360146 0.2637 Ala GCC 551452
0.4037 Ala GCA 308034 0.2255 Ala GCG 146233 0.1071 Total 1365865
Tyr UAU 232240 0.4347 Tyr UAC 301978 0.5653 Total 534218 His CAU
201389 0.4113 His CAC 288200 0.5887 Total 489589 Gln CAA 227742
0.2541 Gln CAG 668391 0.7459 Total 896133 Asn AAU 322271 0.4614 Asn
AAC 376210 0.5386 Total 698481 Lys AAA 462660 0.4212 Lys AAG 635755
0.5788 Total 1098415 Asp GAU 430744 0.4613 Asp GAC 502940 0.5387
Total 933684 Glu GAA 561277 0.4161 Glu GAG 787712 0.5839 Total
1348989 Cys UGU 190962 0.4468 Cys UGC 236400 0.5532 Total 427362
Trp UGG 248083 1.0000 Total 248083 Arg CGU 90899 0.0830 Arg CGC
210931 0.1927 Arg CGA 122555 0.1120 Arg CGG 228970 0.2092 Arg AGA
221221 0.2021 Arg AGG 220119 0.2011 Total 1094695 Gly GGU 209450
0.1632 Gly GGC 441320 0.3438 Gly GGA 315726 0.2459 Gly GGG 317263
0.2471 Total 1283759 Stop UAA 13963 Stop UAG 10631 Stop UGA
24607
TABLE-US-00003 TABLE 3 Codon Usage Table for Mouse Genes (Mus
musculus) Amino Acid Codon Number Frequency Phe UUU 150467 0.4321
Phe UUC 197795 0.5679 Total 348262 Leu UUA 55635 0.0625 Leu UUG
116210 0.1306 Leu CUU 114699 0.1289 Leu CUC 179248 0.2015 Leu CUA
69237 0.0778 Leu CUG 354743 0.3987 Total 889772 Ile AUU 137513
0.3367 Ile AUC 208533 0.5106 Ile AUA 62349 0.1527 Total 408395 Met
AUG 1204546 1.0000 Total 1204546 Val GUU 93754 0.1673 Val GUC
140762 0.2513 Val GUA 64417 0.1150 Val GUG 261308 0.4664 Total
560241 Ser UCU 139576 0.1936 Ser UCC 160313 0.2224 Ser UCA 100524
0.1394 Ser UCG 38632 0.0536 Ser AGU 108413 0.1504 Ser AGC 173518
0.2407 Total 720976 Pro CCU 162613 0.3036 Pro CCC 164796 0.3077 Pro
CCA 151091 0.2821 Pro CCG 57032 0.1065 Total 535532 Thr ACU 119832
0.2472 Thr ACC 172415 0.3556 Thr ACA 140420 0.2896 Thr ACG 52142
0.1076 Total 484809 Ala GCU 178593 0.2905 Ala GCC 236018 0.3839 Ala
GCA 139697 0.2272 Ala GCG 60444 0.0983 Total 614752 Tyr UAU 108556
0.4219 Tyr UAC 148772 0.5781 Total 257328 His CAU 88786 0.3973 His
CAC 134705 0.6027 Total 223491 Gln CAA 101783 0.2520 Gln CAG 302064
0.7480 Total 403847 Asn AAU 138868 0.4254 Asn AAC 187541 0.5746
Total 326409 Lys AAA 188707 0.3839 Lys AAG 302799 0.6161 Total
491506 Asp GAU 189372 0.4414 Asp GAC 239670 0.5586 Total 429042 Glu
GAA 235842 0.4015 Glu GAG 351582 0.5985 Total 587424 Cys UGU 97385
0.4716 Cys UGC 109130 0.5284 Total 206515 Trp UGG 112588 1.0000
Total 112588 Arg CGU 41703 0.0863 Arg CGC 86351 0.1787 Arg CGA
58928 0.1220 Arg CGG 92277 0.1910 Arg AGA 101029 0.2091 Arg AGG
102859 0.2129 Total 483147 Gly GGU 103673 0.1750 Gly GGC 198604
0.3352 Gly GGA 151497 0.2557 Gly GGG 138700 0.2341 Total 592474
Stop UAA 5499 Stop UAG 4661 Stop UGA 10356
TABLE-US-00004 TABLE 4 Codon Usage Table for Domestic Cat Genes
(Felis cattus) Frequency Amino Acid Codon Number of usage Phe UUU
1204.00 0.4039 Phe UUC 1777.00 0.5961 Total 2981 Leu UUA 404.00
0.0570 Leu UUG 857.00 0.1209 Leu CUU 791.00 0.1116 Leu CUC 1513.00
0.2135 Leu CUA 488.00 0.0688 Leu CUG 3035.00 0.4282 Total 7088 Ile
AUU 1018.00 0.2984 Ile AUC 1835.00 0.5380 Ile AUA 558.00 0.1636
Total 3411 Met AUG 1553.00 0.0036 Total 1553 Val GUU 696.00 0.1512
Val GUC 1279.00 0.2779 Val GUA 463.00 0.1006 Val GUG 2164.00 0.4702
Total 4602 Ser UCU 940.00 0.1875 Ser UCC 1260.00 0.2513 Ser UCA
608.00 0.1213 Ser UCG 332.00 0.0662 Ser AGU 672.00 0.1340 Ser AGC
1202.00 0.2397 Total 5014 Pro CCU 958.00 0.2626 Pro CCC 1375.00
0.3769 Pro CCA 850.00 0.2330 Pro CCG 465.00 0.1275 Total 3648 Thr
ACU 822.00 0.2127 Thr ACC 1574.00 0.4072 Thr ACA 903.00 0.2336 Thr
ACG 566.00 0.1464 Total 3865 Ala GCU 1129.00 0.2496 Ala GCC 1951.00
0.4313 Ala GCA 883.00 0.1952 Ala GCG 561.00 0.1240 Total 4524 Tyr
UAU 837.00 0.3779 Tyr UAC 1378.00 0.6221 Total 2215 His CAU 594.00
0.3738 His CAC 995.00 0.6262 Total 1589 Gln CAA 747.00 0.2783 Gln
CAG 1937.00 0.7217 Total 2684 Asn AAU 1109.00 0.3949 Asn AAC
1699.00 0.6051 Total 2808 Lys AAA 1445.00 0.4088 Lys AAG 2090.00
0.5912 Total 3535 Asp GAU 1255.00 0.4055 Asp GAC 1840.00 0.5945
Total 3095 Glu GAA 1637.00 0.4164 Glu GAG 2294.00 0.5836 Total 3931
Cys UGU 719.00 0.4425 Cys UGC 906.00 0.5575 Total 1625 Trp UGG
1073.00 1.0000 Total 1073 Arg CGU 236.00 0.0700 Arg CGC 629.00
0.1865 Arg CGA 354.00 0.1050 Arg CGG 662.00 0.1963 Arg AGA 712.00
0.2112 Arg AGG 779.00 0.2310 Total 3372 Gly GGU 648.00 0.1498 Gly
GGC 1536.00 0.3551 Gly GGA 1065.00 0.2462 Gly GGG 1077.00 0.2490
Total 4326 Stop UAA 55 Stop UAG 36 Stop UGA 110
TABLE-US-00005 TABLE 5 Codon Usage Table for Cow Genes (Bos taunts)
Frequency Amino Acid Codon Number of usage Phe UUU 13002 0.4112 Phe
UUC 18614 0.5888 Total 31616 Leu UUA 4467 0.0590 Leu UUG 9024
0.1192 Leu CUU 9069 0.1198 Leu CUC 16003 0.2114 Leu CUA 4608 0.0609
Leu CUG 32536 0.4298 Total 75707 Ile AUU 12474 0.3313 Ile AUC 19800
0.5258 Ile AUA 5381 0.1429 Total 37655 Met AUG 17770 1.0000 Total
17770 Val GUU 8212 0.1635 Val GUC 12846 0.2558 Val GUA 4932 0.0982
Val GUG 24222 0.4824 Total 50212 Ser UCU 10287 0.1804 Ser UCC 13258
0.2325 Ser UCA 7678 0.1347 Ser UCG 3470 0.0609 Ser AGU 8040 0.1410
Ser AGC 14279 0.2505 Total 57012 Pro CCU 11695 0.2684 Pro CCC 15221
0.3493 Pro CCA 11039 0.2533 Pro CCG 5621 0.1290 Total 43576 Thr ACU
9372 0.2203 Thr ACC 16574 0.3895 Thr ACA 10892 0.2560 Thr ACG 5712
0.1342 Total 42550 Ala GCU 13923 0.2592 Ala GCC 23073 0.4295 Ala
GCA 10704 0.1992 Ala GCG 6025 0.1121 Total 53725 Tyr UAU 9441
0.3882 Tyr UAC 14882 0.6118 Total 24323 His CAU 6528 0.3649 His CAC
11363 0.6351 Total 17891 Gln CAA 8060 0.2430 Gln CAG 25108 0.7570
Total 33168 Asn AAU 12491 0.4088 Asn AAC 18063 0.5912 Total 30554
Lys AAA 17244 0.3897 Lys AAG 27000 0.6103 Total 44244 Asp GAU 16615
0.4239 Asp GAC 22580 0.5761 Total 39195 Glu GAA 21102 0.4007 Glu
GAG 31555 0.5993 Total 52657 Cys UGU 7556 0.4200 Cys UGC 10436
0.5800 Total 17992 Trp UGG 10706 1.0000 Total 10706 Arg CGU 3391
0.0824 Arg CGC 7998 0.1943 Arg CGA 4558 0.1108 Arg CGG 8300 0.2017
Arg AGA 8237 0.2001 Arg AGG 8671 0.2107 Total 41155 Gly GGU 8508
0.1616 Gly GGC 18517 0.3518 Gly GGA 12838 0.2439 Gly GGG 12772
0.2427 Total 52635 Stop UAA 555 Stop UAG 394 Stop UGA 392
[0154] By utilizing these or similar tables, one of ordinary skill
in the art can apply the frequencies to any given polypeptide
sequence, and produce a nucleic acid fragment of a codon-optimized
coding region which encodes the polypeptide, but which uses codons
more optimal for a given species. Codon-optimized coding regions
can be designed by various different methods.
[0155] In another method, termed "full-optimization," the actual
frequencies of the codons are distributed randomly throughout the
coding region. Thus, using this method for optimization, if a
hypothetical polypeptide sequence had 100 leucine residues,
referring to Table 2 for frequency of usage in humans, about 7, or
7% of the leucine codons would be UUA, about 13, or 13% of the
leucine codons would be UUG, about 13, or 13% of the leucine codons
would be CUU, about 20, or 20% of the leucine codons would be CUC,
about 7, or 7% of the leucine codons would be CUA, and about 41, or
41% of the leucine codons would be CUG. These frequencies would be
distributed randomly throughout the leucine codons in the coding
region encoding the hypothetical polypeptide. As will be understood
by those of ordinary skill in the art, the distribution of codons
in the sequence can vary significantly using this method; however,
the sequence always encodes the same polypeptide.
[0156] In using the "full-optimization" method, an entire
polypeptide sequence may be codon-optimized as described above.
With respect to various desired fragments, variants or derivatives
of the complete polypeptide, the fragment variant, or derivative
may first be designed, and is then codon-optimized individually.
Alternatively, a full-length polypeptide sequence is
codon-optimized for a given species resulting in a codon-optimized
coding region encoding the entire polypeptide, and then nucleic
acid fragments of the codon-optimized coding region, which encode
fragments, variants, and derivatives of the polypeptide are made
from the original codon-optimized coding region. As would be well
understood by those of ordinary skill in the art, if codons have
been randomly assigned to the full-length coding region based on
their frequency of use in a given species, nucleic acid fragments
encoding fragments, variants, and derivatives would not necessarily
be fully codon-optimized for the given species. However, such
sequences are still much closer to the codon usage of the desired
species than the native codon usage. The advantage of this approach
is that synthesizing codon-optimized nucleic acid fragments
encoding each fragment, variant, and derivative of a given
polypeptide, although routine, would be time consuming and would
result in significant expense.
[0157] When using the "full-optimization" method, the term "about"
is used precisely to account for fractional percentages of codon
frequencies for a given amino acid. As used herein, "about" is
defined as one amino acid more or one amino acid less than the
value given. The whole number value of amino acids is rounded up if
the fractional frequency of usage is 0.50 or greater, and is
rounded down if the fractional frequency of use is 0.49 or less.
Using again the example of the frequency of usage of leucine in
human genes for a hypothetical polypeptide having 62 leucine
residues, the fractional frequency of codon usage would be
calculated by multiplying 62 by the frequencies for the various
codons. Thus, 7.28 percent of 62 equals 4.51 UUA codons, or "about
5," i.e., 4, 5, or 6 UUA codons, 12.66 percent of 62 equals 7.85
UUG codons or "about 8," i.e., 7, 8, or 9 TUG codons, 12.87 percent
of 62 equals 7.98 CUU codons, or "about 8," i.e., 7, 8, or 9 CTU
codons, 19.56 percent of 62 equals 12.13 CUC codons or "about 12,"
i.e., 11, 12, or 13 CUC codons, 7.00 percent of 62 equals 4.34 CUA
codons or "about 4," i.e., 3, 4, or 5 CUA codons, and 40.62 percent
of 62 equals 25.19 CUG codons, or "about 25," i.e., 24, 25, or 26
CUG codons.
[0158] In a third method termed "minimal optimization," coding
regions are only partially optimized. For example, the invention
includes a nucleic acid fragment of a codon-optimized coding region
encoding a polypeptide in which at least about 1%, 2%, 3%, 4%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95% or 100% of the codon positions have been
codon-optimized for a given species. That is, they contain a codon
that is preferentially used in the genes of a desired species,
e.g., a vertebrate species, e.g., humans, in place of a codon that
is normally used in the native nucleic acid sequence. Codons that
are rarely found in the genes of the vertebrate of interest are
changed to codons more commonly utilized in the coding regions of
the vertebrate of interest.
[0159] This minimal human codon optimization for highly variant
codons has several advantages, which include but are not limited to
the following examples. Since fewer changes are made to the
nucleotide sequence of the gene of interest, fewer manipulations
are required, which leads to reduced risk of introducing unwanted
mutations and lower cost, as well as allowing the use of
commercially available site-directed mutagenesis kits, and reducing
the need for expensive oligonucleotide synthesis. Further,
decreasing the number of changes in the nucleotide sequence
decreases the potential of altering the secondary structure of the
sequence, which can have a significant impact on gene expression in
certain host cells. The introduction of undesirable restriction
sites is also reduced, facilitating the subcloning of the genes of
interest into the plasmid expression vector.
[0160] The present invention also provides isolated polynucleotides
comprising coding regions of herpes simplex virus polypeptides,
e.g., gD, VP 11/12, VP13/14 and/or VP22 or fragments, variants, or
derivatives thereof. The isolated polynucleotides can also be
codon-optimized.
[0161] A human codon-optimized coding region can be designed by any
of the methods discussed herein. For "uniform" optimization, each
amino acid is assigned the most frequent codon used in the human
genome for that amino acid.
[0162] As described above, the term "about" means that the number
of amino acids encoded by a certain codon may be one more or one
less than the number given. It would be understood by those of
ordinary skill in the art that the total number of any amino acid
in the polypeptide sequence must remain constant, therefore, if
there is one "more" of one codon encoding a give amino acid, there
would have to be one "less" of another codon encoding that same
amino acid.
[0163] In another form of minimal optimization, a Codon Usage Table
(CUT) for the specific herpes simplex virus sequence in question is
generated and compared to CUT for human genomic DNA. Amino acids
are identified for which there is a difference of at least 10
percentage points in codon usage between human and herpes simplex
virus DNA (either more or less). Then the wild type herpes simplex
virus codon is modified to conform to predominant human codon for
each such amino acid. Furthermore, the remainder of codons for that
amino acid are also modified such that they conform to the
predominant human codon for each such amino acid.
Compositions and Methods
[0164] In certain embodiments, the present invention is directed to
compositions and methods of enhancing the immune response of a
vertebrate in need of protection against herpes simplex virus
infection by administering in vivo, into a tissue of a vertebrate,
one or more polynucleotides comprising at least one codon-optimized
coding region encoding a herpes simplex virus polypeptide, or a
fragment, variant, or derivative thereof. In addition, the present
invention is directed to compositions and methods of enhancing the
immune response of a vertebrate in need of protection against
herpes simplex virus infection by administering to the vertebrate a
composition comprising one or more polynucleotides as described
herein, and at least one isolated herpes simplex virus polypeptide,
or a fragment, variant, or derivative thereof. The polynucleotide
may be administered either prior to, at the same time
(simultaneously), or subsequent to the administration of the
isolated polypeptide.
[0165] The coding regions encoding herpes simplex virus
polypeptides or fragments, variants, or derivatives thereof may be
codon optimized for a particular vertebrate. Codon optimization is
carried out by the methods described herein, for example, in
certain embodiments codon-optimized coding regions encoding
polypeptides of herpes simplex virus, or nucleic acid fragments of
such coding regions encoding fragments, variants, or derivatives
thereof are optimized according to the codon usage of the
particular vertebrate. The polynucleotides of the invention are
incorporated into the cells of the vertebrate in vivo, and an
immunologically effective amount of a herpes simplex virus
polypeptide or a fragment, variant, or derivative thereof is
produced in vivo. The coding regions encoding a herpes simplex
virus polypeptide or a fragment, variant, or derivative thereof may
be codon optimized for mammals, e.g., humans, apes, monkeys (e.g.,
owl, squirrel, cebus, rhesus, African green, patas, cynomolgus, and
cercopithecus), orangutans, baboons, gibbons, and chimpanzees,
dogs, wolves, cats, lions, and tigers, horses, donkeys, zebras,
cows, pigs, sheep, deer, giraffes, bears, rabbits, mice, ferrets,
seals, whales; birds, e.g., ducks, geese, terns, shearwaters,
gulls, turkeys, chickens, quail, pheasants, geese, starlings and
budgerigars, or other vertebrates.
[0166] In one embodiment, the present invention relates to
codon-optimized coding regions encoding polypeptides of herpes
simplex virus, or nucleic acid fragments of such coding regions
fragments, variants, or derivatives thereof which have been
optimized according to human codon usage. For example, human
codon-optimized coding regions encoding polypeptides of herpes
simplex virus, or fragments, variants, or derivatives thereof are
prepared by substituting one or more codons preferred for use in
human genes for the codons naturally used in the DNA sequence
encoding the herpes simplex virus polypeptide or a fragment,
variant, or derivative thereof. Also provided are polynucleotides,
vectors, and other expression constructs comprising codon-optimized
coding regions encoding polypeptides of herpes simplex virus, or
nucleic acid fragments of such coding regions encoding fragments,
variants, or derivatives thereof, pharmaceutical compositions
comprising polynucleotides, vectors, and other expression
constructs comprising codon-optimized coding regions encoding
polypeptides of herpes simplex virus, or nucleic acid fragments of
such coding regions encoding fragments, variants, or derivatives
thereof, and various methods of using such polynucleotides, vectors
and other expression constructs. Coding regions encoding herpes
simplex virus polypeptides can be uniformly optimized, fully
optimized, minimally optimized, codon-optimized by region and/or
not codon-optimized, as described herein.
[0167] The present invention is further directed towards
polynucleotides comprising codon-optimized coding regions encoding
polypeptides of herpes simplex virus antigens, for example, gD, VP
11/12, VP13/14 and/or VP22 optionally in conjunction with other
antigens. The invention is also directed to polynucleotides
comprising codon-optimized nucleic acid fragments encoding
fragments, variants and derivatives of these polypeptides.
[0168] In certain embodiments, the present invention provides an
isolated polynucleotide comprising a nucleic acid fragment, where
the nucleic acid fragment is a fragment of a codon-optimized coding
region encoding a polypeptide at least 60%, 65%, 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identical to a herpes simplex virus polypeptide, e.g.,
gD, VP 11/12, VP13/14 and/or VP22 and where the nucleic acid
fragment is a variant of a codon-optimized coding region encoding a
herpes simplex virus polypeptide, e.g., gD, VP 11/12, VP13/14
and/or VP22. The human codon-optimized coding region can be
optimized for any vertebrate species and by any of the methods
described herein.
Isolated Herpes Simplex Virus Polypeptides
[0169] The present invention is further drawn to compositions which
include at least one polynucleotide comprising one or more nucleic
acid fragments, where each nucleic acid fragment is optionally a
fragment of a codon-optimized coding region operably encoding a
herpes simplex virus polypeptide or fragment, variant, or
derivative thereof; together with one or more isolated herpes
simplex virus component or isolated polypeptide. The herpes simplex
virus component may be inactivated virus, attenuated virus, a viral
vector expressing an isolated herpes simplex virus polypeptide, or
a herpes simplex virus protein, fragment, variant or derivative
thereof.
[0170] The isolated herpes simplex virus polypeptides of the
invention may be in any form, and are generated using techniques
well known in the art. Examples include isolated herpes simplex
virus proteins produced recombinantly, isolated herpes simplex
virus proteins directly purified from their natural milieu,
recombinant (non-herpes simplex virus) virus vectors expressing an
isolated herpes simplex virus protein, or proteins delivered in the
form of an inactivated herpes simplex virus vaccine, such as
conventional vaccines.
[0171] In the instant invention, the combination of conventional
antigen vaccine compositions with the codon-optimized nucleic acid
compositions provides for therapeutically beneficial effects at
dose sparing concentrations. For example, immunological responses
sufficient for a therapeutically beneficial effect in patients
predetermined for an approved commercial product, such as for the
conventional product described above, can be attained by using less
of the approved commercial product when supplemented or enhanced
with the appropriate amount of codon-optimized nucleic acid. Thus,
dose sparing is contemplated by administration of conventional
herpes simplex virus vaccines administered in combination with the
codon-optimized nucleic acids of the invention
[0172] In particular, the dose of conventional vaccine may be
reduced by at least 5%, at least 10%, at least 20%, at least 30%,
at least 40%, at least 50%, at least 60% or at least 70% when
administered in combination with the codon-optimized nucleic acid
compositions of the invention.
[0173] Similarly, a desirable level of an immunological response
afforded by a DNA based pharmaceutical alone may be attained with
less DNA by including an aliquot of a conventional vaccine.
Further, using a combination of conventional and DNA based
pharmaceuticals may allow both materials to be used in lesser
amounts while still affording the desired level of immune response
arising from administration of either component alone in higher
amounts (e.g. one may use less of either immunological product when
they are used in combination). This may be manifest not only by
using lower amounts of materials being delivered at any time, but
also to reducing the number of administrations points in a
vaccination regime (e.g. 2 versus 3 or 4 injections), and/or to
reducing the kinetics of the immunological response (e.g. desired
response levels are attained in 3 weeks instead of 6 after
immunization).
[0174] In particular, the dose of DNA based pharmaceuticals, may be
reduced by at least 5%, at least 10%, at least 20%, at least 30%,
at least 40%, at least 50%, at least 60% or at least 70% when
administered in combination with conventional herpes simplex virus
vaccines.
[0175] Determining the precise amounts of DNA based pharmaceutical
and conventional antigen is based on a number of factors as
described above, and is readily determined by one of ordinary skill
in the art.
[0176] In addition to dose sparing, the claimed combinatorial
compositions provide for a broadening of the immune response and/or
enhanced beneficial immune responses. Such broadened or enhanced
immune responses are achieved by: adding DNA to enhance cellular
responses to a conventional vaccine; adding a conventional vaccine
to a DNA pharmaceutical to enhance humoral response; using a
combination that induces additional epitopes (both humoral and/or
cellular) to be recognized and/or more desirably responded to
(epitope broadening); employing a DNA-conventional vaccine
combination designed for a particular desired spectrum of
immunological responses; obtaining a desirable spectrum by using
higher amounts of either component. The broadened immune response
is measurable by one of ordinary skill in the art by standard
immunological assay specific for the desirable response
spectrum.
[0177] Both broadening and dose sparing can be obtained
simultaneously.
[0178] The isolated herpes simplex virus polypeptide or fragment,
variant, or derivative thereof to be delivered (either a
recombinant protein, a purified subunit, or viral vector expressing
an isolated herpes simplex virus polypeptide, or in the form of an
inactivated herpes simplex virus vaccine) can be any isolated
herpes simplex virus polypeptide or fragment, variant, or
derivative thereof, including but not limited to the gD, VP 11/12,
VP13/14 and/or VP22 proteins or fragments, variants or derivatives
thereof. It should be noted that any isolated herpes simplex virus
polypeptide or fragment, variant, or derivative thereof described
herein can be combined in a composition with any polynucleotide
comprising a nucleic acid fragment, where the nucleic acid fragment
is optionally a fragment of a codon-optimized coding region
operably encoding a herpes simplex virus polypeptide or fragment,
variant, or derivative thereof. The proteins can be different, the
same, or can be combined in any combination of one or more isolated
herpes simplex virus proteins and one or more polynucleotides.
[0179] In certain embodiments, the isolated herpes simplex virus
polypeptides, or fragments, derivatives or variants thereof can be
fused to or conjugated to a second isolated herpes simplex virus
polypeptide, or fragment, derivative or variant thereof, or can be
fused to other heterologous proteins, including for example,
hepatitis B proteins including, but not limited to the hepatitis B
core antigen (HBcAg), or those derived from diphtheria or tetanus.
The second isolated herpes simplex virus polypeptide or other
heterologous protein can act as a "carrier" that potentiates the
immunogenicity of the herpes simplex virus polypeptide or a
fragment, variant, or derivative thereof to which it is attached.
Hepatitis B virus proteins and fragments and variants thereof
useful as carriers within the scope of the invention are disclosed
in U.S. Pat. Nos. 6,231,864 and 5,143,726. Polynucleotides
comprising coding regions encoding said fused or conjugated
proteins are also within the scope of the invention.
[0180] The use of recombinant particles comprising hepatitis B core
antigen ("HBcAg") and heterologous protein sequences as potent
immunogenic moieties is well documented. For example, addition of
heterologous sequences to the amino terminus of a recombinant HBcAg
results in the spontaneous assembly of particulate structures which
express the heterologous epitope on their surface, and which are
highly immunogenic when inoculated into experimental animals. See
Clarke et al., Nature 330:381-384 (1987). Heterologous epitopes can
also be inserted into HBcAg particles by replacing approximately 40
amino acids of the carboxy terminus of the protein with the
heterologous sequences. These recombinant HBcAg proteins also
spontaneously form immunogenic particles. See Stahl and Murray,
Proc. Natl. Acad. Sci. USA, 86:6283-6287 (1989). Additionally,
chimeric HBcAg particles may be constructed where the heterologous
epitope is inserted in or replaces all or part of the sequence of
amino acid residues in a more central region of the HBcAg protein,
in an immunodominant loop, thereby allowing the heterologous
epitope to be displayed on the surface of the resulting particles.
See EP Patent No. 0421635 B1 and Galibert, F., et al., Nature
281:646-650 (1979); see also U.S. Pat. Nos. 4,818,527, 4,882,145
and 5,143,726.
[0181] Chimaeric HBcAg particles comprising isolated herpes simplex
virus proteins or variants, fragments or derivatives thereof are
prepared by recombinant techniques well known to those of ordinary
skill in the art. A polynucleotide, e.g., a plasmid, which carries
the coding region for the HBcAg operably associated with a promoter
is constructed. Convenient restrictions sites are engineered into
the coding region encoding the N-terminal, central, and/or
C-terminal portions of the HBcAg, such that heterologous sequences
may be inserted. A construct which expresses a HBcAg/herpes simplex
virus fusion protein is prepared by inserting a DNA sequence
encoding a herpes simplex virus protein or variant, fragment or
derivative thereof, in frame, into a desired restriction site in
the coding region of the HBcAg. The resulting construct is then
inserted into a suitable host cell, e.g., E. coli, under conditions
where the chimeric HBcAg will be expressed. The chimaeric HBcAg
self-assembles into particles when expressed, and can then be
isolated, e.g., by ultracentrifugation. The particles formed
resemble the natural 27 nm HBcAg particles isolated from a
hepatitis B virus, except that an isolated herpes simplex virus
protein or fragment, variant, or derivative thereof is contained in
the particle, preferably exposed on the outer particle surface.
[0182] The herpes simplex virus protein or fragment, variant, or
derivative thereof expressed in a chimaeric HBcAg particle may be
of any size which allows suitable particles of the chimeric HBcAg
to self-assemble. As discussed above, even small antigenic epitopes
may be immunogenic when expressed in the context of an immunogenic
carrier, e.g., a HBcAg. Thus, HBcAg particles of the invention may
comprise at least 4, at least 5, at least 6, at least 7, at least
8, at least 9, at least 10, at least 15, at least 20, at least 25,
or between about 15 to about 30 amino acids of a herpes simplex
virus protein fragment of interest inserted therein. HBcAg
particles of the invention may further comprise immunogenic or
antigenic epitopes of at least 5, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues
of a herpes simplex virus protein fragment of interest inserted
therein.
[0183] The immunodominant loop region of HBcAg was mapped to about
amino acid residues 75 to 83, to about amino acids 75 to 85 or to
about amino acids 130 to 140. See Colucci et al., J. Immunol.
141:4376-4380 (1988), and Salfeld et al. J. Virol. 63:798 (1989). A
chimeric HBcAg is still often able to form core particles when
foreign epitopes are cloned into the immunodominant loop. Thus, for
example, amino acids of the herpes simplex virus protein fragment
may be inserted into the sequence of HBcAg amino acids at various
positions, for example, at the N-terminus, from about amino acid 75
to about amino acid 85, from about amino acid 75 to about amino
acid 83, from about amino acid 130 to about amino acid 140, or at
the C-terminus. Where amino acids of the herpes simplex virus
protein fragment replace all or part of the native core protein
sequence, the inserted herpes simplex virus sequence is generally
not shorter, but may be longer, than the HBcAg sequence it
replaces.
[0184] Alternatively, if particle formation is not desired,
full-length herpes simplex virus coding sequences can be fused to
the coding region for the HBcAg. The HBcAg sequences can be fused
either at the N- or C-terminus of any of the Herpes simplex
antigens described herein. Fusions could include flexible protein
linkers. These fusion constructs could be codon optimized by any of
the methods described.
[0185] The chimeric HBcAg can be used in the present invention in
conjunction with a polynucleotide comprising a nucleic acid
fragment, where each nucleic acid fragment is optionally a fragment
of a codon-optimized coding region operably encoding a herpes
simplex virus polypeptide, or a fragment, variant, or derivative
thereof, as a herpes simplex vaccine for a vertebrate.
Methods and Administration
[0186] The present invention also provides methods for delivering a
herpes simplex virus polypeptide or a fragment, variant, or
derivative thereof to a human, which comprise administering to a
human one or more of the compositions described herein; such that
upon administration of compositions such as those described herein,
a herpes simplex virus polypeptide or a fragment, variant, or
derivative thereof is expressed in human cells, in an amount
sufficient to generate an immune response to the herpes simplex
virus or administering the herpes simplex virus polypeptide or a
fragment, variant, or derivative thereof itself to the human in an
amount sufficient to generate an immune response.
[0187] The present invention further provides methods for
delivering a herpes simplex virus polypeptide or a fragment,
variant, or derivative thereof to a human, which comprise
administering to a vertebrate one or more of the compositions
described herein; such that upon administration of compositions
such as those described herein, an immune response is generated in
the vertebrate.
[0188] The term "vertebrate" is intended to encompass a singular
"vertebrate" as well as plural "vertebrates" and comprises mammals
and birds, as well as fish, reptiles, and amphibians.
[0189] The term "mammal" is intended to encompass a singular
"mammal" and plural "mammals," and includes, but is not limited to
humans; primates such as apes, monkeys (e.g., owl, squirrel, cebus,
rhesus, African green, patas, cynomolgus, and cercopithecus),
orangutans, baboons, gibbons, and chimpanzees; canids such as dogs
and wolves; felids such as cats, lions, and tigers; equines such as
horses, donkeys, and zebras, food animals such as cows, pigs, and
sheep; ungulates such as deer and giraffes; ursids such as bears;
and others such as rabbits, mice, ferrets, seals, whales. In
particular, the mammal can be a human subject, a food animal or a
companion animal.
[0190] The term "bird" is intended to encompass a singular "bird"
and plural "birds," and includes, but is not limited to, feral
water birds such as ducks, geese, terns, shearwaters, and gulls; as
well as domestic avian species such as turkeys, chickens, quail,
pheasants, geese, and ducks. The term "bird" also encompasses
passerine birds such as starlings and budgerigars.
[0191] The present invention further provides a method for
generating, enhancing or modulating an immune response to a herpes
simplex virus comprising administering to a vertebrate one or more
of the compositions described herein. In this method, the
compositions may include one or more isolated polynucleotides
comprising at least one nucleic acid fragment where the nucleic
acid fragment is optionally a fragment of a codon-optimized coding
region encoding a herpes simplex virus polypeptide, or a fragment,
variant, or derivative thereof. In another embodiment, the
compositions may include both a polynucleotide as described above,
and also an isolated herpes simplex virus polypeptide, or a
fragment, variant, or derivative thereof, wherein the protein is
provided as a recombinant protein, in particular, a fusion protein,
a purified subunit, viral vector expressing the protein, or in the
form of an inactivated herpes simplex virus vaccine. Thus, the
latter compositions include both a polynucleotide encoding a herpes
simplex virus polypeptide or a fragment, variant, or derivative
thereof and an isolated herpes simplex virus polypeptide or a
fragment, variant, or derivative thereof. The herpes simplex virus
polypeptide or a fragment, variant, or derivative thereof encoded
by the polynucleotide of the compositions need not be the same as
the isolated herpes simplex virus polypeptide or a fragment,
variant, or derivative thereof of the compositions. Compositions to
be used according to this method may be univalent, bivalent,
trivalent or multivalent.
[0192] The polynucleotides of the compositions may comprise a
fragment of a human (or other vertebrate) codon-optimized coding
region encoding a protein of the herpes simplex virus, or a
fragment, variant, or derivative thereof. The polynucleotides are
incorporated into the cells of the vertebrate in vivo, and an
antigenic amount of the herpes simplex virus polypeptide, or
fragment, variant, or derivative thereof, is produced in vivo. Upon
administration of the composition according to this method, the
herpes simplex virus polypeptide or a fragment, variant, or
derivative thereof is expressed in the vertebrate in an amount
sufficient to elicit an immune response. Such an immune response
might be used, for example, to generate antibodies to the herpes
simplex virus for use in diagnostic assays or as laboratory
reagents, or as therapeutic or preventative vaccines as described
herein.
[0193] The present invention further provides a method for
generating, enhancing, or modulating a protective and/or
therapeutic immune response to herpes simplex virus in a
vertebrate, comprising administering to a vertebrate in need of
therapeutic and/or preventative immunity one or more of the
compositions described herein. In this method, the compositions
include one or more polynucleotides comprising at least one nucleic
acid fragment, where the nucleic acid fragment is optionally a
fragment of a codon-optimized coding region encoding a herpes
simplex virus polypeptide, or a fragment, variant, or derivative
thereof. In a further embodiment, the composition used in this
method includes both an isolated polynucleotide comprising at least
one nucleic acid fragment, where the nucleic acid fragment is
optionally a fragment of a codon-optimized coding region encoding a
herpes simplex virus polypeptide, or a fragment, variant, or
derivative thereof; and at least one isolated herpes simplex virus
polypeptide, or a fragment, variant, or derivative thereof. Thus,
the latter composition includes both an isolated polynucleotide
encoding a herpes simplex virus polypeptide or a fragment, variant,
or derivative thereof and an isolated herpes simplex virus
polypeptide or a fragment, variant, or derivative thereof, for
example, a recombinant protein, a purified subunit, viral vector
expressing the protein, or an inactivated virus vaccine. Upon
administration of the composition according to this method, the
herpes simplex virus polypeptide or a fragment, variant, or
derivative thereof is expressed in the human in a therapeutically
or prophylactically effective amount.
[0194] As used herein, an "immune response" refers to the ability
of a vertebrate to elicit an immune reaction to a composition
delivered to that vertebrate. Examples of immune responses include
an antibody response or a cellular, e.g., cytotoxic T-cell,
response. One or more compositions of the present invention may be
used to prevent herpes simplex infection in vertebrates, e.g., as a
prophylactic vaccine, to establish or enhance immunity to herpes
simplex virus in a healthy individual prior to exposure to herpes
simplex or contraction of herpes simplex disease, thus preventing
the disease or reducing the severity of disease symptoms.
[0195] As mentioned above, compositions of the present invention
can be used both to prevent herpes simplex virus infection, and
also to therapeutically treat herpes simplex virus infection. In
individuals already exposed to herpes simplex, or already suffering
from herpes simplex disease, the present invention is used to
further stimulate the immune system of the vertebrate, thus
reducing or eliminating the symptoms associated with that disease
or disorder. As defined herein, "treatment" refers to the use of
one or more compositions of the present invention to prevent, cure,
retard, or reduce the severity of herpes simplex disease symptoms
in a vertebrate, and/or result in no worsening of herpes simplex
disease over a specified period of time in a vertebrate which has
already been exposed to herpes simplex virus and is thus in need of
therapy. The term "prevention" refers to the use of one or more
compositions of the present invention to generate immunity in a
vertebrate which has not yet been exposed to a particular strain of
herpes simplex virus, thereby preventing or reducing disease
symptoms if the vertebrate is later exposed to the particular
strain of herpes simplex virus. The methods of the present
invention therefore may be referred to as therapeutic vaccination
or preventative or prophylactic vaccination. It is not required
that any composition of the present invention provide total
immunity to herpes simplex or totally cure or eliminate all herpes
simplex disease symptoms. As used herein, a "vertebrate in need of
therapeutic and/or preventative immunity" refers to an individual
for whom it is desirable to treat, i.e., to prevent, cure, retard,
or reduce the severity of herpes simplex disease symptoms, and/or
result in no worsening of herpes simplex disease over a specified
period of time.
[0196] One or more compositions of the present invention are
utilized in a "prime boost" regimen. An example of a "prime boost"
regimen may be found in Yang, Z. et al. J. Virol. 77:799-803
(2002). In these embodiments, one or more polynucleotide vaccine
compositions of the present invention are delivered to a
vertebrate, thereby priming the immune response of the vertebrate
to a herpes simplex virus, and then a second immunogenic
composition is utilized as a boost vaccination. One or more
compositions of the present invention are used to prime immunity,
and then a second immunogenic composition, e.g., a recombinant
viral vaccine or vaccines, a different polynucleotide vaccine, or
one or more purified subunit isolated herpes simplex virus
polypeptides or fragments, variants or derivatives thereof is used
to boost the anti-herpes simplex virus immune response.
[0197] In one embodiment, a priming composition and a boosting
composition are combined in a single composition or single
formulation. For example, a single composition may comprise an
isolated herpes simplex virus polypeptide or a fragment, variant,
or derivative thereof as the priming component and a polynucleotide
encoding a herpes simplex protein as the boosting component. In
this embodiment, the compositions may be contained in a single vial
where the priming component and boosting component are mixed
together. In general, because the peak levels of expression of
protein from the polynucleotide does not occur until later (e.g.
7-10 days) after administration, the polynucleotide component may
provide a boost to the isolated protein component. Compositions
comprising both a priming component and a boosting component are
referred to herein as "combinatorial vaccine compositions" or
"single formulation heterologous prime-boost vaccine compositions."
In addition, the priming composition may be administered before the
boosting composition, or even after the boosting composition, if
the boosting composition is expected to take longer to act.
[0198] In another embodiment, the priming composition may be
administered simultaneously with the boosting composition, but in
separate formulations where the priming component and the boosting
component are separated.
[0199] The terms "priming" or "primary" and "boost" or "boosting"
as used herein may refer to the initial and subsequent
immunizations, respectively, i.e., in accordance with the
definitions these terms normally have in immunology. However, in
certain embodiments, e.g., where the priming component and boosting
component are in a single formulation, initial and subsequent
immunizations may not be necessary as both the "prime" and the
"boost" compositions are administered simultaneously.
[0200] In certain embodiments, one or more compositions of the
present invention are delivered to a vertebrate by methods
described herein, thereby achieving an effective therapeutic and/or
an effective preventative immune response. More specifically, the
compositions of the present invention may be administered to any
tissue of a vertebrate, including, but not limited to, muscle,
skin, brain tissue, lung tissue, liver tissue, spleen tissue, bone
marrow tissue, thymus tissue, heart tissue, e.g., myocardium,
endocardium, and pericardium, lymph tissue, blood tissue, bone
tissue, pancreas tissue, kidney tissue, gall bladder tissue,
stomach tissue, intestinal tissue, testicular tissue, ovarian
tissue, uterine tissue, vaginal tissue, rectal tissue, nervous
system tissue, eye tissue, glandular tissue, tongue tissue, and
connective tissue, e.g., cartilage.
[0201] Furthermore, the compositions of the present invention may
be administered to any internal cavity of a vertebrate, including,
but not limited to, the lungs, the mouth, the nasal cavity, the
stomach, the peritoneal cavity, the intestine, any heart chamber,
veins, arteries, capillaries, lymphatic cavities, the uterine
cavity, the vaginal cavity, the rectal cavity, joint cavities,
ventricles in brain, spinal canal in spinal cord, the ocular
cavities, the lumen of a duct of a salivary gland or a liver. When
the compositions of the present invention is administered to the
lumen of a duct of a salivary gland or liver, the desired
polypeptide is expressed in the salivary gland and the liver such
that the polypeptide is delivered into the blood stream of the
vertebrate from each of the salivary gland or the liver. Certain
modes for administration to secretory organs of a gastrointestinal
system using the salivary gland, liver and pancreas to release a
desired polypeptide into the bloodstream is disclosed in U.S. Pat.
Nos. 5,837,693 and 6,004,944.
[0202] In certain embodiments, the compositions are administered to
muscle, either skeletal muscle or cardiac muscle, or to lung
tissue. Specific, but non-limiting modes for administration to lung
tissue are disclosed in Wheeler, C. J., et al., Proc. Natl. Acad.
Sci. USA 93:11454-11459 (1996), which is incorporated herein by
reference in its entirety.
[0203] According to the disclosed methods, compositions of the
present invention can be administered by intramuscular (i.m.),
interdermal (i.d.), subcutaneous (s.c), or intrapulmonary routes.
Other suitable routes of administration include, but are not
limited to intratracheal, transdermal, intraocular, intranasal,
inhalation, intracavity, intravenous (i.v.), intraductal (e.g.,
into the pancreas) and intraparenchymal (i.e., into any tissue)
administration. Transdermal delivery includes, but not limited to
intradermal (e.g., into the dermis or epidermis), transdermal
(e.g., percutaneous) and transmucosal administration (i.e., into or
through skin or mucosal tissue). Intracavity administration
includes, but not limited to administration into oral, vaginal,
rectal, nasal, peritoneal, or intestinal cavities as well as,
intrathecal (i.e., into spinal canal), intraventricular (i.e., into
the brain ventricles or the heart ventricles), intraatrial (i.e.,
into the heart atrium) and sub arachnoid (i.e., into the sub
arachnoid spaces of the brain) administration.
[0204] For oral indications, the present invention may be
administered in the form of tongue strips wherein the composition
is embedded or applied to the strip. The user places the strip on
the tongue and the strip melts or dissolves in the mouth thereby
releasing the composition.
[0205] Any mode of administration can be used so long as the mode
results in the expression of the desired peptide or protein, in the
desired tissue, in an amount sufficient to generate an immune
response to herpes simplex virus and/or to generate a
prophylactically or therapeutically effective immune response to
herpes simplex virus in a human in need of such response.
Administration means of the present invention include needle
injection, catheter infusion, biolistic injectors, particle
accelerators (e.g., "gene guns" or pneumatic "needleless"
injectors) Med-E-Jet (Vahlsing, H., et al., J. Immunol. Methods
171:11-22 (1994)), Pigjet (Schrijver, R., et al., Vaccine 15:
1908-1916 (1997)), Biojector (Davis, H., et al., Vaccine 12:
1503-1509 (1994); Gramzinski, R., et al., Mol. Med. 4: 109-118
(1998)), AdvantaJet (Linmayer, I., et al., Diabetes Care 9:294-297
(1986)), Medi-jector (Martins, J., and Roedl, E. J. Occup. Med.
21:821-824 (1979)), U.S. Pat. No. 5,399,163; U.S. Pat. No.
5,383,851; gelfoam sponge depots, other commercially available
depot materials (e.g., hydrogels), osmotic pumps (e.g., Alza
minipumps), oral or suppositorial solid (tablet or pill)
pharmaceutical formulations, topical skin creams, and decanting,
use of polynucleotide coated suture (Qin, Y., et al., Life Sciences
65:2193-2203 (1999)) or topical applications during surgery.
Certain modes of administration are intramuscular or intradermal
needle-based injection and pulmonary application via catheter
infusion. Energy-assisted plasmid delivery (EAPD) methods may also
be employed to administer the compositions of the invention. One
such method involves the application of brief electrical pulses to
injected tissues, a procedure commonly known as electroporation.
See generally Mir, L. M. et al., Proc. Natl. Acad. Sci USA
96:4262-7 (1999); Hartikka, J. et al., Mol. Ther. 4:407-15 (2001);
Mathiesen, I., Gene Ther. 6:508-14 (1999); Rizzuto G. et al., Hum.
Gen. Ther. 11:1891-900 (2000).
[0206] Determining an effective amount of one or more compositions
of the present invention depends upon a number of factors
including, for example, the antigen being expressed or administered
directly, e.g., gD, VP 11/12, VP13/14 and/or VP22, or fragments,
variants, or derivatives thereof, the age and weight of the
subject, the precise condition requiring treatment and its
severity, and the route of administration. Based on the above
factors, determining the precise amount, number of doses, and
timing of doses are within the ordinary skill in the art and will
be readily determined by the attending physician or
veterinarian.
[0207] Compositions of the present invention may include various
salts, excipients, delivery vehicles and/or auxiliary agents as are
disclosed, e.g., in U.S. patent application Publication No.
2002/0019358, published Feb. 14, 2002.
[0208] Furthermore, compositions of the present invention may
include one or more transfection facilitating compounds that
facilitate delivery of polynucleotides to the interior of a cell,
and/or to a desired location within a cell. As used herein, the
terms "transfection facilitating compound," "transfection
facilitating agent," and "transfection facilitating material" are
synonymous, and may be used interchangeably. It should be noted
that certain transfection facilitating compounds may also be
"adjuvants" as described infra, i.e., in addition to facilitating
delivery of polynucleotides to the interior of a cell, the compound
acts to alter or increase the immune response to the antigen
encoded by that polynucleotide. Examples of the transfection
facilitating compounds include, but are not limited to, inorganic
materials such as calcium phosphate, alum (aluminum sulfate), and
gold particles (e.g., "powder" type delivery vehicles); peptides
that are, for example, canonic, intercell targeting (for selective
delivery to certain cell types), intracell targeting (for nuclear
localization or endosomal escape), and ampipathic (helix forming or
pore forming); proteins that are, for example, basic (e.g.,
positively charged) such as histories, targeting (e.g.,
asialoprotein), viral (e.g., Sendai virus coat protein), and
pore-forming; lipids that are, for example, cationic (e.g., DMRIE,
DOSPA, DC-Chol), basic (e.g., steryl amine), neutral (e.g.,
cholesterol), anionic (e.g., phosphatidyl serine), and zwitterionic
(e.g., DOPE, DOPC); and polymers such as dendrimers, star-polymers,
"homogenous" poly-amino acids (e.g., poly-lysine, poly-arginine),
"heterogeneous" poly-amino acids (e.g., mixtures of lysine &
glycine), co-polymers, polyvinylpyrrolidinone (PVP), poloxamers
(e.g. CRL 1005) and polyethylene glycol (PEG). A transfection
facilitating material can be used alone or in combination with one
or more other transfection facilitating materials. Two or more
transfection facilitating materials can be combined by chemical
bonding (e.g., covalent and ionic such as in lipidated polylysine,
PEGylated polylysine) (Toncheva, et al., Biochim. Biophys. Acta
1380(3):354-368 (1988)), mechanical mixing (e.g., tree moving
materials in liquid or solid phase such as "polylysine+ cationic
lipids") (Gao and Huang, Biochemistry 35:1027-1036 (1996);
Trubetskoy, et al., Biochem. Biophys. Acta 1131:311-313 (1992)),
and aggregation (e.g., co-precipitation, gel forming such as in
cationic lipids+ poly-lactide, and polylysine+ gelatin).
[0209] One category of transfection facilitating materials is
cationic lipids. Examples of cationic lipids are
5-carboxyspermylglycine dioctadecylamide (DOGS) and
dipalmitoyl-phophatidylethanolamine-5-carboxyspermylamide (DPPES).
Cationic cholesterol derivatives are also useful, including
{3.beta.-[N-N',N'-dimethylamino)ethane]-carbomoyl}-cholesterol
(DC-Chol). Dimethyldioctdecyl-ammonium bromide (DDAB),
N-(3-aminopropyl)-N,N-(bis-(2-tetradecyloxyethyl))-N-methyl-ammonium
bromide (PA-DEMO),
N-(3-aminopropyl)-N,N-(bis-(2-dodecyloxyethyl))-N-methyl-ammonium
bromide (PA-DELO),
N,N,N-tris-(2-dodecyloxy)ethyl-N-(3-amino)propyl-ammonium bromide
(PA-TELO), and
N1-(3-aminopropyl)((2-dodecyloxy)ethyl)-N2-(2-dodecyloxy)ethyl-1-piperazi-
naminium bromide (GA-LOE-BP) can also be employed in the present
invention.
[0210] Non-diether cationic lipids, such as
DL-1,2-doleoyl-3-dimethylaminopropyl-.beta.-hydroxyethylammonium
(DORI diester),
1-O-oleyl-2-oleoyl-3-dimethylaminopropyl-p-hydroxyethylammonium
(DORI ester/ether), and their salts promote in vivo gene delivery.
In some embodiments, cationic lipids comprise groups attached via a
heteroatom attached to the quaternary ammonium moiety in the head
group. A glycyl spacer can connect the linker to the hydroxyl
group.
[0211] Specific, but non-limiting cationic lipids for use in
certain embodiments of the present invention include DMRIE
((.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanam-
inium bromide), GAP-DMORIE
((.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1--
propanaminium bromide), and
GAP-DMRIE((.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-1-pr-
opaniminium bromide).
[0212] Other specific but non-limiting cationic surfactants for use
in certain embodiments of the present invention include Bn-DHRIE,
DhxRIE, DhxRIE-OAc, DhxRIE-OBz and Pr-DOctRIE-OAc. These lipids are
disclosed in copending U.S. patent application Ser. No. 10/725,015.
In another aspect of the present invention, the cationic surfactant
is Pr-DOctRIE-OAc.
[0213] Other cationic lipids include
(.+-.)-N,N-dimethyl-N-[2-(sperminecarboxamido)ethyl]-2,3-bis(dioleyloxy)--
1-propaniminium pentahydrochloride (DOSPA),
(.+-.)-N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanimini-
um bromide (.beta.-aminoethyl-DMRIE or .beta.AE-DMRIE) (Wheeler, et
al., Biochim. Biophys. Acta 1280:1-11 (1996), and
(.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminium
bromide (GAP-DLRIE) (Wheeler, et al., Proc. Natl. Acad. Sci. USA
93:11454-11459 (1996)), which have been developed from DMRIE.
[0214] Other examples of DMRIE-derived cationic lipids that are
useful for the present invention are
(.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-decyloxy)-1-propanaminium
bromide (GAP-DDRIE),
(.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-tetradecyloxy)-1-propanami-
nium bromide (GAP-DMRIE),
(.+-.)-N-((N''-methyl)-N'-ureyl)propyl-N,N-dimethyl-2,3-bis(tetradecyloxy-
-)-1-propanaminium bromide (GMU-DMRIE),
(.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminiu-
m bromide (DLRIE), and
(.+-.)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis-([Z]-9-octadecenyloxy)prop-
yl-1-propaniminium bromide (HP-DORIE).
[0215] In the embodiments where the immunogenic composition
comprises a cationic lipid, the cationic lipid may be mixed with
one or more co-lipids. For purposes of definition, the term
"co-lipid" refers to any hydrophobic material which may be combined
with the cationic lipid component and includes amphipathic lipids,
such as phospholipids, and neutral lipids, such as cholesterol.
Cationic lipids and co-lipids may be mixed or combined in a number
of ways to produce a variety of non-covalently bonded macroscopic
structures, including, for example, liposomes, multilamellar
vesicles, unilamellar vesicles, micelles, and simple films. One
non-limiting class of co-lipids are the zwitterionic phospholipids,
which include the phosphatidylethanolamines and the
phosphatidylcholines. Examples of phosphatidylethanolamines,
include DOPE, DMPE and DPyPE. In certain embodiments, the co-lipid
is DPyPE which comprises two phytanoyl substituents incorporated
into the diacylphosphatidylethanolamine skeleton and the cationic
lipid is GAP-DMORIE, (resulting in VAXFECTIN adjuvant). In other
embodiments, the co-lipid is DOPE, the CAS name is
1,2-diolyeoyl-sn-glycero-3-phosphoethanolamine.
[0216] When a composition of the present invention comprises a
cationic lipid and co-lipid, the cationic lipid:co-lipid molar
ratio may be from about 9:1 to about 1:9, from about 4:1 to about
1:4, from about 2:1 to about 1:2, or about 1:1.
[0217] In order to maximize homogeneity, the cationic lipid and
co-lipid components may be dissolved in a solvent such as
chloroform, followed by evaporation of the cationic lipid/co-lipid
solution under vacuum to dryness as a film on the inner surface of
a glass vessel (e.g., a Rotovap round-bottomed flask). Upon
suspension in an aqueous solvent, the amphipathic lipid component
molecules self-assemble into homogenous lipid vesicles. These lipid
vesicles may subsequently be processed to have a selected mean
diameter of uniform size prior to complexing with, for example, a
codon-optimized polynucleotide of the present invention, according
to methods known to those skilled in the art. For example, the
sonication of a lipid solution is described in Felgner et al.,
Proc. Natl. Acad. Sci. USA 8:7413-7417 (1987) and in U.S. Pat. No.
5,264,618.
[0218] In those embodiments where the composition includes a
cationic lipid, polynucleotides of the present invention are
complexed with lipids by mixing, for example, a plasmid in aqueous
solution and a solution of cationic lipid:co-lipid as prepared
herein are mixed. The concentration of each of the constituent
solutions can be adjusted prior to mixing such that the desired
final plasmid/cationic lipid:co-lipid ratio and the desired plasmid
final concentration will be obtained upon mixing the two solutions.
The cationic lipid:co-lipid mixtures are suitably prepared by
hydrating a thin film of the mixed lipid materials in an
appropriate volume of aqueous solvent by vortex mixing at ambient
temperatures for about 1 minute. The thin films are prepared by
admixing chloroform solutions of the individual components to
afford a desired molar solute ratio followed by aliquoting the
desired volume of the solutions into a suitable container. The
solvent is removed by evaporation, first with a stream of dry,
inert gas (e.g. argon) followed by high vacuum treatment.
[0219] Other hydrophobic and amphiphilic additives, such as, for
example, sterols, fatty acids, gangliosides, glycolipids,
lipopeptides, liposaccharides, neobees, niosomes, prostaglandins
and sphingolipids, may also be included in compositions of the
present invention. In such compositions, these additives may be
included in an amount between about 0.1 mol % and about 99.9 mol %
(relative to total lipid), about 1-50 mol %, or about 2-25 mol
%.
[0220] Additional embodiments of the present invention are drawn to
compositions comprising an auxiliary agent which is administered
before, after, or concurrently with the polynucleotide. As used
herein, an "auxiliary agent" is a substance included in a
composition for its ability to enhance, relative to a composition
which is identical except for the inclusion of the auxiliary agent,
the entry of polynucleotides into vertebrate cells in vivo, and/or
the in vivo expression of polypeptides encoded by such
polynucleotides. Certain auxiliary agents may, in addition to
enhancing entry of polynucleotides into cells, enhance an immune
response to an immunogen encoded by the polynucleotide. Auxiliary
agents of the present invention include nonionic, anionic, canonic,
or zwitterionic surfactants or detergents, with nonionic
surfactants or detergents being preferred, chelators, DNase
inhibitors, poloxamers, agents that aggregate or condense nucleic
acids, emulsifying or solubilizing agents, wetting agents,
gel-forming agents, and buffers.
[0221] Auxiliary agents for use in compositions of the present
invention include, but are not limited to non-ionic detergents and
surfactants IGEPAL CA 6300 octylphenyl-polyethylene glycol, NONIDET
NP-40 nonylphenoxypolyethoxyethanol, NONIDET P-40
octylphenoxypolyethoxyethanol, TWEEN-20 polysorbate 20, TWEEN-80
polysorbate 80, PLURONIC F68 poloxamer (ave. MW: 8400; approx. MW
of hydrophobe, 1800; approx. wt. % of hydrophile, 80%), PLURONIC
F77 poloxamer (ave. MW: 6600; approx. MW of hydrophobe, 2100;
approx. wt. % of hydrophile, 70%), PLURONIC P65 poloxamer_(ave. MW:
3400; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile,
50%), TRITON X-100 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene
glycol, and TRITON X-114
(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol; the anionic
detergent sodium dodecyl sulfate (SDS); the sugar stachyose; the
condensing agent DMSO; and the chelator/DNAse inhibitor EDTA, CRL
1005 (12 kDa, 5% POE), and BAK (Benzalkonium chloride 50% solution,
available from Ruger Chemical Co. Inc.). In certain specific
embodiments, the auxiliary agent is DMSO, NONIDET P-40
octylphenoxypolyethoxyethanol, PLURONIC F68 poloxamer (ave. MW:
8400; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile,
80%), PLURONIC F77 poloxamer (ave. MW: 6600; approx. MW of
hydrophobe, 2100; approx. wt. % of hydrophile, 70%), PLURONIC P65
(ave. MW: 3400; approx. MW of hydrophobe, 1800; approx. wt. % of
hydrophile, 50%), PLURONIC L64 poloxamer (ave. MW: 2900; approx. MW
of hydrophobe, 1800; approx. wt. % of hydrophile, 40%), and
PLURONIC F108 poloxamer (ave. MW: 14600; approx. MW of hydrophobe,
3000; approx. wt. % of hydrophile, 80%). See, e.g., U.S. patent
application Publication No. 2002/0019358, published Feb. 14,
2002.
[0222] Certain compositions of the present invention can further
include one or more adjuvants before, after, or concurrently with
the polynucleotide. The term "adjuvant" refers to any material
having the ability to (1) alter or increase the immune response to
a particular antigen or (2) increase or aid an effect of a
pharmacological agent. It should be noted, with respect to
polynucleotide vaccines, that an "adjuvant," can be a transfection
facilitating material. Similarly, certain "transfection
facilitating materials" described supra, may also be an "adjuvant."
An adjuvant maybe used with a composition comprising a
polynucleotide of the present invention. In a prime-boost regimen,
as described herein, an adjuvant may be used with either the
priming immunization, the booster immunization, or both. Suitable
adjuvants include, but are not limited to, cytokines and growth
factors; bacterial components (e.g., endotoxins, in particular
superantigens, exotoxins and cell wall components); aluminum-based
salts; calcium-based salts; silica; polynucleotides; toxoids; serum
proteins, viruses and virally-derived materials, poisons, venoms,
imidazoquiniline compounds, poloxamers, and cationic lipids.
[0223] A great variety of materials have been shown to have
adjuvant activity through a variety of mechanisms. Any compound
which may increase the expression, antigenicity or immunogenicity
of the polypeptide is a potential adjuvant. The present invention
provides an assay to screen for improved immune responses to
potential adjuvants. Potential adjuvants which may be screened for
their ability to enhance the immune response according to the
present invention include, but are not limited to: inert carriers,
such as alum, bentonite, latex, and acrylic particles; PLURONIC
block polymers, such as TITERMAX (block copolymer CRL-8941,
squalene (a metabolizable oil) and a microparticulate silica
stabilizer); depot formers, such as Freunds adjuvant, surface
active materials, such as saponin, lysolecithin, retinal, Quil A,
liposomes, and PLURONIC polymer formulations; macrophage
stimulators, such as bacterial lipopolysaccharide; alternate
pathway complement activators, such as insulin, zymosan, endotoxin,
and levamisole; and non-ionic surfactants, such as poloxamers,
poly(oxyethylene)-poly(oxypropylene) tri-block copolymers. Also
included as adjuvants are transfection-facilitating materials, such
as those described above.
[0224] Poloxamers which may be screened for their ability to
enhance the immune response according to the present invention
include, but are not limited to, commercially available poloxamers
such as PLURONIC surfactants, which are block copolymers of
propylene oxide and ethylene oxide in which the propylene oxide
block is sandwiched between two ethylene oxide blocks. Examples of
PLURONIC surfactants include PLURONIC L121 poloxamer (ave. MW:
4400; approx. MW of hydrophobe, 3600; approx. wt % of hydrophile,
10%), PLURONIC L101 poloxamer (ave. MW: 3800; approx. MW of
hydrophobe, 3000; approx. wt. % of hydrophile, 10%), PLURONIC L81
poloxamer (ave. MW: 2750; approx. MW of hydrophobe, 2400; approx.
wt. % of hydrophile, 10%), PLURONIC L61 poloxamer (ave. MW: 2000;
approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile, 10%),
PLURONIC L31 poloxamer (ave. MW: 1100; approx. MW of hydrophobe,
900; approx. wt. % of hydrophile, 10%), PLURONIC L122 poloxamer
(ave. MW: 5000; approx. MW of hydrophobe, 3600; approx. wt. % of
hydrophile, 20%), PLURONIC L92 poloxamer (ave. MW: 3650; approx. MW
of hydrophobe, 2700; approx. wt. % of hydrophile, 20%), PLURONIC
L72 poloxamer (ave. MW: 2750; approx. MW of hydrophobe, 2100;
approx. wt. % of hydrophile, 20%), PLURONIC L62 poloxamer (ave. MW:
2500; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile,
20%), PLURONIC L42 poloxamer (ave. MW: 1630; approx. MW of
hydrophobe, 1200; approx. wt. % of hydrophile, 20%), PLURONIC L63
poloxamer (ave. MW: 2650; approx. MW of hydrophobe, 1800; approx.
wt. % of hydrophile, 30%), PLURONIC L43 poloxamer (ave. MW: 1850;
approx. MW of hydrophobe, 1200; approx. wt. % of hydrophile, 30%),
PLURONIC L64 poloxamer (ave. MW: 2900; approx. MW of hydrophobe,
1800; approx. wt. % of hydrophile, 40%), PLURONIC L44 poloxamer
(ave. MW: 2200; approx. MW of hydrophobe, 1200; approx. wt. % of
hydrophile, 40%), PLURONIC L35 poloxamer (ave. MW: 1900; approx. MW
of hydrophobe, 900; approx. wt. % of hydrophile, 50%), PLURONIC
P123 poloxamer (ave. MW: 5750; approx. MW of hydrophobe, 3600;
approx. wt. % of hydrophile, 30%), PLURONIC P103 poloxamer (ave.
MW: 4950; approx. MW of hydrophobe, 3000; approx. wt. % of
hydrophile, 30%), PLURONIC P104 poloxamer (ave. MW: 5900; approx.
MW of hydrophobe, 3000; approx. wt. % of hydrophile, 40%), PLURONIC
P84 poloxamer (ave. MW: 4200; approx. MW of hydrophobe, 2400;
approx. wt. % of hydrophile, 40%), PLURONIC P105 poloxamer (ave.
MW: 6500; approx. MW of hydrophobe, 3000; approx. wt. % of
hydrophile, 50%), PLURONIC P85 poloxamer (ave. MW: 4600; approx. MW
of hydrophobe, 2400; approx. wt. % of hydrophile, 50%), PLURONIC
P75 poloxamer (ave. MW: 4150; approx. MW of hydrophobe, 2100;
approx. wt. % of hydrophile, 50%), PLURONIC P65 poloxamer (ave. MW:
3400; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile,
50%), PLURONIC F127 poloxamer (ave. MW: 12600; approx. MW of
hydrophobe, 3600; approx. wt. % of hydrophile, 70%), PLURONIC F98
poloxamer (ave. MW: 13000; approx. MW of hydrophobe, 2700; approx.
wt. % of hydrophile, 80%), PLURONIC F87 poloxamer (ave. MW: 7700;
approx. MW of hydrophobe, 2400; approx. wt. % of hydrophile, 70%),
PLURONIC F77 poloxamer (ave. MW: 6600; approx. MW of hydrophobe,
2100; approx. wt. % of hydrophile, 70%), PLURONIC F108 poloxamer
(ave. MW: 14600; approx. MW of hydrophobe, 3000; approx. wt. % of
hydrophile, 80%), PLURONIC F98 poloxamer (ave. MW: 13000; approx.
MW of hydrophobe, 2700; approx. wt. % of hydrophile, 80%), PLURONIC
F88 poloxamer (ave. MW: 11400; approx. MW of hydrophobe, 2400;
approx. wt. % of hydrophile, 80%), PLURONIC F68 poloxamer (ave. MW:
8400; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile,
80%), PLURONIC F38 poloxamer (ave. MW: 4700; approx. MW of
hydrophobe, 900; approx. wt. % of hydrophile, 80%).
[0225] Reverse poloxamers which may be screened for their ability
to enhance the immune response according to the present invention
include, but are not limited to PLURONIC R 31R1 reverse poloxamer
(ave. MW: 3250; approx. MW of hydrophobe, 3100; approx. wt. % of
hydrophile, 10%), PLURONIC R25R1 reverse poloxamer (ave. MW: 2700;
approx. MW of hydrophobe, 2500; approx. wt. % of hydrophile, 10%),
PLURONIC R 17R1 reverse poloxamer (ave. MW: 1900; approx. MW of
hydrophobe, 1700; approx. wt. % of hydrophile, 10%), PLURONIC R
31R2 reverse poloxamer (ave. MW: 3300; approx. MW of hydrophobe,
3100; approx. wt. % of hydrophile, 20%), PLURONIC R 25R2 reverse
poloxamer (ave. MW: 3100; approx. MW of hydrophobe, 2500; approx.
wt. % of hydrophile, 20%), PLURONIC R 17R2 reverse poloxamer (ave.
MW: 2150; approx. MW of hydrophobe, 1700; approx. wt. % of
hydrophile, 20%), PLURONIC R 12R3 reverse poloxamer (ave. MW: 1800;
approx. MW of hydrophobe, 1200; approx. wt. % of hydrophile, 30%),
PLURONIC R 31R4 reverse poloxamer (ave. MW: 4150; approx. MW of
hydrophobe, 3100; approx. wt. % of hydrophile, 40%), PLURONIC R
25R4 reverse poloxamer (ave. MW: 3600; approx. MW of hydrophobe,
2500; approx. wt. % of hydrophile, 40%), PLURONIC R 22R4 reverse
poloxamer (ave. MW: 3350; approx. MW of hydrophobe, 2200; approx.
wt. % of hydrophile, 40%), PLURONIC R17R4 reverse poloxamer (ave.
MW: 3650; approx. MW of hydrophobe, 1700; approx. wt. % of
hydrophile, 40%), PLURONIC R 25R5 reverse poloxamer (ave. MW: 4320;
approx. MW of hydrophobe, 2500; approx. wt. % of hydrophile, 50%),
PLURONIC R10R5 reverse poloxamer (ave. MW: 1950; approx. MW of
hydrophobe, 1000; approx. wt. % of hydrophile, 50%), PLURONIC R
25R8 reverse poloxamer (ave. MW: 8550; approx. MW of hydrophobe,
2500; approx. wt. % of hydrophile, 80%), PLURONIC R 17R8 reverse
poloxamer (ave. MW: 7000; approx. MW of hydrophobe, 1700; approx.
wt. % of hydrophile, 80%), and PLURONIC R 10R8 reverse poloxamer
(ave. MW: 4550; approx. MW of hydrophobe, 1000; approx. wt. % of
hydrophile, 80%).
[0226] Other commercially available poloxamers which may be
screened for their ability to enhance the immune response according
to the present invention include compounds that are block copolymer
of polyethylene and polypropylene glycol such as SYNPERONIC L121
(ave. MW: 4400), SYNPERONIC L122 (ave. MW: 5000), SYNPERONIC P104
(ave. MW: 5850), SYNPERONIC P105 (ave. MW: 6500), SYNPERONIC P123
(ave. MW: 5750), SYNPERONIC P85 (ave. MW: 4600) and SYNPERONIC P94
(ave. MW: 4600), in which L indicates that the surfactants are
liquids, P that they are pastes, the first digit is a measure of
the molecular weight of the polypropylene portion of the surfactant
and the last digit of the number, multiplied by 10, gives the
percent ethylene oxide content of the surfactant; and compounds
that are nonylphenyl polyethylene glycol such as SYNPERONIC NP10
(nonylphenol ethoxylated surfactant--10% solution), SYNPERONIC NP30
(condensate of 1 mole of nonylphenol with 30 moles of ethylene
oxide) and SYNPERONIC NP5 (condensate of 1 mole of nonylphenol with
5.5 moles of naphthalene oxide).
[0227] Other poloxamers which may be screened for their ability to
enhance the immune response according to the present invention
include: (a) a polyether block copolymer comprising an A-type
segment and a B-type segment, wherein the A-type segment comprises
a linear polymeric segment of relatively hydrophilic character, the
repeating units of which contribute an average Hansch-Leo
fragmental constant of about -0.4 or less and have molecular weight
contributions between about 30 and about 500, wherein the B-type
segment comprises a linear polymeric segment of relatively
hydrophobic character, the repeating units of which contribute an
average Hansch-Leo fragmental constant of about -0.4 or more and
have molecular weight contributions between about 30 and about 500,
wherein at least about 80% of the linkages joining the repeating
units for each of the polymeric segments comprise an ether linkage;
(b) a block copolymer having a polyether segment and a polycation
segment, wherein the polyether segment comprises at least an A-type
block, and the polycation segment comprises a plurality of cationic
repeating units; and (c) a polyether-polycation copolymer
comprising a polymer, a polyether segment and a polycationic
segment comprising a plurality of cationic repeating units of
formula --NH--R.sup.0, wherein R.sup.0 is a straight chain
aliphatic group of 2 to 6 carbon atoms, which may be substituted,
wherein said polyether segments comprise at least one of an A-type
of B-type segment. See U.S. Pat. No. 5,656,611. Other poloxamers of
interest include CRL1005 (12 kDa, 5% POE), CRL8300 (11 kDa, 5%
POE), CRL2690 (12 kDa, 10% POE), CRL4505 (15 kDa, 5% POE) and
CRL1415 (9 kDa, 10% POE).
[0228] Other auxiliary agents which may be screened for their
ability to enhance the immune response according to the present
invention include, but are not limited to, Acacia (gum arabic); the
poloxyethylene ether R--O--(C.sub.2H.sub.4O).sub.x--H (BRIJ), e.g.,
polyethylene glycol dodecyl ether (BRIJ 35, x=23), polyethylene
glycol dodecyl ether (BRIJ 30, x=4), polyethylene glycol hexadecyl
ether (BRIJ 52 x=2), polyethylene glycol hexadecyl ether (BRIJ 56,
x=10), polyethylene glycol hexadecyl ether (BRIJ 58P, x=20),
polyethylene glycol octadecyl ether (BRIJ 72, x=2), polyethylene
glycol octadecyl ether (BRIJ 76, x=10), polyethylene glycol
octadecyl ether (BRIJ 78P, x=20), polyethylene glycol oleyl ether
(BRIJ 92V, x=2), and polyoxyl 10 oleyl ether (BRIJ 97, x=10);
poly-D-glucosamine (chitosan); chlorbutanol; cholesterol;
diethanolamine; digitonin; dimethylsulfoxide (DMSO),
ethylenediamine tetraacetic acid (EDTA); glyceryl monosterate;
lanolin alcohols; mono- and di-glycerides; monoethanolamine;
nonylphenol polyoxyethylene ether (NP-40);
octylphenoxypolyethoxyethanol (NONIDET NP-40 from Amresco); ethyl
phenol poly (ethylene glycol ether).sup.n, n=1 1 (NONIDET P40 from
Roche); octyl phenol ethylene oxide condensate with about 9
ethylene oxide units (NONIDET P40); IGEPAL CA 630 ((octyl phenoxy)
polyethoxyethanol; structurally same as NONIDET NP-40); oleic acid;
oleyl alcohol; polyethylene glycol 8000; polyoxyl 20 cetostearyl
ether; polyoxyl 35 castor oil; polyoxyl 40 hydrogenated castor oil;
polyoxyl 40 stearate; polyoxyethylene sorbitan monolaurate
(polysorbate 20, or TWEEN-20; polyoxyethylene sorbitan monooleate
(polysorbate 80, or TWEEN-80); propylene glycol diacetate;
propylene glycol monstearate; protamine sulfate; proteolytic
enzymes; sodium dodecyl sulfate (SDS); sodium monolaurate; sodium
stearate; sorbitan derivatives (SPAN), e.g., sorbitan monopalmitate
(SPAN 40), sorbitan monostearate (SPAN.RTM. 60), sorbitan
tristearate (SPAN 65), sorbitan monooleate (SPAN 80), and sorbitan
trioleate (SPAN 85);
2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosa-hexaene
(squalene); stachyose; stearic acid; sucrose; surfactin
(lipopeptide antibiotic from Bacillus subtilis);
dodecylpoly(ethyleneglycolether)9 (THESIT) MW 582.9; octyl phenol
ethylene oxide condensate with about 9-10 ethylene oxide units
(TRITON X-100); octyl phenol ethylene oxide condensate with about
7-8 ethylene oxide units (TRITON X-114); tris(2-hydroxyethyl)amine
(trolamine); and emulsifying wax.
[0229] In certain adjuvant compositions, the adjuvant is a
cytokine. A composition of the present invention can comprise one
or more cytokines, chemokines, or compounds that induce the
production of cytokines and chemokines, or a polynucleotide
encoding one or more cytokines, chemokines, or compounds that
induce the production of cytokines and chemokines. Examples
include, but are not limited to, granulocyte macrophage colony
stimulating factor (GM-CSF), granulocyte colony stimulating factor
(G-CSF), macrophage colony stimulating factor (M-CSF), colony
stimulating factor (CSF), erythropoietin (EPO), interleukin 2
(IL-2), interleukin-3 (IL-3), interleukin 4 (IL-4), interleukin 5
(IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8
(IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interleukin
15 (IL-15), interleukin 18 (IL-18), interferon alpha (IFN.alpha.),
interferon beta (IFN.beta.), interferon gamma (IFN.gamma.),
interferon omega (IFN.OMEGA.), interferon tau (IFN.tau.),
interferon gamma inducing factor I (IGIF), transforming growth
factor beta (TGF-.beta.), RANTES (regulated upon activation, normal
T-cell expressed and presumably secreted), macrophage inflammatory
proteins (e.g., MIP-1 alpha and M3P-1 beta), Leishmania elongation
initiating factor (LEIF), and Flt-3 ligand.
[0230] In certain compositions of the present invention, the
polynucleotide construct may be complexed with an adjuvant
composition comprising
(.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-p-
ropanaminium bromide (GAP-DMORIE). The composition may also
comprise one or more co-lipids, e.g.,
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE), and/or
1,2-dimyristoyl-glycer-3-phosphoethanolamine (DMPE). An adjuvant
composition comprising GAP-DMORIE and DPyPE at a 1:1 molar ratio is
referred to herein as VAXFECTIN adjuvant. See, e.g., PCT
Publication No. WO 00/57917.
[0231] In other embodiments, the polynucleotide itself may function
as an adjuvant as is the case when the polynucleotides of the
invention are derived, in whole or in part, from bacterial DNA.
Bacterial DNA containing motifs of unmethylated CpG-dinucleotides
(CpG-DNA) triggers innate immune cells in vertebrates through a
pattern recognition receptor (including toll receptors such as TLR
9) and thus possesses potent immunostimulatory effects on
macrophages, dendritic cells and B-lymphocytes. See, e.g., Wagner,
H., Curr. Opin. Microbiol. 5:62-69 (2002); Jung, J. et al., J.
Immunol. 169: 2368-73 (2002); see also Klinman, D. M. et al., Proc.
Natl Acad. Sci. U.S.A. 93:2879-83 (1996). Methods of using
unmethylated CpG-dinucleotides as adjuvants are described in, for
example, U.S. Pat. Nos. 6,207,646, 6,406,705 and 6,429, 199.
[0232] The ability of an adjuvant to increase the immune response
to an antigen is typically manifested by a significant increase in
immune-mediated protection. For example, an increase in humoral
immunity is typically manifested by a significant increase in the
titer of antibodies raised to the antigen, and an increase in
T-cell activity is typically manifested in increased cell
proliferation, or cellular cytotoxicity, or cytokine secretion. An
adjuvant may also alter an immune response, for example, by
changing a primarily humoral or Th.sub.2 response into a primarily
cellular, or Th.sub.I response.
[0233] Nucleic acid molecules and/or polynucleotides of the present
invention, e.g., plasmid DNA, mRNA, linear DNA or oligonucleotides,
may be solubilized in any of various buffers. Suitable buffers
include, for example, phosphate buffered saline (PBS), normal
saline, Tris buffer, and sodium phosphate (e.g., 150 mM sodium
phosphate). Insoluble polynucleotides may be solubilized in a weak
acid or weak base, and then diluted to the desired volume with a
buffer. The pH of the buffer may be adjusted as appropriate. In
addition, a pharmaceutically acceptable additive can be used to
provide an appropriate osmolarity. Such additives are within the
purview of one skilled in the art. For aqueous compositions used in
vivo, sterile pyrogen-free water can be used. Such formulations
will contain an effective amount of a polynucleotide together with
a suitable amount of an aqueous solution in order to prepare
pharmaceutically acceptable compositions suitable for
administration to a human.
[0234] Compositions of the present invention can be formulated
according to known methods. Suitable preparation methods are
described, for example, in Remington's Pharmaceutical Sciences,
16th Edition, A. Osol, ed., Mack Publishing Co., Easton, Pa.
(1980), and Remington's Pharmaceutical Sciences, 19th Edition, A.
R. Gennaro, ed., Mack Publishing Co., Easton, Pa. (1995). Although
the composition may be administered as an aqueous solution, it can
also be formulated as an emulsion, gel, solution, suspension,
lyophilized form, or any other form known in the art. In addition,
the composition may contain pharmaceutically acceptable additives
including, for example, diluents, binders, stabilizers, and
preservatives.
[0235] The following examples are included for purposes of
illustration only and are not intended to limit the scope of the
present invention, which is defined by the appended claims.
Examples
Materials and Methods
[0236] The following materials and methods apply generally to all
the examples disclosed herein. Specific materials and methods are
disclosed in each example, as necessary.
[0237] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology (including PCR), vaccinology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Molecular Cloning A Laboratory
Manual, 2nd Ed., Sambrook et al., ed., Cold Spring Harbor
Laboratory Press: (1989); DNA Cloning, Volumes I and II (D. N.
Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,
1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.
154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And
Molecular Biology (Mayer and Walker, eds., Academic Press, London,
1987); and in Ausubel et al., Current Protocols in Molecular
Biology, John Wiley and Sons, Baltimore, Md. (1989).
Gene Construction
[0238] Constructs of the present invention are constructed based on
the sequence information provided herein or in the art utilizing
standard molecular biology techniques, including, but not limited
to, the following. First, a series complementary oligonucleotide
pairs of 80-90 nucleotides each in length and spanning the length
of the construct are synthesized by standard methods. These
oligonucleotide pairs are synthesized such that upon annealing,
they form double stranded fragments of 80-90 base pairs, containing
cohesive ends. The single-stranded ends of each pair of
oligonucleotides are designed to anneal with a single-stranded end
of an adjacent oligonucleotide duplex. Several adjacent
oligonucleotide pairs prepared in this manner are allowed to
anneal, and approximately five to six adjacent oligonucleotide
duplex fragments are then allowed to anneal together via the
cohesive single stranded ends. This series of annealed
oligonucleotide duplex fragments is then ligated together and
cloned into a suitable plasmid, such as the TOPO.RTM. vector
available from Invitrogen Corporation, Carlsbad, Calif. The
construct is then sequenced by standard methods. Constructs
prepared in this manner, comprising 5 to 6 adjacent 80 to 90 base
pair fragments ligated together, i.e., fragments of about 500 base
pairs, are prepared, such that the entire desired sequence of the
construct is represented in a series of plasmid constructs. The
inserts of these plasmids are then cut with appropriate restriction
enzymes and ligated together to form the final construct. The final
construct is then cloned into a standard bacterial cloning vector,
and sequenced. The oligonucleotides and primers referred to herein
can easily be designed by a person of skill in the art based on the
sequence information provided herein and in the art, and such can
be synthesized by any of a number of commercial nucleotide
providers, for example Retrogen, San Diego, Calif., and GENEART,
Regensburg, Germany.
Plasmid Vectors
[0239] Constructs of the present invention can be inserted, for
example, into eukaryotic expression vectors VR1012 or VR10551.
These vectors are built on a modified pUC18 background (see
Yanisch-Perron, C., et al. Gene 33:103-119 (1985)), and contain a
kanamycin resistance gene, the human cytomegalovirus immediate
early promoter/enhancer and intron A, and the bovine growth hormone
transcription termination signal, and a polylinker for inserting
foreign genes. See Hartikka, J., et al., Hum. Gene Ther.
7:1205-1217 (1996). However, other standard commercially available
eukaryotic expression vectors may be used in the present invention,
including, but not limited to: plasmids pcDNA3, pHCMV/Zeo, pCR3.1,
pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV,
pUB6/V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San
Diego, Calif.), and plasmid pCI (available from Promega, Madison,
Wis.).
[0240] An optimized backbone plasmid, termed VR10551, has minor
changes from the VR1012 backbone described above. The VR10551
vector is derived from and similar to VR1012 in that it uses the
human cytomegalovirus immediate early (hCMV-IE) gene
enhancer/promoter and 5' untranslated region (UTR), including the
hCMV-IE Intron A. The changes from the VR1012 to the VR10551
include some modifications to the multiple cloning site, and a
modified rabbit 13 globin 3' untranslated region/polyadenylation
signal sequence/transcriptional terminator has been substituted for
the same functional domain derived from the bovine growth hormone
gene.
Plasmid DNA Purification
[0241] Plasmid DNA may be transformed into competent cells of an
appropriate Escherichia coli strain (including but not limited to
the DH5.alpha. strain) and highly purified covalently closed
circular plasmid DNA was isolated by a modified lysis procedure
(Horn, N. A., et al., Hum. Gene Ther. 6:565-573 (1995)) followed by
standard double CsCl-ethidium bromide gradient ultracentrifugation
(Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd
Ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989)).
Alternatively, plasmid DNAs are purified using Giga columns from
Qiagen (Valencia, Calif.) according to the kit instructions. All
plasmid preparations were free of detectable chromosomal DNA, RNA
and protein impurities based on gel analysis and the bicinchoninic
protein assay (Pierce Chem. Co., Rockford Ill.). Endotoxin levels
were measured using Limulus Amebocyte Lysate assay (LAL, Associates
of Cape Cod, Falmouth, Mass.) and were less than 0.6 Endotoxin
Units/mg of plasmid DNA. The spectrophotometric A.sub.260/A.sub.280
ratios of the DNA solutions were typically above 1.8. Plasmids were
ethanol precipitated and resuspended in an appropriate solution,
e.g., 150 mM sodium phosphate (for other appropriate excipients and
auxiliary agents, see U.S. patent application Publication
2002/0019358, published Feb. 14, 2002). DNA was stored at
-20.degree. C. until use. DNA was diluted by mixing it with 300 mM
salt solutions and by adding appropriate amount of USP water to
obtain 1 mg/ml plasmid DNA in the desired salt at the desired molar
concentration.
Plasmid Expression in Mammalian Cell Lines
[0242] The expression plasmids are analyzed in vitro by
transfecting the plasmids into a well characterized mouse melanoma
cell line (VM-92, also known as UM-449). See, e.g., Wheeler, C. J.,
Sukhu, L., Yang, G., Tsai, Y., Bustamente, C. Felgner, P. Norman, J
& Manthorpe, M. "Converting an Alcohol to an Amine in a
Cationic Lipid Dramatically Alters the Co-lipid Requirement,
Cellular Transfection Activity and the Ultrastructure of
DNA-Cytofectin Complexes," Biochim. Biophys. Acta. 1280:1-11
(1996). Other well-characterized human cell lines can also be used,
e.g. MRC-5 cells, American Type Culture Collection (ATCC) Accession
No. CCL-171 or human rhabdomyosarcoma cell line RD (ATCC CCL-136).
The transfection is performed using cationic lipid-based
transfection procedures well known to those of skill in the art.
Other transfection procedures are well known in the art and may be
used, for example electroporation and calcium chloride-mediated
transfection (Graham F. L. and A. J. van der Eb Virology 52:456-67
(1973)). Following transfection, cell lysates and culture
supernatants of transfected cells are evaluated to compare relative
levels of expression of herpes simplex virus antigen proteins. The
samples are assayed by western blots and ELISAs, using commercially
available polyclonal and/or monoclonal antibodies (available, e.g.,
from Research Diagnostics Inc., Flanders N.J.), so as to compare
both the quality and the quantity of expressed antigen.
[0243] In addition to plasmids encoding single herpes simplex virus
proteins, single plasmids which contain two or more herpes simplex
virus coding regions are constructed according to standard methods.
For example, a polycistronic construct, where two or more herpes
simplex virus coding regions are transcribed as a single transcript
in eukaryotic cells may be constructed by separating the various
coding regions with IRES sequences. Alternatively, two or more
coding regions may be inserted into a single plasmid, each with
their own promoter sequence.
Codon Optimization Algorithm
[0244] The following is an outline of the algorithm used to derive
human codon-optimized sequences of herpes simplex antigens.
[0245] Back Translation
[0246] Starting with the amino acid sequence, one can either (a)
manually backtranslate using the human codon usage table on the
world wide web at kazusa.or.ip/codon/
[0247] Homo sapiens [gbpri]: 55194 CDS's (24298072 codons)
[0248] Fields: [triplet] [frequency: per thousand] ([number])
TABLE-US-00006 TABLE 6 UUU 17.1(415589) UCU 14.7(357770) UAU
12.1(294182) UGU 10.0(243198) UUC 20.6(500964) UCC 17.6(427664) UAC
15.5(377811) UGC 12.2(297010) UUA 7.5(182466) UCA 12.0(291788) UAA
0.7(17545) UGA 1.5(36163) UUG 12.6(306793) UCG 4.4(107809) UAG
0.6(13416) UGG 12.7(309683) CUU 13.0(315804) CCU 17.3(419521) CAU
10.5(255135) CGU 4.6(112673) CUC 19.8(480790) CCC 20.1(489224) CAC
15.0(364828) CGC 10.7(259950) CUA 7.8(189383) CCA 16.7(405320) CAA
12.0(292745) CGA 6.3(152905) CUG 39.8(967277) CCG 6.9(168542) CAG
34.1(827754) CGG 11.6(281493) AUU 16.1(390571) ACU 13.0(315736) AAU
16.7(404867) AGU 11.9(289294) AUC 21.6(525478) ACC 19.4(471273) AAC
19.5(473208) AGC 19.3(467869) AUA 7.7(186138) ACA 15.1(366753) AAA
24.1(585243) AGA 11.5(278843) AUG 22.2(538917) ACG 6.1(148277) AAG
32.2(781752) AGG 11.4(277693) GUU 11.0(266493) GCU 18.6(451517) GAU
21.9(533009) GGU 10.8(261467) GUC 14.6(354537) GCC 28.4(690382) GAC
25.6(621290) GGC 22.5(547729) GUA 7.2(174572) GCA 16.1(390964) GAA
29.0(703852) GGA 16.4(397574) GUG 28.4(690428) GCG 7.5(181803) GAG
39.9(970417) GGG 16.3(396931) * Coding GC 52.45% 1st letter GC
56.04% 2nd letter GC 42.37% 3rd letter GC 58.93% (Table as of Nov.
6, 2003)
[0249] Or (b) log on to the world wide web at syntheticgenes.com
and use the backtranslation tool, as follows:
[0250] (1) Under Protein tab, paste amino acid sequence;
[0251] (2) Under download codon usage tab, highlight homo sapiens
and then download CUT.
TABLE-US-00007 TABLE 7 UUU 17.1(415589) UCU 14.7(357770) UAU
12.1(294182) UGU 10.0(243198) UUC 20.6(500964) UCC 17.6(427664) UAC
15.5(377811) UGC 12.2(297010) UUA 7.5(182466) UCA 12.0(291788) UAA
0.7(17545) UGA 1.5(36163) UUG 12.6(306793) UCG 4.4(107809) UAG
0.6(13416) UGG 12.7(309683) CUU 13.0(315804) CCU 17.3(419521) CAU
10.5(255135) CGU 4.6(112673) CUC 19.8(480790) CCC 20.1(489224) CAC
15.0(364828) CGC 10.7(259950) CUA 7.8(189383) CCA 16.7(405320) CAA
12.0(292745) CGA 6.3(152905) CUG 39.8(967277) CCG 6.9(168542) CAG
34.1(827754) CGG 11.6(281493) AUU 16.1(390571) ACU 13.0(315736) AAU
16.7(404867) AGU 11.9(289294) AUC 21.6(525478) ACC 19.4(471273) AAC
19.5(473208) AGC 19.3(467869) AUA 7.7(186138) ACA 15.1(366753) AAA
24.1(585243) AGA 11.5(278843) AUG 22.2(538917) ACG 6.1(148277) AAG
32.2(781752) AGG 11.4(277693) GUU 11.0(266493) GCU 18.6(451517) GAU
21.9(533009) GGU 10.8(261467) GUC 14.6(354537) GCC 28.4(690382) GAC
25.6(621290) GGC 22.5(547729) GUA 7.2(174572) GCA 16.1(390964) GAA
29.0(703852) GGA 16.4(397574) GUG 28.4(690428) GCG 7.5(181803) GAG
39.9(970417) GGG 16.3(396931)
(Table as of Nov. 6, 2003)
[0252] (3) Hit Apply button.
[0253] (4) Under Optimize TAB, open General TAB.
[0254] (5) Check use only most frequent codon box.
[0255] (6) Hit Apply button.
[0256] (7) Under Optimize TAB, open Motif TAB.
[0257] (8) Load desired cloning restriction sites into bad motifs;
load any undesirable sequences, such as Pribnow Box sequences
(TATAA), Chi sequences (GCTGGCGG), and restriction sites into bad
motifs.
[0258] (9) Under Output TAB, click on Start box. Output will
include sequence, motif search results (under Report TAB), and
codon usage report.
[0259] The program did not always use the most frequent codon for
amino acids such as cysteine proline, and arginine. To change this,
go back to the Edit CUT TAB and manually drag the rainbow colored
bar to 100% for the desired codon. Then re-do start under the
Output TAB.
[0260] The use of CGG for arginine can lead to very high GC
content, so AGA can be used for arginine as an alternative. The
difference in codon usage is 11.6 per thousand for CGG vs. 11.5 per
thousand for AGA.
[0261] Splice Donor and Acceptor Site Search
[0262] (1) Log on to Berkeley Drosophila Genome Project Website on
the world wide web at fruitfly.org/seg_tools/spice.html\.
[0263] (2) Check boxes for Human or other and both splice
sites.
[0264] (3) Select minimum scores for 5' and 3' splice sites between
0 and 1. [0265] Used the default setting at 0.4 where: [0266]
Default minimum score is 0.4, where:
TABLE-US-00008 [0266] % splice % false sites recognized positives
Human 5' Splice sites 93.2% 5.2% Human 3' Splice sites 83.8%
3.1%
[0267] (4) Paste in sequence.
[0268] (5) Submit.
[0269] (6) Based on predicted donors or acceptors, change the
individual codons until the sites are no longer predicted.
[0270] Add in 5' and 3' Sequences.
[0271] On the 5' end of the gene sequence, the restriction enzyme
site and Kozak sequence (gccacc) was added before ATG. On 3' end of
the sequence, tca was added following the stop codon (tga on
opposite strand) and then a restriction enzyme site. The GC content
and Open Reading Frames were then checked in SEC Central.
Preparation of Vaccine Formulations
[0272] Plasmid constructs comprising codon-optimized and
non-codon-optimized coding regions encoding gD, VP 11/12, VP13/14
and/or VP22; or alternatively coding regions (either
codon-optimized or non-codon optimized) encoding various herpes
simplex virus proteins or fragments, variants or derivatives either
alone or as fusions with a carrier protein, e.g., HBcAg, as well as
various controls, e.g., empty vector, are formulated with the
poloxamer CRL 1005 and BAK (Benzalkonium chloride 50% solution,
available from Ruger Chemical Co. Inc.) by the following methods.
Specific final concentrations of each component of the formulae are
described in the following methods, but for any of these methods,
the concentrations of each component maybe varied by basic
stoichiometric calculations known by those of ordinary skill in the
art to make a final solution having the desired concentrations.
[0273] For example, the concentration of CRL 1005 is adjusted
depending on, for example, transfection efficiency, expression
efficiency, or immunogenicity, to achieve a final concentration of
between about 1 mg/ml to about 75 mg/ml, for example, about 1
mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml,
about 6.5 mg/ml, about 7 mg/ml, about 7.5 mg/ml, about 8 mg/ml,
about 9 mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml,
about 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml,
about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml,
about 65 mg/ml, about 70 mg/ml, or about 75 mg/ml of CRL 1005.
[0274] Similarly the concentration of DNA is adjusted depending on
many factors, including the amount of a formulation to be
delivered, the age and weight of the subject, the delivery method
and route and the immunogenicity of the antigen being delivered. In
general, formulations of the present invention are adjusted to have
a final concentration from about 1 ng/ml to about 30 mg/ml of
plasmid (or other polynucleotide). For example, a formulation of
the present invention may have a final concentration of about 1
ng/ml, about 5 ng/ml, about 10 ng/ml, about 50 ng/ml, about 100
ng/ml, about 500 ng/ml, about 1 .mu.g/ml, about 5 .mu.g/ml, about
10 .mu.g/ml, about 50 .mu.g/ml, about 200 .mu.g/ml, about 400
.mu.g/ml, about 600 .mu.g/ml, about 800 .mu.g/ml, about 1 mg/ml,
about 2 mg/ml, about 2.5, about 3 mg/ml, about 3.5, about 4 mg/ml,
about 4.5, about 5 mg/ml, about 5.5 mg/ml, about 6 mg/ml, about 7
mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 20
mg/ml, or about 30 mg mg/ml of a plasmid.
[0275] Certain formulations of the present invention include a
cocktail of plasmids of the present invention, e.g., comprising
coding regions encoding herpes simplex virus proteins gD, VP 11/12,
VP13/14 and/or VP22 and optionally, plasmids encoding immunity
enhancing proteins, e.g., cytokines. Various plasmids desired in a
cocktail are combined together in PBS or other diluent prior to the
addition to the other ingredients. Furthermore, plasmids may be
present in a cocktail at equal proportions, or the ratios may be
adjusted based on, for example, relative expression levels of the
antigens or the relative immunogenicity of the encoded antigens.
Thus, various plasmids in the cocktail may be present in equal
proportion, or up to twice or three times as much of one plasmid
may be included relative to other plasmids in the cocktail.
[0276] Additionally, the concentration of BAK may be adjusted
depending on, for example, a desired particle size and improved
stability. Indeed, in certain embodiments, formulations of the
present invention include CRL 1005 and DNA, but are free of BAK. In
general BAK-containing formulations of the present invention are
adjusted to have a final concentration of BAK from about 0.05 mM to
about 0.5 mM. For example, a formulation of the present invention
may have a final BAK concentration of about 0.05 mM, 0.1 mM, 0.2
mM, 0.3 mM, 0.4 mM or 0.5 mM.
[0277] The total volume of the formulations produced by the methods
below may be scaled up or down, by choosing apparatus of
proportional size. Finally, in carrying out any of the methods
described below, the three components of the formulation, BAK, CRL
1005, and plasmid DNA, may be added in any order. In each of these
methods described below the term "cloud point" refers to the point
in a temperature shift, or other titration, at which a clear
solution becomes cloudy, i.e., when a component dissolved in a
solution begins to precipitate out of solution.
Thermal Cycling of a Pre-Mixed Formulation
[0278] This example describes the preparation of a formulation
comprising 0.3 mM BAK, 7.5 mg/ml CRL 1005, and 5 mg/ml of DNA in a
total volume of 3.6 ml. The ingredients are combined together at a
temperature below the cloud point and then the formulation is
thermally cycled to room temperature (above the cloud point)
several times.
[0279] A 1.28 mM solution of BAK is prepared in PBS, 846 .mu.l of
the solution is placed into a 15 ml round bottom flask fitted with
a magnetic stirring bar, and the solution is stirred with moderate
speed, in an ice bath on top of a stirrer/hotplate (hotplate off)
for 10 minutes. CRL 1005 (27 .mu.l) is then added using a 100 .mu.l
positive displacement pipette and the solution is stirred for a
further 60 minutes on ice. Plasmids comprising codon-optimized
coding regions, and optionally, additional plasmids comprising
codon-optimized or non-codin-optimized coding regions encoding,
e.g., additional herpes simplex virus proteins, and or other
proteins, e.g., cytokines, are mixed together at desired
proportions in PBS to achieve 6.4 mg/ml total DNA. This plasmid
cocktail is added drop wise, slowly, to the stirring solution over
1 min using a 5 ml pipette. The solution at this point (on ice) is
clear since it is below the cloud point of the poloxamer and is
further stirred on ice for 15 min. The ice bath is then removed,
and the solution is stirred at ambient temperature for 15 minutes
to produce a cloudy solution as the poloxamer passes through the
cloud point.
[0280] The flask is then placed back into the ice bath and stirred
for a further 15 minutes to produce a clear solution as the mixture
is cooled below the poloxamer cloud point. The ice bath is again
removed and the solution stirred at ambient temperature for a
further 15 minutes. Stirring for 15 minutes above and below the
cloud point (total of 30 minutes), is defined as one thermal cycle.
The mixture is cycled six more times. The resulting formulation may
be used immediately, or may be placed in a glass vial, cooled below
the cloud point, and frozen at -80.degree. C. for use at a later
time.
Animal Immunizations
[0281] The immunogenicity of the various herpes simplex virus
expression products encoded by the codon-optimized polynucleotides
described herein are initially evaluated based on each plasmid's
ability to mount an immune response in vivo. Plasmids are tested
individually and in combinations by injecting single constructs as
well as multiple constructs. Immunizations are initially carried
out in animals, such as mice, rabbits, goats, sheep, non-human
primates, or other suitable animal, by intramuscular (IM) or
intradermal (ID) injections. Serum is collected from immunized
animals, and the antigen specific antibody response is quantified
by ELISA assay using purified immobilized antigen proteins in a
protein--immunized subject antibody--anti-species antibody type
assay, according to standard protocols. The tests of immunogenicity
further include measuring antibody titer, neutralizing antibody
titer, T-cell proliferation, T-cell secretion of cytokines,
cytolytic T cell responses, and by direct enumeration of antigen
specific CD4+ and CD8+ T-cells. Correlation to protective levels of
the immune responses in humans are made according to methods well
known by those of ordinary skill in the art.
DNA Formulations
[0282] Plasmid DNA is formulated with a poloxamer. Alternatively,
plasmid DNA is prepared and dissolved at a concentration of about
0.1 mg/ml to about 10 mg/ml, preferably about 1 mg/ml, in PBS with
or without transfection-facilitating cationic lipids, e.g.,
DMRIE/DOPE at a 4:1 DNA:lipid mass ratio. Alternative DNA
formulations include 150 mM sodium phosphate instead of PBS,
adjuvants, e.g., VAXFECTIN adjuvant at a 4:1 DNA: VAXFECTIN
adjuvant mass ratio, mono-phosphoryl lipid A (detoxified endotoxin)
from S. Minnesota (MPL) and trehalosedicorynomycolateAF (TDM), in
2% oil (squalene)-TWEEN-80 polysorbate 80-water (MPL+TDM, available
from Sigma/Aldrich, St. Louis, Mo., (catalog # M6536)), a
solubilized mono-phosphoryl lipid A formulation (AF, available from
Corixa), or
(.+-.)-N-(3-Acetoxypropyl)-N,N-dimethyl-2,3-bis(octyloxy)-1-propanaminium
chloride (compound # VC1240) (see Shriver, J. W. et al., Nature
415:331-335 (2002), and P.C.T. Publication No. WO 02/00844 A2.
Animal Immunizations
[0283] Plasmid constructs comprising codon-optimized and
non-codon-optimized coding regions encoding gD, VP 11/12, VP13/14
and/or VP22; or alternatively coding regions (either
codon-optimized or non-codon optimized) encoding various herpes
simplex virus proteins or fragments, variants or derivatives either
alone or as fusions with a carrier protein, e.g., HBcAg, as well as
various controls, e.g., empty vector, are injected into BALB/c mice
as single plasmids or as cocktails of two or more plasmids, as
either DNA in PBS or formulated with the poloxamer-based delivery
system: 2 mg/ml DNA, 3 mg/ml CRL 1005, and 0.1 mM BAK. Groups of 10
mice are immunized three times, at biweekly intervals, and serum is
obtained to determine antibody titers to each of the antigens.
Groups are also included in which mice are immunized with a
trivalent preparation, containing each of the three plasmid
constructs in equal mass.
[0284] The immunization schedule is as follows:
[0285] Day 3 Pre-bleed
[0286] Day 0 Plasmid injections, intramuscular, bilateral in rectus
femoris, 5-50 .mu.g/leg
[0287] Day 21 Plasmid injections, intramuscular, bilateral in
rectus femoris, 5-50 .mu.g/leg
[0288] Day 49 Plasmid injections, intramuscular, bilateral in
rectus femoris, 5-50 .mu.g/leg
[0289] Day 59 Serum collection
[0290] Serum antibody titers are determined by ELIS A with
recombinant proteins, peptides or transfection supernatants and
lysates from transfected VM-92 cells live, inactivated, or lysed
virus.
Immunization of Mice with Vaccine Formulations Using a VAXFECTIN
Adjuvant
[0291] VAXFECTIN adjuvant (a 1:1 molar ratio of the cationic lipid
VC 1052 and the neutral co-lipid DPyPE) is a synthetic cationic
lipid formulation which enhances antibody titers against when
administered with DNA intramuscularly to mice.
[0292] VAXFECTIN adjuvant mixtures are prepared by mixing
chloroform solutions of VC1052 cationic lipid with chloroform
solutions of DpyPE neutral co-lipid. Dried films are prepared in 2
ml sterile glass vials by evaporating the chloroform under a stream
of nitrogen, and placing the vials under vacuum overnight to remove
solvent traces. Each vial contains 1.5 .mu.mole each of VC1052 and
DPyPE. Liposomes are prepared by adding sterile water followed by
vortexing. The resulting liposome solution is mixed with DNA at a
phosphate mole:cationic lipid mole ratio of 4:1.
[0293] Plasmid constructs comprising codon-optimized and
non-codon-optimized coding regions encoding gD, VP 11/12, VP13/14
and/or VP22; or alternatively coding regions (either
codon-optimized or non-codon optimized) encoding various herpes
simplex virus proteins or fragments, variants or derivatives either
alone or as fusions with a carrier protein, e.g., HBcAg, as well as
various controls, e.g., empty vector, are mixed together at desired
proportions in PBS to achieve a final concentration of 1.0 mg/ml.
The plasmid cocktail, as well as the controls, are formulated with
VAXFECTIN adjuvant. Groups of 5 BALB/c female mice are injected
bilaterally in the rectus femoris muscle with 50 .mu.l of DNA
solution (100 .mu.l total/mouse), on days 1 and 21 and 49 with each
formulation. Mice are bled for serum on days 0 (prebleed), 20
(bleed 1), and 41 (bleed 2), and 62 (bleed 3), and up to 40 weeks
post-injection. Antibody titers to the various herpes simplex virus
proteins encoded by the plasmid DNAs are measured by ELISA.
[0294] Cytolytic T-cell responses are measured as described in
Hartikka et al. "Vaxfectin Enhances the Humoral Response to Plasmid
DNA-encoded Antigens," Vaccine 19:1911-1923 (2001). Standard
ELISPOT technology is used for the CD4+ and CD8+ T-cell assays.
Production of Antisera in Animals
[0295] Plasmid constructs comprising codon-optimized and
non-codon-optimized coding regions encoding gD, VP 11/12, VP13/14
and/or VP22; or alternatively coding regions (either
codon-optimized or non-codon optimized) encoding various herpes
simplex virus proteins or fragments, variants or derivatives either
alone or as fusions with a carrier protein, e.g., HBcAg, as well as
various controls, e.g., empty vector, are prepared according to the
immunization scheme described above and injected into a suitable
animal for generating polyclonal antibodies. Serum is collected and
the antibody titered as above.
[0296] Monoclonal antibodies are also produced using hybridoma
technology (Kohler, et al., Nature 256:495 (1975); Kohler, et al.,
Eur. J. Immunol. 6:511 (1976); Kohler, et al., Eur. J. Immunol.
6:292 (1976); Hammerling, et al., in Monoclonal Antibodies and
T-Cell Hybridomas, Elsevier, N.Y., (1981), pp. 563-681. In general,
such procedures involve immunizing an animal (preferably a mouse)
as described above. The splenocytes of such mice are extracted and
fused with a suitable myeloma cell line. Any suitable myeloma cell
line may be employed in accordance with the present invention;
however, it is preferable to employ the parent myeloma cell line
(SP2O), available from the American Type Culture Collection,
Rockville, Md. After fusion, the resulting hybridoma cells are
selectively maintained in HAT medium, and then cloned by limiting
dilution as described by Wands et al., Gastroenterology 80:225-232
(1981), incorporated herein by reference in its entirety. The
hybridoma cells obtained through such a selection are then assayed
to identify clones which secrete antibodies capable of binding the
various herpes simplex virus proteins.
[0297] Alternatively, additional antibodies capable of binding to
herpes simplex virus proteins described herein may be produced in a
two-step procedure through the use of anti-idiotypic antibodies.
Such a method makes use of the fact that antibodies are themselves
antigens, and that, therefore, it is possible to obtain an antibody
which binds to a second antibody. In accordance with this method,
various herpes simplex virus-specific antibodies are used to
immunize an animal, preferably a mouse. The splenocytes of such an
animal are then used to produce hybridoma cells, and the hybridoma
cells are screened to identify clones which produce an antibody
whose ability to bind to the herpes simplex virus protein-specific
antibody can be blocked by the cognate herpes simplex virus
protein. Such antibodies comprise anti-idiotypic antibodies to the
herpes simplex virus protein-specific antibody and can be used to
immunize an animal to induce formation of further herpes simplex
virus-specific antibodies.
[0298] It will be appreciated that Fab and F(ab')2 and other
fragments of the antibodies of the present invention maybe used.
Such fragments are typically produced by proteolytic cleavage,
using enzymes such as papain (to produce Fab fragments) or pepsin
(to produce F(ab')2 fragments). Alternatively, gD, VP 11/12,
VP13/14 and/or VP22 binding fragments can be produced through the
application of recombinant DNA technology or through synthetic
chemistry.
[0299] It may be preferable to use "humanized" chimeric monoclonal
antibodies. Such antibodies can be produced using genetic
constructs derived from hybridoma cells producing the monoclonal
antibodies described above. Methods for producing chimeric
antibodies are known in the art. See, for review, Morrison, Science
229:1202 (1985); Oi, et al., BioTechniques 4:214 (1986); Cabilly,
et al., U.S. Pat. No. 4,816,567; Taniguchi, et al., EP 171496;
Morrison, et al., EP 173494; Neuberger, et al., WO 8601533;
Robinson, et al, WO 8702671; Boulianne, et al., Nature 312:643
(1984); Neuberger, et al., Nature 314:268 (1985).
[0300] These antibodies are used, for example, in diagnostic
assays, as a research reagent, to screen animals for identification
of the vaccine's effectiveness, or to further immunize animals to
generate herpes simplex virus-specific anti-idiotypic antibodies.
Non-limiting examples of uses for anti-herpes simplex virus
antibodies include use in Western blots, ELISA (competitive,
sandwich, and direct), immunofluorescence, immunoelectron
microscopy, radioimmunoassay, immunoprecipitation, agglutination
assays, immunodiffusion, immunoelectrophoresis, and epitope mapping
(Weir, D. Ed. Handbook of Experimental Immunology, 4th ed. Vols. I
and II, Blackwell Scientific Publications (1986)).
Mucosal Vaccination and Electrically Assisted Plasmid Delivery
Mucosal DNA Vaccination
[0301] Plasmid constructs comprising codon-optimized and
non-codon-optimized coding regions encoding gD, VP 11/12, VP13/14
and/or VP22; or alternatively coding regions (either
codon-optimized or non-codon optimized) encoding various herpes
simplex virus proteins or fragments, variants or derivatives either
alone or as fusions with a carrier protein, e.g., HBcAg, as well as
various controls, e.g., empty vector, (100 .mu.g/50 .mu.l total
DNA) are delivered to BALB/c mice at 0, 2 and 4 weeks via i.m.,
intranasal (i.n.), intravenous (i.v.), intravaginal (i.vag.),
intrarectal (i.r.) or oral routes. The DNA is delivered
unformulated or formulated with the cationic lipids DMRIE/DOPE (DD)
or GAP-DLRIE/DOPE (GD). As endpoints, serum IgG titers against the
various herpes simplex virus antigens are measured by ELISA and
splenic T-cell responses are measured by antigen-specific
production of IFN-gamma and IL-4 in ELISPOT assays. Standard
chromium release assays are used to measure specific cytotoxic T
lymphocyte (CTL) activity against the various herpes simplex virus
antigens. Tetramer assays are used to detect and quantify antigen
specific T-cells, with quantification being confirmed and
phenotypic characterization accomplished by intracellular cytokine
staining. In addition, IgG and IgA responses against the various
herpes simplex virus antigens are analyzed by ELISA of vaginal
washes.
Electrically-Assisted Plasmid Delivery
[0302] In vivo gene delivery may be enhanced through the
application of brief electrical pulses to injected tissues, a
procedure referred to herein as electrically-assisted plasmid
delivery (EAPD). See, e.g., Aihara, H. & Miyazaki, J. Nat.
Biotechnol. 16:867-70 (1998); Mir, L. M. et al., Proc. Natl Acad.
Sci. USA 96:4262-67 (1999); Hartikka, J. et al., Mol. Ther.
4:407-15 (2001); and Mir, L. M. et al.; Rizzuto, G. et al., Hum
Gene Ther 11:1891-900 (2000); Widera, G. et al, J. of Immuno.
164:4635-4640 (2000). The use of electrical pulses for cell
electropermeabilization has been used to introduce foreign DNA into
prokaryotic and eukaryotic cells in vitro. Cell permeabilization
can also be achieved locally, in vivo, using electrodes and optimal
electrical parameters that are compatible with cell survival.
[0303] The electroporation procedure can be performed with various
electroporation devices. These devices include external plate type
electrodes or invasive needle/rod electrodes and can possess two
electrodes or multiple electrodes placed in an array. Distances
between the plate or needle electrodes can vary depending upon the
number of electrodes, size of target area and treatment
subject.
[0304] The TriGrid needle array is a three electrode array
comprising three elongate electrodes in the approximate shape of a
geometric triangle. Needle arrays may include single, double,
three, four, five, six or more needles arranged in various array
formations. The electrodes are connected through conductive cables
to a high voltage switching device that is connected to a power
supply.
[0305] The electrode array is placed into the muscle tissue, around
the site of nucleic acid injection, to a depth of approximately 3
mm to 3 cm. The depth of insertion varies depending upon the target
tissue and size of patient receiving electroporation. After
injection of foreign nucleic acid, such as plasmid DNA, and a
period of time sufficient for distribution of the nucleic acid,
square wave electrical pulses are applied to the tissue. The
amplitude of each pulse ranges from about 100 volts to about 1500
volts, e.g., about 100 volts, about 200 volts, about 300 volts,
about 400 volts, about 500 volts, about 600 volts, about 700 volts,
about 800 volts, about 900 volts, about 1000 volts, about 1100
volts, about 1200 volts, about 1300 volts, about 1400 volts, or
about 1500 volts or about 1-1.5 kV/cm, based on the spacing between
electrodes. Each pulse has a duration of about 1 .mu.s to about
1000 .mu.s, e.g., about 1 .mu.s, about 10 .mu.s, about 50 .mu.s,
about 100 .mu.s, about 200 .mu.s, about 300 .mu.s, about 400 .mu.s,
about 500 .mu.s, about 600 .mu.s, about 700 .mu.s, about 800 .mu.s,
about 900 .mu.s, or about 1000 .mu.s, and a pulse frequency on the
order of about 1-10 Hz. The polarity of the pulses may be reversed
during the electroporation procedure by switching the connectors to
the pulse generator. Pulses are repeated multiple times. The
electroporation parameters (e.g. voltage amplitude, duration of
pulse, number of pulses, depth of electrode insertion and
frequency) will vary based on target tissue type, number of
electrodes used and distance of electrode spacing, as would be
understood by one of ordinary skill in the art.
[0306] Immediately after completion of the pulse regimen, subjects
receiving electroporation can be optionally treated with membrane
stabilizing agents to prolong cell membrane permeability as a
result of the electroporation. Examples of membrane stabilizing
agents include, but are not limited to, steroids (e.g.
dexamethasone, methylprednisone and progesterone), angiotensin II
and vitamin E. A single dose of dexamethasone, approximately 0.1 mg
per kilogram of body weight, should be sufficient to achieve a
beneficial affect.
[0307] EAPD techniques such as electroporation can also be used for
plasmids contained in liposome formulations. The liposome-plasmid
suspension is administered to the animal or patient and the site of
injection is treated with a safe but effective electrical field
generated, for example, by a TriGrid needle array. The
electroporation may aid in plasmid delivery to the cell by
destabilizing the liposome bilayer so that membrane fusion between
the liposome and the target cellular structure occurs.
Electroporation may also aid in plasmid delivery to the cell by
triggering the release of the plasmid, in high concentrations, from
the liposome at the surface of the target cell so that the plasmid
is driven across the cell membrane by a concentration gradient via
the pores created in the cell membrane as a result of the
electroporation.
[0308] To test the effect of electroporation on therapeutic protein
expression in non-human primates, male or female rhesus monkeys are
given either 2 or 6 i.m. injections of plasmid constructs
comprising codon-optimized and non-codon-optimized coding regions
encoding gD, VP 11/12, VP13/14 and/or VP22; or alternatively coding
regions (either codon-optimized or non-codon optimized) encoding
various herpes simplex virus proteins or fragments, variants or
derivatives either alone or as fusions with a carrier protein,
e.g., HBcAg, as well as various controls, e.g., empty vector, (0.1
to 10 mg DNA total per animal). Target muscle groups include, but
are not limited to, bilateral rectus fermoris, cranial tibialis,
biceps, gastrocenemius or deltoid muscles. The target area is
shaved and a needle array, comprising between 4 and 10 electrodes,
spaced between 0.5-1.5 cm apart, is implanted into the target
muscle. Once injections are complete, a sequence of brief
electrical pulses are applied to the electrodes implanted in the
target muscle using an Ichor TGP-2 pulse generator. The pulses have
an amplitude of approximately 120-200V. The pulse sequence is
completed within one second. During this time, the target muscle
may make brief contractions or twitches. The injection and
electroporation may be repeated.
[0309] Sera are collected from vaccinated monkeys at various time
points. As endpoints, serum IgG titers against the various herpes
simplex virus antigens are measured by ELISA and PBMC T-cell
proliferative responses are measured by antigen-specific production
of IFN-gamma and IL-4 in ELISPOT assays or by tetramer assays to
detect and quantify antigen specific T-cells, with quantification
being confirmed and phenotypic characterization accomplished by
intracellular cytokine staining. Standard chromium release assays
are used to measure specific cytotoxic T lymphocyte (CTL) activity
against the various TV antigens.
Vaccine Construction and Evaluation
[0310] UL46, 47, and 49 open reading frames (ORFs) from seven to
eight HSV-2 strains isolated from primary genital infections were
PCR-amplified and sequenced. The consensus was used for vaccine
design.
[0311] Plasmids were constructed by cloning synthetic, full-length,
codon-optimized UL46, UL47, or UL49 DNA into backbone plasmid
VR1012.
[0312] Codon-optimized UL46, 47, and 49 constructs were compared
with full length wild-type UL46, 47 and 49 by co-transfecting Cos-7
cells with the different individual constructs plus full-length
cDNA encoding HLA A*0201, A*0101, or B*0702. CD8.sup.+ T-cell
clones known to react to UL46, UL47, or UL49 epitopes were
incubated with the transfected cells, with an ELISA for IFN-.gamma.
secretion employed as readout.
[0313] DNA vaccines were formulated with PBS, with VAXFECTIN
adjuvant at 1 .mu.g DNA/1.09 .mu.g VAXFECTIN adjuvant, or with
poloxamer, such as, but not limited to, CRL1005, at 1 .mu.g DNA/1.5
.mu.g poloxamer in PBS.
Animals and Immunization Regimen
[0314] Female, 4-8 week old BALB/c mice (10 per group) were
immunized with 50 .mu.g DNA in 50 .mu.L by IM injection in
bilateral quadriceps (100 .mu.g/immunization) on Days 0, 14, and
28.
[0315] Sera were collected from each animal Days -1 (pre-bleed),
13, 27, and 42. Splenocytes were isolated at terminal sacrifice Day
42.
EUSA Evaluation of Antibody Response
[0316] Antigens for ELISA were derived from recombinant full-length
tegument proteins made by transiently transfecting VM92 cells with
the vaccine constructs and collecting supernatants.
[0317] Sera were serially diluted in TBS with 0.1% BSA and 0.05%
TWEEN-20 polysorbate 20. Standard reagents measured
antigen-specific IgG by absorbance at OD450 nm. A positive (Day 42)
serum pool for each antigen was run on every ELISA plate. An
exponential curve was generated from values in the mid portion of
dilution vs. OD.sub.450 (corrected for background absorbance).
Antibody titers for each animal at a time point were calculated as
the mean titer determined from the individual dilutions (calculated
from the standard curve). If the OD.sub.450 for all dilutions of
experimental sera were very low, the titer was assigned as 1:1.
Evaluation of Cellular Response by ELISPOT Assay
[0318] IFN-.gamma. secretion by T-cells was assessed by ELISPOT
assay with standard reagents. Plates were read by computer.
[0319] Splenocytes (0.5-1.times.10.sup.6 cells/well in duplicate
wells) from individual animals were stimulated using peptide pools
(18-24 13-mer peptides overlapping by 9 amino acids, at 0.42-0.56
.mu.g/ml each), or positive (Con A) or negative (media) controls.
Pooled responder splenocytes were also tested with single 13-mer
peptides at 10 .mu.g/ml.
[0320] Peptides found to produce responses were further evaluated
using shorter (9-11 aa) peptides, dose-response curves, and/or
CD4.sup.+ or CD8.sup.+ responders (negative selection, Miltenyi,
>80% pure) back-mixed with naive splenocyte APC. Plates with
too-numerous-to-count spots were arbitrarily assigned 103
spots.
Discussion
[0321] HSV-2 UL46, UL47, and UL49 DNA vaccines are immunogenic in
BALB/c (H-2d) mice.
[0322] H-2d CD8+ epitopes were found with all vaccines, and
CD4.sup.+ epitopes were found for UL46 and UL49.
[0323] CD8.sup.+ splenocytes respond to peptides at concentrations
from 10.sup.-3 to 10.sup.-6 .mu.M; CD4.sup.+ splenocytes require
peptide concentrations of 10.sup.-1 .mu.M or higher.
[0324] Cellular responses in BALB/c mice are greatest for UL47,
followed by UL49 and UL46, when expressed as SFU/106
splenocytes.
[0325] Relative antibody titers are UL49>UL47>UL46.
[0326] The poloxamer-based formulation boosted humoral immunity on
Day 42 for all three tegument DNA vaccines by 3-5-fold compared to
no adjuvant. Formulations based on Vaxfectin.TM. boosted antibody
responses to UL46 and UL49 DNA about 2-fold by Day 42 relative to
no adjuvant.
[0327] Poloxamer significantly boosted cellular responses to UL47
DNA vaccine (p=0.03), but not to UL46 or UL49 DNA vaccines.
VAXFECTIN adjuvant had no statistically significant adjuvant effect
on any tested vaccine (p>0.05).
[0328] Cellular immunity to three HSV-2 tegument proteins was
detectable after DNA vaccination. Responses to both UL47 and UL49
were particularly strong. Due to difficulties with peptide
synthesis, 19% of the UL46 peptides were missing in the assay, as
compared to 1 or 2 peptides missing for assays involving UL47 and
UL49. Nonetheless, multiple CD4.sup.+ and CD8.sup.+ epitopes have
been identified that should assist animal pathogenesis studies in
BALB/c mice.
[0329] The adjuvant effects of poloxamer and VAXFECTIN adjuvant
based formulations were moderate and inconsistent between antigens.
Adjuvant effect may be more apparent with a lower dose of antigen,
or when used in higher species.
Identify the HSV-2 Genes of Interest.
[0330] HSV-2 encodes .about.85 proteins (Roizman, B., Knipe, D. M.,
Whitley, R. J., Herpes simplex viruses, in Fields Virology, D. M.
Knipe, Howley, P. M., Editor. 2007, Lippincott, Williams, and
Wilkins: Philadelphia, p. 2501-2602). The pDNA approach is limited
to one or a few ORFs. Criteria for choosing ORFs include quick
recognition of infected cells, immunodominance (within-person and
within-population), antiviral effector functions, and localization
to lesions.
[0331] Tegument proteins UL46, UL47, UL49, and UL7 are CD8+
antigens (Koelle, D. M., et al., CD8 CTL from genital herpes
simplex lesions: recognition of viral tegument and immediate early
proteins and lysis of infected cutaneous cells. Journal of
Immunology, 2001. 166: p. 4049-4058). CD8.sup.+ T-cells specific
for UL47 and UL49 are abundant enough to be detected by "direct"
PBMC staining with HLA-peptide tetramers (Koelle, D. M., et al.,
Expression of cutaneous lymphocyte-associated antigen by
CD8+T-cells specific for a skin-tropic virus. Journal of Clinical
Investigation, 2002. 110: p. 537-548). CD8.sup.+ cells specific for
glycoprotein, capsid, or immediate early proteins are less
abundant, and have never been detected in "direct" PBMC staining
(Koelle, unpublished). Studies of up to 95 independently derived
HSV-2-specific CD8+ clones per subject showed that responses to
tegument proteins were immunodominant (Koelle, D. M., Liu Z.,
McClurkan C. L., Cevallos R. C., Vieira J., Hosken N. A., Meseda C.
A., Snow D. C., Wald A., Corey L., Immunodominance among herpes
simplex virus-specific CD8 T-cells expressing a tissue-specific
homing receptor. Proc Natl Acad Sci USA, 2003. 100: p.
12899-12904).
[0332] We then measured human CD8.sup.+ responses to 48 HSV-2 ORFs
(57% of the total) by making 5,230 synthetic peptides covering
these ORFs. CD8.sup.+ T-cells from peripheral blood were probed by
IFN-.gamma. ELISPOT as the readout (Hosken, N., McGowan P., Meier
A., Koelle D. M., Sleath P., Wegener F., Elliott M., Grabstein L.,
Posavad C., Corey L., Diversity of the CD8+T cell response to
herpes simpolex virus type 2 proteins among persons with genital
herpes. Journal of Virology, 2006. 80: p. 5509-5515). Among 24
HSV-2-infected subjects, 50% recognized the tegument proteins UL46
and UL47. Responses to UL49 were slightly lower (.about.40%).
[0333] A trial of a HSV-2 therapeutic vaccine comprised of an HLA
A*0201-restricted epitope in envelope glycoprotein B and an
adjuvant is being conducted. At baseline (n=42 persons), the
population prevalence of responses to an HLA A*0201 epitope in gB
is 45% compared to 68% for an A*0201 epitope in the tegument
protein UL47 (p=0.012, Fisher's exact test).
[0334] One advantage of tegument-specific CD8.sup.+ T-cells, over
cells specific for immediate early, capsid, or envelope proteins,
is that tegument-specific CD8.sup.+ T-cells can kill target cells
immediately after they are infected. This is due to recognition of
virion input protein, as proven in CTL assays with the
transcriptional blocker actinomycin D, or target cells sensitized
with UV-treated virus (Koelle, D. M., et al., CD8 CTL from genital
herpes simplex lesions: recognition of viral tegument and immediate
early proteins and lysis of infected cutaneous cells. Journal of
Immunology, 2001. 166: p. 4049-4058).
Recognition of Processed Tegument Input Protein Bypasses the Immune
Evasion by HSV TAP Inhibitor Protein ICP47.
[0335] In addition, tetramer in situ stain of human skin biopsies
in healing/post-healing phases of genital HSV-2 lesions, show that
tetramer-specific CD8+ T-cells localize adjacent to HSV-2-infected
cells during recurrences, and monitor the region of peripheral
nerve endings at the dermal-epidermal junction after healing (Zhu,
J., Koelle, D. M., Cao, J., Vezquez, J., Huang, M X., Hladik, F.,
Wald, A., Corey, L., Peripheral virus-specific CD8+T cells
contiguous to sensory nerve endings limit HSV-2 reactivation in
human genital skin. Journal of Experimental Medicine, 2007. epub
Feb. 26 2007: p. epub Feb. 26 2007).
[0336] Taken together, within-population and within-subject
dominance, high absolute numerical levels, localization to lesional
and post-healing skin, antiviral effector functions, and immediate
recognition of infected cells all argue that HSV-2 tegument
proteins are rational targets for CD8+-directed therapeutic
approach to decreasing HSV-2 lesions, symptoms, and shedding.
Determine the Amino Acid Sequence for Candidate Vaccin Tegument
HSV-2 Genes.
[0337] We sequenced tegument proteins UL46, UL47, and UL49 in
circulating North American HSV-2 strains. We isolated HSV-2 from
persons with documented primary genital herpes (Ashley, R. A., et
al., Comparison of Western blot (immunoblot) and glycoprotein
G-specific immunoblot for detecting antibodies to herpes simplex
types 1 and 2 in human sera. Journal of Clinical Microbiology,
1988. 26: p. 662-667), passaged them minimally in vitro,
PCR-amplified tegument genes with an accurate polymerase, and
sequenced them bi-directionally (Martin, E., Koelle D M, Byrd B,
Huang M L, Vieira J, Corey L, Wald A, Sequence-based methods for
identifying epidemiologically linked herpes simplex virus type 2
strains. J Clin Microbiol, 2006. 44(7): p. 2541-6). We identified
many loci at which all the wild-type strains shared the same coding
difference from HG52 (Dolan, A., et al., The genome sequence of
herpes simplex virus type 2. Journal of Virology, 1998. 72: p.
2010-2021). At other loci, there were polymorphic amino acids that
differed between wild-type strains. For these, we picked the
prevalent alleles for our vaccine composition. (Table 8)
TABLE-US-00009 TABLE 8 Selected coding polymorphisms in eight
wild-type HSV-2 isolates. Amino acid (AA) numbers per HG52
nomenclature (Dolan, A., et al., The genome sequence of herpes
simplex virus type 2. Journal of Virology, 1998. 72: p. 2010-2021).
Table entries list HG52 AA followed by wild-type. Vaccine sequences
VR2145, VR2144, and VR2143 are also shown. UL46 amino acid strain
78 110 364 425 436 471 474 587 594 613 634 637 638 644 672 673 346
TR CF delA EK PL RQ LD LP RP RG 2589 AS TR CF HP PL RQ LD LP RP RG
2899 KN TR CF delA EK GS PL RQ LD LP RP RG 7124 TR CF delA GS PL RQ
LD LP RP RG 7566 KN TR CF delA EK GS PL RQ LD LP RP RG 10875 KN TR
CF delA EK GS PL RQ LD LP RP RG 11449 TR AV VG CF delA EK GS PL RQ
LD LP RP RG 16293 KN TR CF delA EK GS PL RQ LD LP RP RG VR2145 A K
R A V F H delA K S L Q D P P G UL47 amino acid UL49 amino acid
strain 3 38 69 82 156 172 177 529 strain 73-74 76 87 94 134 346 VA
PS PA SP 346 SE insert SA SP 2589 NS PA SP RH 2589 SE insert DN RH
SP 2899 VG NS PA SP 2899 7566 VG NS PA SP 7566 10875 VG RQ NS PA SP
10875 11449 VG NS PA SP 11449 16293 VG NS PA SP 16293 VR2144 V G P
R S A P R VR2143 no insert D R S S
[0338] We were concerned that CD8+ T-cell epitopes might be under
selective pressure to mutate and "escape" CD8.sup.+ CTL, as proven
for HIV (Nolan, D., I. James, and S. Mallal, HIV/AIDS. HIV:
experiencing the pressures of modern life. Science, 2005.
307(5714): p. 1422-4). HSV-2 has an accurate DNA polymerase, but
mutations do occur during acyclovir therapy (Czartoski, T., et al.,
Fulminant, acyclovir-resistant, herpes simplex virus type 2
hepatitis in an immunocompetent woman. J Clin Microbiol, 2006.
44(4): p. 1584-6). We sequenced more than 100 additional wild-type
HSV-2 strains, focusing on known CD8.sup.+ epitopes regions in
UL46, UL47, and UL49. We found no coding polymorphisms in or near
CD8+ epitopes (Koelle, D. M., et al., CD8 CTL from genital herpes
simplex lesions: recognition of viral tegument and immediate early
proteins and lysis of infected cutaneous cells. Journal of
Immunology, 2001. 166: p. 4049-4058; Koelle, D. M., Liu Z.,
McClurkan C. L., Cevallos R. C., Vieira J., Hosken N. A., Meseda C.
A., Snow D. C., Wald A., Corey L., Immunodominance among herpes
simplex virus-specific CD8 T-cells expressing a tissue-specific
homing receptor. Proc Natl Acad Sci USA, 2003. 100: p. 12899-12904)
in UL46 or UL47, or in or near CD4.sup.+ epitopes (Koelle, D. M.,
et al., Recognition of herpes simplex virus type 2 tegument
proteins by CD4 T cells infiltrating human genital herpes lesions.
Journal of Virology, 1998. 72: p. 7476-7483; Posavad, C M., et al.,
T cell immunity to herpes simplex virus in seronegative persons:
silent infection or acquired immunity. Journal of Immunology, 2003.
170: p. 4380-4388; Koelle, D. M., et al., Tegument-specific,
virus-reactive CD4 T-cells localize to the cornea in herpes simplex
virus interstitial keratitis in humans. Journal of Virology, 2000.
74: p. 10930-10938) in any protein.
[0339] In contrast, there is marked heterogeneity in the HLA
B*0702-restricted CD8.sup.+ epitope in UL49 (AA 49-57). While 70%
of isolates have the majority sequence RPRGEVREFL, 29% have the
minority RPMREVRFL, and 1% have the rare RPRGKVRFL. Our immune
studies (Koelle, D. M., Liu, C., Byrd, B., Sette, A., Sidney, J.,
Wald, A. HSV-2 VP22 sequences from wild-type isolates that escape a
dominant CD8 CTL response in linkage with a polymorphism at an
adjacent casein kinase II substrate domain, in 30th International
Herpesvirus Workshop, 2005. Turku, Finland), in brief, show that
while all 3 variants bind well to recombinant HLA B*0702
[IC.sub.50<2 nM, assays as per (Southwood, S., et al., Several
common HLA-DR types share largely overlapping peptide binding
repertoires. J Immunol, 1998. 160(7): p. 3363-73)], CD8.sup.+
T-cells specific for the "majority" are not cross-reactive with
"minority" or "rare" variants. Vaccination with the 70% consensus
"majority" would miss 30% of circulating strains.
Optimize the Genes for Protein Expression and Stability, Synthesize
them, and Clone them into a pDNA Backbone.
[0340] After establishing the AA sequences for the pDNA vaccines,
we used proprietary codon optimization algorithms with the goals of
high eukaryotic expression. The genes were synthesized by GeneArt.
Versions of UL46, UL47, and UL49 were made with or without the
gD.sub.1 epitope tag QPELAPEDPED. Each was cloned into plasmid
VR1012 (Hartikka, J., et al., An improved plasmid DNA expression
vector for direct injection into skeletal muscle. Hum Gene Ther,
1996. 7(10): p. 1205-17). VR1012 encodes kanamycin resistance, and
contains a promoter/enhancer and intron A of CMV immediate early
gene 1, and a bovine growth hormone-based terminator. VR1012 was
chosen because 1) it achieves high level expression in many cells,
animal species, and tissues; 2) there has been no stability
problems in in-house studies; 3) high plasmid yields are obtained
in E. coli; and most importantly 4) VR1012-based products are in
clinical trials. The inserts were sequence verified to an average
redundancy of 4-fold.
[0341] VAXFECTIN adjuvant was used for intramuscular injection
studies. VAXFECTIN adjuvant is an equimolar mixture of VC1052
((.+-.)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis
(myristoleyloxy)-1-propanaminium bromide) and DPyPE
(diphytanoylphosphatidyl-ethanolamine) (Hartikka, J., et al., An
improved plasmid DNA expression vector for direct injection into
skeletal muscle. Hum Gene Ther, 1996. 7(10): p. 1205-17). A lipid
film was prepared by mixing chloroform solutions of VC1052 and
DPyPE in glass vials, evaporating the chloroform, and vacuum
packing. At the time of vaccination, the lipid film was
reconstituted to 2.18 mg/mL with 1 mL of 0.9% saline. Vaccines were
prepared at a final pDNA (phosphate): cationic lipid molar ratio of
4:1 by adding an equal volume of lipid to pDNA (2 mg/mL in 0.9%
saline, 20 mM sodium phosphate). Reconstituted vaccine was held at
room temperature and used within 60 minutes.
[0342] Initial experiments expressed the gD-tagged versions of
UL46, UL47, and UL49 by transient transfection of VM92 cells
(Kumar, S., et al., A DNA vaccine encoding the 42 kDa C-terminus of
merozoite surface protein 1 of Plasmodium falciparum induces
antibody, interferon-gamma and cytotoxic T cell responses in rhesus
monkeys: immuno-stimulatory effects of granulocyte
macrophage-colony stimulating factor. Immunol Lett, 2002. 81(1): p.
13-24). Immunoblots showed bands at the predicted MW (not shown).
All subsequent experiments used the full-length tegument constructs
without the epitope tag. We used human CD8+CTL clones specific for
UL46, UL47, and UL49 (Koelle, D. M., et al., CD8 CTL from genital
herpes simplex lesions: recognition of viral tegument and immediate
early proteins and lysis of infected cutaneous cells. Journal of
Immunology, 2001. 166: p. 4049-4058; Koelle, D. M., Liu Z.,
McClurkan C. L., Cevallos R. C., Vieira J., Hosken N. A, Meseda C.
A., Snow D. C., Wald A., Corey L., Immunodominance among herpes
simplex virus-specific CD8 T-cells expressing a tissue-specific
homing receptor. Proc Natl Acad Sci USA, 2003. 100: p. 12899-12904)
to establish that the pDNA vaccines encoded proteins that are
processed and presented to CD8+ T-cells. COS-7 cells were
co-transfected with (1) candidate vaccine plasmid and (2) cDNA
encoding a specific human HLA class I .alpha.-chain. The human HLA
class I heavy chains form a functional heterodimer with non-human
primate (COS-7 cell) .beta..sub.2 microglobulin (.beta..sub.2M). If
the vaccine construct encodes a protein that can be processed to
antigenic peptides, some HLA class I-.beta..sub.2M heterodimers
will translocate to the cell surface loaded with HSV-2 peptides.
After two days, human CD8.sup.+ T cell clones specific for relevant
tegument proteins were added. T-cell activation was detected by
IFN-.gamma. ELISA of the supernatant (Koelle, D. M., et al., CD8
CTL from genital herpes simplex lesions: recognition of viral
tegument and immediate early proteins and lysis of infected
cutaneous cells. Journal of Immunology, 2001. 166: p. 4049-4058;
Koelle, D. M., et al., Expression of cutaneous
lymphocyte-associated antigen by CD8+T-cells specific for a
skin-tropic virus. Journal of Clinical Investigation, 2002. 110: p.
537-548). Specific responses were detected (FIG. 3). T cell clones
did not recognize COS-7 cells transfected with HLA class I cDNA
alone, HSV-2 plasmid DNA alone, or HSV-2 DNA plus the "wrong" HLA
(not shown).
[0343] The proteins encoded by the candidate vaccines were also
recognized by human anti-HSV antibodies. To make vaccine-encoded
protein, VM92 cells (Kumar, S., et al., A DNA vaccine encoding the
42 kDa C-terminus of merozoite surf ace protein 1 of Plasmodium
falciparum induces antibody, interferon-gamma and cytotoxic T cell
responses in rhesus monkeys: immuno-stimulatory effects of
granulocyte macrophage-colony stimulating factor. Immunol Lett,
2002. 81(1): p. 13-24) were transfected with the vaccine plasmids,
and supernatants collected. These were used as antigen (1:5
dilution) to coat ELISA plates. Pooled sera from HSV-2 seropositive
individuals bound recombinant tegument proteins (FIG. 4), while
pooled sera from HSV-2 seronegative individuals did not. These
tests show that bona fide HSV-2-specific T-cells and antibodies
recognize vaccine-encoded HSV-2 tegument proteins.
Measure Immune Responses to HSV-2 Tegument Plasmids Alone or in
Combination in Mice.
[0344] We chose the female BALB/c mouse (H-2.sup.d) so we could
combine immunogenicity and protective efficacy tests. The only
previously known HSV-2 CD8+ epitope in BALB/c mice is in protein
ICP27 (Haynes, J., Arrington J, Dong L, Braun R P, Payne L G,
Potent protective cellular immune responses generated by a DNA
vaccine encoding HSV-2 ICP27 and the E. coli heat labile
enterotoxin. Vaccine, 2006. 24(23): p. 5016-26). Several
type-common regions of gD are CD4+ epitopes in these animals
(BenMohamed, L., et al., Identification of novel immunodominant
CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus
glycoprotein D that confer protective immunity. J Virol, 2003.
77(17): p. 9463-73). BALB/c mice are very susceptible to
intravaginal infection with HSV-2 (Lopez, C., Genetics of natural
resistance to herpes virus infections in mice. Nature, 1975. 258:
p. 1352-1353).
[0345] Humoral response. We immunized mice with 100 .mu.g pDNA on
days 0, 14, and 28 as 50 .mu.M per quadriceps. We compared
VAXFECTIN adjuvant and a CRL 1005 poloxamer as shown in 6,844,001
as Example 1 (Selinsky, C, et al., A DNA-based vaccine for the
prevention of human cytomegahvirus-associated diseases. Hum Vaccin,
2005.1(1): p. 16-23) as adjuvants versus PBS. As we have focused on
VAXFECTIN adjuvant, and results (titers, speed to antibody, titers
at each time point for antibody, and IFN-.gamma. sfu/10.sup.6
splenocytes for T-cells) were generally similar for the adjuvants
and PBS at this high pDNA dose, only VAXFECTIN adjuvant data are
shown.
[0346] Every mouse produced antibodies against the relevant protein
by the second immunization (FIGS. 20A through 20I). Antibody titers
were significantly higher from one measurement to the next
(p<0.03, paired two-tailed t-test) for each vaccine and every
time-point comparison. We also verified that vaccine-elicited
antibodies bound to a whole HSV-2 lysate (FIG. 4). These data again
show the plasmids encode bona fide HSV-2 proteins.
CD8.sup.+ and CD4.sup.+ Responses to Tegument DNA Vaccines in
BALB/c Mice.
[0347] Overlapping peptides 13 AA long, offset by four AA, were
synthesized to match the predicted vaccine sequences (Table 8).
Initial assays used peptide pools (18-24 peptides/pool,
concentration for each peptide 0.5 .mu.g/mL). Splenocytes from
individual mice (FIG. 6A through 6C), harvested two weeks after the
third immunization, were assayed. The readout was IFN-.gamma.
ELISPOT (Haynes, J., Arrington J, Dong L, Braun R P, Payne L G,
Potent protective cellular immune responses generated by a DNA
vaccine encoding HSV-2 ICP27 and the E. coli heat labile
enterotoxin. Vaccine, 2006. 24(23): p. 5016-26). Responses to pools
were summed for each animal to give cumulative responses, expressed
as spot forming units (sfu)/10.sup.6 splenocytes. For UL47 and
UL49, responses were higher than from naive mice (p<0.01,
two-tailed t-test). For UL46, responses were not statistically
different from naive (p=0.37), due to high responses in two naive
mice. However, testing of single peptides from UL46 still disclosed
antigenic peptides.
[0348] The individual peptides in positive pools were tested in
follow-up ELISPOT, and in each case, single or neighboring
overlapping peptides were positive. We used overlap regions (when
present) and MHC-peptide epitope prediction algorithms (Bui, H. H.,
et al., Automated generation and evaluation of specific MHC binding
predictive tools: ARB matrix applications. Immunogenetics, 2005;
Parker, K. C., M. A. Bednarek, and J. E. Coligan, Scheme for
ranking potential HLA-A2 binding peptides based on independent
binding of individual peptide side-chains. Journal of Immunology,
1994. 152: p. 163-168; Rammensee, H., et al., SYFPEITHI: database
for MHC ligands and peptide motifs. Immunogenetics, 1999. 50: p.
213-319) to pick shorter peptides for further tests. We used
negative selection with magnetic bead-conjugated antibodies to
enrich CD4.sup.+ or CD8.sup.+ splenocytes, and back-mixed these
with naive congenic splenocytes as APC. In IFN-.gamma. ELISPOT,
potent CD8.sup.+ epitopes were found for each vaccine protein.
CD4.sup.+ responses were detected in UL46 and UL49. CD4.sup.+
responses were generally weaker than CD8.sup.+ responses when
quantified as sfu/10.sup.6 splenocytes or EC.sub.50 (the
concentration giving 50% of the maximum response) (FIG. 21). Some
CD8.sup.+ epitopes were active at 10.sup.-12 M (FIG. 21). Such
potent CD8+ epitopes typically bind tightly to relevant MCH class I
molecules. The IC.sub.50 for binding UL46 183-191 (KYAAAVAGL) to
H-2K.sup.d was 9.91 nM (very tight binding) (Sette, A., et al., A
roadmap for the immunomics of category A-C pathogens. Immunity,
2005. 22(2): p. 155-61). Overall, the tegument protein vaccines
elicited high avidity, and often multi-epitope and combined
(CD4.sup.+ and CD8.sup.+) T cell responses.
HSV-2-Infected Mice Generate T cells Against Tegument Protein
Epitopes.
[0349] T cells primed in vivo by HSV-2 infection would be boosted
by vaccination. In this context, it was important to test if
tegument-specific T cells were primed in vivo by HSV-2 infection,
as well as by vaccine (above). We infected BALB/c mice with an
attenuated HSV-2 strain 333 variant deficient in thymidine kinase
(333tk-) (Milligan, G. N. and D. I. Bernstein, Generation of
humoral responses against herpes simplex virus type 2 in the murine
female genital tract. Virology, 1995. 206: p. 234-241). Mice were
made susceptible to intravaginal infection by subcutaneous
Depo-provera (progestin) 6 days before infection. Splenocytes from
day 14 (FIGS. 22 and 23) mice showed T cell responses to CD8+
tegument epitopes previously discovered using pDNA vaccines
(above). In both humans and mice, tegument proteins UL46, UL47, and
UL49 are processed and presented via the MHC class I pathway during
viral infection.
Tegument Vaccines Provide Partial Protection Against Lethal
Intravaginal Challenge with HSV-2.
[0350] "CD8.sup.+-only" vaccines can protect mice from lethal
intracerebral or footpad HSV-1 challenge (Blaney, J. E., et al.,
Immunization with a single major histocompatibility class
I-restricted cytotoxic T-lymphocyte recognition epitope of herpes
simplex virus type 2 confers protective immunity. Journal of
Virology, 1998. 72: p. 9567-9574; Orr, M. T., Orgun, N. N., Wilson,
C. B., Way, S. S., Cutting edge: recombinant listeria monocytogenes
expressing a single immune-dominant peptide confers immunity to
herpes simplex virus-1 infection. Journal of Immunology, 2007. 178:
p. In Press Apr. 15, 2007 edition), but have never been studied in
the intravaginal HSV-2 model. We found that univalent tegument
vaccines provided partial protection in the intravaginal model. We
used the virulent HSV-2 strain 186 (Nishiyama, Y. and F. Rapp,
Latency in vitro using irradiated herpes simplex virus. J Gen
Virol, 1981. 52(Pt 1): p. 113-9) for lethal challenge.
3.times.10.sup.3 pfu. The endpoints were measured twice a day, day
14 survival, and vaginal HSV-2 titers on day 1-5. All-dacron swabs
were placed into 1 mL PCR buffer, extracted, and analyzed for HSV-2
DNA copy number by high-throughput real-time PCR as described
(Ryncarz, A. J., et al., Development of a high throughput
quantitative assay for detecting HSV DNA in clinical samples.
Journal of Clinical Microbiology, 1999. 37: p. 1941-1947). Positive
vaccine controls were intravaginal infection with 10.sup.6 pfu of
attenuated HSV-2 333tk-(after Depo-provera), or three injections of
a truncated gD.sub.2 pDNA vaccine (please see below). Negative
control was empty plasmid. pDNA vaccines were given as 3 doses on
days 0, 14, and 28 at 100 .mu.g/dose IM in PBS. Mice were
challenged 14 days after vaccination with 50 times LD.sub.50
(50.times.(3.times.10.sup.3)=1.5.times.10.sup.5 pfu) after
Depo-provera.
[0351] HSV-2 333tk- and gD.sub.2 protected all animals (FIG. 24).
UL47 pDNA provided 44% protection (4 of 9 animals), with possible
slight protection for UL49. The UL47 and UL49 survivors were
confirmed to have been infected by ELISA using whole HSV-2 lysate;
they had much higher OD.sub.450 values (data not shown) than could
be explained by immunity to the immunizing construct alone (FIGS.
20A through 21). The tegument vaccines were non-sterilizing: HSV-2
replication occurred in the vagina after challenge (FIG. 24). The
gD.sub.2 vaccine (below) did lead to a measurable reduction in
HSV-2 replication (FIG. 24). The clinical severity score was
reduced after UL47, UL49, and gD.sub.2 vaccination.
gD.sub.2 Shows Minimal Sequence Variation and is an Effective
Preventative Vaccine that Lowers HSV-2 Replication.
[0352] We sequenced six wild-type gD.sub.2 genes. We found few
changes from HG52: one had V169A and a second had V353A and L375P
changes. No changes were detected in known gD.sub.2 CD8+ or
neutralizing epitopes (Koelle, D. M., Liu Z., McClurkan C. L.,
Cevallos R. C., Vieira J., Hosken N. A, Meseda C. A., Snow D. C.,
Wald A, Corey L., Immunodominance among herpes simplex
virus-specific CD8 T-cells expressing a tissue-specific homing
receptor. Proc Natl Acad Sci USA, 2003. 100: p. 12899-12904;
Tigges, M. A., et al., Human CD8+ herpes simplex virus-specific
cytotoxic T lymphocyte clones recognize diverse virion protein
antigens. Journal of Virology, 1992. 66: p. 1622-1634; Spear, P.
G., R. J. Eisenberg, and G. H. Cohen. Three classes of surface
receptors for alphaherpesvirus entry. Virology, 2000. 275: p.
1-8.). Our candidate pDNA gD.sub.2 vaccine, VR2139, encodes AA
1-340 of gD.sub.2 using the HG52 sequence. AA 341-393 were omitted
because they contain a leader and transmembrane domain. Humoral
responses were detected by ELISA using commercially available
gD.sub.1 as coating antigen and a commercially available mAb
against a type-common gD epitope as a calibrator (FIG. 25).
Cellular responses were detected with overlapping 13-mer peptides
exactly as described above for tegument proteins. After three
vaccinations of 100 .mu.g gD.sub.2 pDNA vaccine on days 0, 14, and
28 with VAXFECTIN adjuvant, brisk humoral and total splenocyte
IFN-.gamma. ELISPOT responses were noted in most animals (FIG. 25).
Survival, clinical severity and intravaginal HSV-2 DNA viral load
benefits were described above.
SUMMARY
[0353] CD8.sup.+ T cell responses control HSV infection in mice and
humans in the skin and ganglia. Tegument proteins are important
targets of the CD8+ human immune response to HSV-2 (Koelle, D. M.,
et al., CD8 CTL from genital herpes simplex lesions: recognition of
viral tegument and immediate early proteins and lysis of infected
cutaneous cells. Journal of Immunology, 2001. 166: p. 4049-4058;
Koelle, D. M., et al., Recognition of herpes simplex virus type 2
tegument proteins by CD4 T cells infiltrating human genital herpes
lesions. Journal of Virology, 1998. 72: p. 7476-7483; Posavad, C.
M., et al., T cell immunity to herpes simplex virus in seronegative
persons: silent infection or acquired immunity. Journal of
Immunology, 2003. 170: p. 4380-4388; Koelle, D. M., Liu Z.,
McClurkan C. L., Cevallos R. C., Vieira J., Hosken N. A, Meseda C.
A., Snow D. C., Wald A., Corey L., Immunodominance among herpes
simplex virus-specific CD8 T-cells expressing a tissue-specific
homing receptor. Proc Natl Acad Sci USA, 2003. 100: p. 12899-12904;
Koelle, D. M., et al., Tegument-specific, virus-reactive CD4
T-cells localize to the cornea in herpes simplex virus interstitial
keratitis in humans. Journal of Virology, 2000. 74: p. 10930-10938;
Verjans, G. M., et al., Intraocular T cells of patients with herpes
simplex (HSV)-induced acute retinal necrosis recognize HSV tegument
proteins VP11/12 and VP13/14. Journal of Infectious Diseases, 2000.
182: p. 923-927). DNA vaccines encoding HSV-2 tegument proteins
were found to stimulate CD8.sup.+, CD4.sup.+, and antibody
responses, and selected univalent vaccines were partially
protective in an intravaginal challenge model.
OTHER EMBODIMENTS
[0354] The foregoing description is considered as illustrative only
of the principles of the invention. Further, since numerous
modifications and changes will readily occur to those skilled in
the art, it is not desired to limit the invention to the exact
construction and process as described above. Accordingly, all
suitable modifications and equivalents may be resorted to falling
within the scope of the invention as defined by the claims that
follow. The words "comprise," "comprising," "include," "including,"
and "includes" when used in this specification and in the following
claims are intended to specify the presence of stated features,
integers, components, or steps, but they do not preclude the
presence or addition of one or more other features, integers,
components, steps, or groups thereof.
[0355] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
[0356] All patent documents and references cited herein are
incorporated by reference as if fully set forth.
Sequence CWU 1
1
121339PRTherpes simplex virus 2 gD 1Met Gly Arg Leu Thr Ser Gly Val
Gly Thr Ala Ala Leu Leu Val Val 1 5 10 15 Ala Val Gly Leu Arg Val
Val Cys Ala Lys Tyr Ala Leu Ala Asp Pro 20 25 30 Ser Leu Lys Met
Ala Asp Pro Asn Arg Phe Arg Gly Lys Asn Leu Pro 35 40 45 Val Leu
Gln Leu Thr Asp Pro Pro Gly Val Lys Arg Val Tyr His Ile 50 55 60
Gln Pro Ser Leu Glu Asp Pro Phe Gln Pro Pro Ser Ile Pro Ile Thr 65
70 75 80 Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu
Leu His 85 90 95 Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala
Ser Asp Glu Ala 100 105 110 Arg Lys His Thr Tyr Asn Leu Thr Ile Ala
Trp Tyr Arg Met Gly Asp 115 120 125 Asn Cys Ala Ile Pro Ile Thr Val
Met Glu Tyr Thr Glu Cys Pro Tyr 130 135 140 Asn Lys Ser Leu Gly Val
Cys Pro Ile Arg Thr Gln Pro Arg Trp Ser 145 150 155 160 Tyr Tyr Asp
Ser Phe Ser Ala Val Ser Glu Asp Asn Leu Gly Phe Leu 165 170 175 Met
His Ala Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu Val 180 185
190 Lys Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His Arg
195 200 205 Ala Arg Ala Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro
Pro Ala 210 215 220 Ala Cys Leu Thr Ser Lys Ala Tyr Gln Gln Gly Val
Thr Val Asp Ser 225 230 235 240 Ile Gly Met Leu Pro Arg Phe Ile Pro
Glu Asn Gln Arg Thr Val Ala 245 250 255 Leu Tyr Ser Leu Lys Ile Ala
Gly Trp His Gly Pro Lys Pro Pro Tyr 260 265 270 Thr Ser Thr Leu Leu
Pro Pro Glu Leu Ser Asp Thr Thr Asn Ala Thr 275 280 285 Gln Pro Glu
Leu Val Pro Glu Asp Pro Glu Asp Ser Ala Leu Leu Glu 290 295 300 Asp
Pro Ala Gly Thr Val Ser Ser Gln Ile Pro Pro Asn Trp His Ile 305 310
315 320 Pro Ser Ile Gln Asp Val Ala Pro His His Ala Pro Ala Ala Pro
Ser 325 330 335 Asn Pro Gly 24400DNAherpes simplex virus 2 gD
2ggccgccgcc accatgggca gactgactag cggagtgggc acagccgccc tgctcgtggt
60ggctgtgggc ctgagagtgg tgtgtgctaa gtacgccctg gctgacccta gcctgaagat
120ggctgatcct aataggttta ggggcaagaa cctgcccgtg ctggaccagc
tgactgaccc 180ccctggcgtg aagagagtgt accacatcca gcctagcctg
gaggacccct tccagccccc 240tagcatccct atcaccgtgt actacgccgt
gctggagaga gcctgtagaa gcgtgctgct 300gcacgcccct agtgaggccc
ctcagattgt gagaggcgct agtgacgagg ctaggaagca 360cacctataac
ctgaccatcg cctggtatag gatgggcgat aactgcgcca tccccatcac
420agtgatggag tacactgagt gcccctataa taagagcctg ggcgtgtgtc
ccattaggac 480ccagcctagg tggagctact acgatagctt tagcgccgtg
agtgaggata acctgggctt 540cctgatgcac gccccagcct ttgagaccgc
cggcacctac ctgagactgg tgaagattaa 600cgactggact gagatcaccc
agttcatcct ggagcatagg gctagggcta gctgtaaata 660cgccctgccc
ctgagaatcc cccctgccgc ctgcctgact agtaaggcct accagcaagg
720cgtgaccgtg gatagcatcg gcatgctgcc tagattcatc cctgagaacc
agagaaccgt 780ggccctgtat agcctgaaaa tcgccggctg gcacggccct
aagcctcctt acactagcac 840cctgctgccc cctgagctga gtgataccac
taacgccacc cagcctgagc tggtgcctga 900ggaccctgag gatagcgctc
tgctggaaga tcctgccggc accgtgagta gccagatccc 960ccctaactgg
cacatcccta gcattcagga cgtggccccc caccacgccc ctgccgctcc
1020tagtaaccct ggctgatgag gatccagatc tgctgtgcct tctagttgcc
agccatctgt 1080tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
gccactccca ctgtcctttc 1140ctaataaaat gaggaaattg catcgcattg
tctgagtagg tgtcattcta ttctgggggg 1200tggggtgggg cagcacagca
agggggagga ttgggaagac aatagcaggc atgctgggga 1260tgcggtgggc
tctatgggta cccaggtgct gaagaattga cccggttcct cctgggccag
1320aaagaagcag gcacatcccc ttctctgtga cacaccctgt ccacgcccct
ggttcttagt 1380tccagcccca ctcataggac actcatagct caggagggct
ccgccttcaa tcccacccgc 1440taaagtactt ggagcggtct ctccctccct
catcagccca ccaaaccaaa cctagcctcc 1500aagagtggga agaaattaaa
gcaagatagg ctattaagtg cagagggaga gaaaatgcct 1560ccaacatgtg
aggaagtaat gagagaaatc atagaatttc ttccgcttcc tcgctcactg
1620actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
aaggcggtaa 1680tacggttatc cacagaatca ggggataacg caggaaagaa
catgtgagca aaaggccagc 1740aaaaggccag gaaccgtaaa aaggccgcgt
tgctggcgtt tttccatagg ctccgccccc 1800ctgacgagca tcacaaaaat
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 1860aaagatacca
ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
1920cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
tctcaatgct 1980cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
caagctgggc tgtgtgcacg 2040aaccccccgt tcagcccgac cgctgcgcct
tatccggtaa ctatcgtctt gagtccaacc 2100cggtaagaca cgacttatcg
ccactggcag cagccactgg taacaggatt agcagagcga 2160ggtatgtagg
cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
2220ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
agagttggta 2280gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
tttttttgtt tgcaagcagc 2340agattacgcg cagaaaaaaa ggatctcaag
aagatccttt gatcttttct acggggtctg 2400acgctcagtg gaacgaaaac
tcacgttaag ggattttggt catgagatta tcaaaaagga 2460tcttcaccta
gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
2520agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
tcagcgatct 2580gtctatttcg ttcatccata gttgcctgac tccggggggg
gggggcgctg aggtctgcct 2640cgtgaagaag gtgttgctga ctcataccag
gcctgaatcg ccccatcatc cagccagaaa 2700gtgagggagc cacggttgat
gagagctttg ttgtaggtgg accagttggt gattttgaac 2760ttttgctttg
ccacggaacg gtctgcgttg tcgggaagat gcgtgatctg atccttcaac
2820tcagcaaaag ttcgatttat tcaacaaagc cgccgtcccg tcaagtcagc
gtaatgctct 2880gccagtgtta caaccaatta accaattctg attagaaaaa
ctcatcgagc atcaaatgaa 2940actgcaattt attcatatca ggattatcaa
taccatattt ttgaaaaagc cgtttctgta 3000atgaaggaga aaactcaccg
aggcagttcc ataggatggc aagatcctgg tatcggtctg 3060cgattccgac
tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
3120tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
aaaagcttat 3180gcatttcttt ccagacttgt tcaacaggcc agccattacg
ctcgtcatca aaatcactcg 3240catcaaccaa accgttattc attcgtgatt
gcgcctgagc gagacgaaat acgcgatcgc 3300tgttaaaagg acaattacaa
acaggaatcg aatgcaaccg gcgcaggaac actgccagcg 3360catcaacaat
attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
3420cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
tgcttgatgg 3480tcggaagagg cataaattcc gtcagccagt ttagtctgac
catctcatct gtaacatcat 3540tggcaacgct acctttgcca tgtttcagaa
acaactctgg cgcatcgggc ttcccataca 3600atcgatagat tgtcgcacct
gattgcccga cattatcgcg agcccattta tacccatata 3660aatcagcatc
catgttggaa tttaatcgcg gcctcgagca agacgtttcc cgttgaatat
3720ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
gttcatgatg 3780atatattttt atcttgtgca atgtaacatc agagattttg
agacacaacg tggctttccc 3840ccccccccca ttattgaagc atttatcagg
gttattgtct catgagcgga tacatatttg 3900aatgtattta gaaaaataaa
caaatagggg ttccgcgcac atttccccga aaagtgccac 3960ctgacgtcta
agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
4020ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
atgcagctcc 4080cggagacggt cacagcttgt ctgtaagcgg atgccgggag
cagacaagcc cgtcagggcg 4140cgtcagcggg tgttggcggg tgtcggggct
ggcttaacta tgcggcatca gagcagattg 4200tactgagagt gcaccatatg
cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 4260gcatcagatt
ggctattggc cattgcatac gttgtatcca tatcataata tgtacattta
4320tattggctca tgtccaacat taccgccatg ttgacattga ttattgacta
gttattaata 4380gtaatcaatt acggggtcat 44003300PRTherpes simplex
virus 2 UL49 3Met Thr Ser Arg Arg Ser Val Lys Ser Cys Pro Arg Glu
Ala Pro Arg 1 5 10 15 Gly Thr His Glu Glu Leu Tyr Tyr Gly Pro Val
Ser Pro Ala Asp Pro 20 25 30 Glu Ser Pro Arg Asp Asp Phe Arg Arg
Gly Ala Gly Pro Met Arg Ala 35 40 45 Arg Pro Arg Gly Glu Val Arg
Phe Leu His Tyr Asp Glu Ala Gly Tyr 50 55 60 Ala Leu Tyr Arg Asp
Ser Ser Ser Asp Asp Asp Glu Ser Arg Asp Thr 65 70 75 80 Ala Arg Pro
Arg Arg Ser Ala Ser Val Ala Gly Ser His Gly Pro Gly 85 90 95 Pro
Ala Arg Ala Pro Pro Pro Pro Gly Gly Pro Val Gly Ala Gly Gly 100 105
110 Arg Ser His Ala Pro Pro Ala Arg Thr Pro Lys Met Thr Arg Gly Ala
115 120 125 Pro Lys Ala Ser Ala Thr Pro Ala Thr Asp Pro Ala Arg Gly
Arg Arg 130 135 140 Pro Ala Gln Ala Asp Ser Ala Val Leu Leu Asp Ala
Pro Ala Pro Thr 145 150 155 160 Ala Ser Gly Arg Thr Lys Thr Pro Ala
Gln Gly Leu Ala Lys Lys Leu 165 170 175 His Phe Ser Thr Ala Pro Pro
Ser Pro Thr Ala Pro Trp Thr Pro Arg 180 185 190 Val Ala Gly Phe Asn
Lys Arg Val Phe Cys Ala Ala Val Gly Arg Leu 195 200 205 Ala Ala Thr
His Ala Arg Leu Ala Ala Val Gln Leu Trp Asp Met Ser 210 215 220 Arg
Pro His Thr Asp Glu Asp Leu Asn Glu Leu Leu Asp Leu Thr Thr 225 230
235 240 Ile Arg Val Thr Val Cys Glu Gly Lys Asn Leu Leu Gln Arg Ala
Asn 245 250 255 Glu Leu Val Asn Pro Asp Ala Ala Gln Asp Val Asp Ala
Thr Ala Ala 260 265 270 Ala Arg Gly Arg Pro Ala Gly Arg Ala Ala Ala
Thr Ala Arg Ala Pro 275 280 285 Ala Arg Ser Ala Ser Arg Pro Arg Arg
Pro Leu Glu 290 295 300 45817DNAherpes simplex virus 2 UL49
4atcccaccat gacctctagg cggagcgtga agagctgccc tagagaggcc cctagaggca
60cccacgagga gctgtactac ggccctgtgt cccctgccga ccctgagagc cctagagatg
120actttagacg gggagccggc cctatgagag ccagacctag aggcgaagtg
agattcctgc 180actacgacga ggccggctac gccctgtatc gggatagcag
ctctgacgac gacgagtcta 240gggataccgc caggcctaga agaagcgcca
gcgtggccgg cagccacggc cctggccctg 300ccagagcccc ccctcctcct
ggcggccctg tgggagccgg cggaagaagc cacgcccctc 360ccgcccggac
ccctaagatg accagaggcg cccctaaggc cagcgccacc cccgccaccg
420atcccgccag aggcaggaga cccgcccagg ccgatagcgc cgtgctgctg
gacgcccctg 480cccccaccgc ctccggcaga accaagaccc ctgcccaggg
cctggccaag aagctgcact 540ttagcaccgc ccctccttcc cccaccgccc
cctggacccc tagagtggcc ggctttaata 600agcgcgtgtt ctgtgccgct
gtgggcagac tggccgccac ccacgccagg ctggccgccg 660tgcagctgtg
ggatatgagc agaccccaca ccgacgagga cctgaacgag ctgctggacc
720tgaccacaat tagagtgacc gtgtgtgagg gcaagaacct gctgcagagg
gccaacgagc 780tggtgaaccc tgacgccgcc caggacgtgg acgccaccgc
cgccgccagg ggcagacctg 840ccggcagagc cgccgccaca gccagggccc
ctgccagaag cgcctctagg ccaagacggc 900ccctggagcc taggtaatct
agaccaggcc ctggatccag atctgctgtg ccttctagtt 960gccagccatc
tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
1020ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
aggtgtcatt 1080ctattctggg gggtggggtg gggcaggaca gcaaggggga
ggattgggaa gacaatagca 1140ggcatgctgg ggatgcggtg ggctctatgg
gtacccaggt gctgaagaat tgacccggtt 1200cctcctgggc cagaaagaag
caggcacatc cccttctctg tgacacaccc tgtccacgcc 1260cctggttctt
agttccagcc ccactcatag gacactcata gctcaggagg gctccgcctt
1320caatcccacc cgctaaagta cttggagcgg tctctccctc cctcatcagc
ccaccaaacc 1380aaacctagcc tccaagagtg ggaagaaatt aaagcaagat
aggctattaa gtgcagaggg 1440agagaaaatg cctccaacat gtgaggaagt
aatgagagaa atcatagaat ttcttccgct 1500tcctcgctca ctgactcgct
gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 1560tcaaaggcgg
taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
1620gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
gtttttccat 1680aggctccgcc cccctgacga gcatcacaaa aatcgacgct
caagtcagag gtggcgaaac 1740ccgacaggac tataaagata ccaggcgttt
ccccctggaa gctccctcgt gcgctctcct 1800gttccgaccc tgccgcttac
cggatacctg tccgcctttc tcccttcggg aagcgtggcg 1860ctttctcata
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
1920ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
taactatcgt 1980cttgagtcca acccggtaag acacgactta tcgccactgg
cagcagccac tggtaacagg 2040attagcagag cgaggtatgt aggcggtgct
acagagttct tgaagtggtg gcctaactac 2100ggctacacta gaagaacagt
atttggtatc tgcgctctgc tgaagccagt taccttcgga 2160aaaagagttg
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
2220gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
tttgatcttt 2280tctacggggt ctgacgctca gtggaacgaa aactcacgtt
aagggatttt ggtcatgaga 2340ttatcaaaaa ggatcttcac ctagatcctt
ttaaattaaa aatgaagttt taaatcaatc 2400taaagtatat atgagtaaac
ttggtctgac agttaccaat gcttaatcag tgaggcacct 2460atctcagcga
tctgtctatt tcgttcatcc atagttgcct gactcggggg gggggggcgc
2520tgaggtctgc ctcgtgaaga aggtgttgct gactcatacc aggcctgaat
cgccccatca 2580tccagccaga aagtgaggga gccacggttg atgagagctt
tgttgtaggt ggaccagttg 2640gtgattttga acttttgctt tgccacggaa
cggtctgcgt tgtcgggaag atgcgtgatc 2700tgatccttca actcagcaaa
agttcgattt attcaacaaa gccgccgtcc cgtcaagtca 2760gcgtaatgct
ctgccagtgt tacaaccaat taaccaattc tgattagaaa aactcatcga
2820gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
ttttgaaaaa 2880gccgtttctg taatgaagga gaaaactcac cgaggcagtt
ccataggatg gcaagatcct 2940ggtatcggtc tgcgattccg actcgtccaa
catcaataca acctattaat ttcccctcgt 3000caaaaataag gttatcaagt
gagaaatcac catgagtgac gactgaatcc ggtgagaatg 3060gcaaaagctt
atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
3120caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
gcgagacgaa 3180atacgcgatc gctgttaaaa ggacaattac aaacaggaat
cgaatgcaac cggcgcagga 3240acactgccag cgcatcaaca atattttcac
ctgaatcagg atattcttct aatacctgga 3300atgctgtttt cccggggatc
gcagtggtga gtaaccatgc atcatcagga gtacggataa 3360aatgcttgat
ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
3420ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
ggcgcatcgg 3480gcttcccata caatcgatag attgtcgcac ctgattgccc
gacattatcg cgagcccatt 3540tatacccata taaatcagca tccatgttgg
aatttaatcg cggcctcgag caagacgttt 3600cccgttgaat atggctcata
acaccccttg tattactgtt tatgtaagca gacagtttta 3660ttgttcatga
tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa
3720cgtggctttc cccccccccc cattattgaa gcatttatca gggttattgt
ctcatgagcg 3780gatacatatt tgaatgtatt tagaaaaata aacaaatagg
ggttccgcgc acatttcccc 3840gaaaagtgcc acctgacgtc taagaaacca
ttattatcat gacattaacc tataaaaata 3900ggcgtatcac gaggcccttt
cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac 3960acatgcagct
cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
4020cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
tatgcggcat 4080cagagcagat tgtactgaga gtgcaccata tgcggtgtga
aataccgcac agatgcgtaa 4140ggagaaaata ccgcatcaga ttggctattg
gccattgcat acgttgtatc catatcataa 4200tatgtacatt tatattggct
catgtccaac attaccgcca tgttgacatt gattattgac 4260tagttattaa
tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
4320cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
cccgcccatt 4380gacgtcaata atgacgtatg ttcccatagt aacgccaata
gggactttcc attgacgtca 4440atgggtggag tatttacggt aaactgccca
cttggcagta catcaagtgt atcatatgcc 4500aagtacgccc cctattgacg
tcaatgacgg taaatggccc gcctggcatt atgcccagta 4560catgacctta
tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
4620catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
actcacgggg 4680atttccaagt ctccacccca ttgacgtcaa tgggagtttg
ttttggcacc aaaatcaacg 4740ggactttcca aaatgtcgta acaactccgc
cccattgacg caaatgggcg gtaggcgtgt 4800acggtgggag gtctatataa
gcagagctcg tttagtgaac cgtcagatcg cctggagacg 4860ccatccacgc
tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg
4920ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta
ccgcctatag 4980actctatagg cacacccctt tggctcttat gcatgctata
ctgtttttgg cttggggcct 5040atacaccccc gcttccttat gctataggtg
atggtatagc ttagcctata ggtgtgggtt 5100attgaccatt attgaccact
cccctattgg tgacgatact ttccattact aatccataac 5160atggctcttt
gccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac
5220tgacacggac tctgtatttt tacaggatgg ggtcccattt attatttaca
aattcacata 5280tacaacaacg ccgtcccccg tgcccgcagt ttttattaaa
catagcgtgg gatctccacg 5340cgaatctcgg gtacgtgttc cggacatggg
ctcttctccg gtagcggcgg agcttccaca 5400tccgagccct ggtcccatgc
ctccagcggc tcatggtcgc tcggcagctc cttgctccta 5460acagtggagg
ccagacttag gcacagcaca atgcccacca ccaccagtgt gccgcacaag
5520gccgtggcgg tagggtatgt gtctgaaaat gagcgtggag attgggctcg
cacggctgac 5580gcagatggaa gacttaaggc agcggcagaa gaagatgcag
gcagctgagt tgttgtattc 5640tgataagagt cagaggtaac tcccgttgcg
gtgctgttaa cggtggaggg cagtgtagtc 5700tgagcagtac tcgttgctgc
cgcgcgcgcc accagacata atagctgaca gactaacaga 5760ctgttccttt
ccatgggtct tttctgcagt caccgtcgtc gacacgtgtg atcagat
58175696PRTherpes simplex virus 2 UL47 5Met Ser Val Arg Gly His Ala
Val Arg Arg Arg Arg Ala Ser Thr Arg 1 5 10 15 Ser His Ala Pro Ser
Ala His Arg Ala Asp Ser Pro Val Glu Asp Glu 20 25 30 Pro Glu Gly
Gly Gly Gly Gly Leu Met Gly Tyr Leu Arg Ala Val Phe 35 40 45 Asn
Val Asp Asp Asp Ser Glu Val Glu Ala Ala Gly Glu Met Ala Ser 50 55
60 Glu Glu Pro Pro Pro Arg Arg Arg
Arg Glu Ala Arg Gly His Pro Gly 65 70 75 80 Ser Arg Arg Ala Ser Glu
Ala Arg Ala Ala Ala Pro Pro Arg Arg Ala 85 90 95 Ser Phe Pro Arg
Pro Arg Ser Val Thr Ala Arg Ser Gln Ser Val Arg 100 105 110 Gly Arg
Arg Asp Ser Ala Ile Thr Arg Ala Pro Arg Gly Gly Tyr Leu 115 120 125
Gly Pro Met Asp Pro Arg Asp Val Leu Gly Arg Val Gly Gly Ser Arg 130
135 140 Val Val Pro Ser Pro Leu Phe Leu Asp Glu Leu Ser Tyr Glu Glu
Asp 145 150 155 160 Asp Tyr Pro Ala Ala Val Ala His Asp Asp Gly Ala
Gly Ala Arg Pro 165 170 175 Pro Ala Thr Val Glu Ile Leu Ala Gly Arg
Val Ser Gly Pro Glu Leu 180 185 190 Gln Ala Ala Phe Pro Leu Asp Arg
Leu Thr Pro Arg Val Ala Ala Trp 195 200 205 Asp Glu Ser Val Arg Ser
Ala Leu Ala Leu Gly His Pro Ala Gly Phe 210 215 220 Tyr Pro Cys Pro
Asp Ser Ala Phe Gly Leu Ser Arg Val Gly Val Met 225 230 235 240 His
Phe Ala Ser Pro Ala Asp Pro Lys Val Phe Phe Arg Gln Thr Leu 245 250
255 Gln Gln Gly Glu Ala Leu Ala Trp Tyr Ile Thr Gly Asp Ala Ile Leu
260 265 270 Asp Leu Thr Asp Arg Arg Ala Lys Thr Ser Pro Ser Arg Ala
Met Gly 275 280 285 Phe Leu Val Asp Ala Ile Val Arg Val Ala Ile Asn
Gly Trp Val Cys 290 295 300 Gly Thr Arg Leu His Thr Glu Gly Arg Gly
Ser Glu Leu Asp Asp Arg 305 310 315 320 Ala Ala Glu Leu Arg Arg Gln
Phe Ala Ser Leu Thr Ala Leu Arg Pro 325 330 335 Val Gly Ala Ala Ala
Val Pro Leu Leu Ser Ala Gly Gly Ala Ala Pro 340 345 350 Pro His Pro
Gly Pro Asp Ala Ala Val Phe Arg Ser Ser Leu Gly Ser 355 360 365 Leu
Leu Tyr Trp Pro Gly Val Arg Ala Leu Leu Gly Arg Asp Cys Arg 370 375
380 Val Ala Ala Arg Tyr Ala Gly Arg Met Thr Tyr Ile Ala Thr Gly Ala
385 390 395 400 Leu Leu Ala Arg Phe Asn Pro Gly Ala Val Lys Cys Val
Leu Pro Arg 405 410 415 Glu Ala Ala Phe Ala Gly Arg Val Leu Asp Val
Leu Ala Val Leu Ala 420 425 430 Glu Gln Thr Val Gln Trp Leu Ser Val
Val Val Gly Ala Arg Leu His 435 440 445 Pro His Ser Ala His Pro Ala
Phe Ala Asp Val Glu Gln Glu Ala Leu 450 455 460 Phe Arg Ala Leu Pro
Leu Gly Ser Pro Gly Val Val Ala Ala Glu His 465 470 475 480 Glu Ala
Leu Gly Asp Thr Ala Ala Arg Arg Leu Leu Ala Thr Ser Gly 485 490 495
Leu Asn Ala Val Leu Gly Ala Ala Val Tyr Ala Leu His Thr Ala Leu 500
505 510 Ala Thr Val Thr Leu Lys Tyr Ala Leu Ala Cys Gly Asp Ala Arg
Arg 515 520 525 Arg Arg Asp Asp Ala Ala Ala Ala Arg Ala Val Leu Ala
Thr Gly Leu 530 535 540 Ile Leu Gln Arg Leu Leu Gly Leu Ala Asp Thr
Val Val Ala Cys Val 545 550 555 560 Ala Leu Ala Ala Phe Asp Gly Gly
Ser Thr Ala Pro Glu Val Gly Thr 565 570 575 Tyr Thr Pro Leu Arg Tyr
Ala Cys Val Leu Arg Ala Thr Gln Pro Leu 580 585 590 Tyr Ala Arg Thr
Thr Pro Ala Lys Phe Trp Ala Asp Val Arg Ala Ala 595 600 605 Ala Glu
His Val Asp Leu Arg Pro Ala Ser Ser Ala Pro Arg Ala Pro 610 615 620
Val Ser Gly Thr Ala Asp Pro Ala Phe Leu Leu Glu Asp Leu Ala Ala 625
630 635 640 Phe Pro Pro Ala Pro Leu Asn Ser Glu Ser Val Leu Gly Pro
Arg Val 645 650 655 Arg Val Val Asp Ile Met Ala Gln Phe Arg Lys Leu
Leu Met Gly Asp 660 665 670 Glu Glu Thr Ala Ala Leu Arg Ala His Val
Ser Gly Arg Arg Ala Thr 675 680 685 Gly Leu Gly Gly Pro Pro Arg Pro
690 695 67005DNAherpes simplex virus 2 UL47 6ctagaccagg ccctggatcc
agatctgctg tgccttctag ttgccagcca tctgttgttt 60gcccctcccc cgtgccttcc
ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 120aaaatgagga
aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
180tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
ggggatgcgg 240tgggctctat gggtacccag gtgctgaaga attgacccgg
ttcctcctgg gccagaaaga 300agcaggcaca tccccttctc tgtgacacac
cctgtccacg cccctggttc ttagttccag 360ccccactcat aggacactca
tagctcagga gggctccgcc ttcaatccca cccgctaaag 420tacttggagc
ggtctctccc tccctcatca gcccaccaaa ccaaacctag cctccaagag
480tgggaagaaa ttaaagcaag ataggctatt aagtgcagag ggagagaaaa
tgcctccaac 540atgtgaggaa gtaatgagag aaatcataga atttcttccg
cttcctcgct cactgactcg 600ctgcgctcgg tcgttcggct gcggcgagcg
gtatcagctc actcaaaggc ggtaatacgg 660ttatccacag aatcagggga
taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 720gccaggaacc
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
780gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
actataaaga 840taccaggcgt ttccccctgg aagctccctc gtgcgctctc
ctgttccgac cctgccgctt 900accggatacc tgtccgcctt tctcccttcg
ggaagcgtgg cgctttctca tagctcacgc 960tgtaggtatc tcagttcggt
gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 1020cccgttcagc
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
1080agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
agcgaggtat 1140gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
acggctacac tagaagaaca 1200gtatttggta tctgcgctct gctgaagcca
gttaccttcg gaaaaagagt tggtagctct 1260tgatccggca aacaaaccac
cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 1320acgcgcagaa
aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
1380cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
aaggatcttc 1440acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
tctaaagtat atatgagtaa 1500acttggtctg acagttacca atgcttaatc
agtgaggcac ctatctcagc gatctgtcta 1560tttcgttcat ccatagttgc
ctgactcggg gggggggggc gctgaggtct gcctcgtgaa 1620gaaggtgttg
ctgactcata ccaggcctga atcgccccat catccagcca gaaagtgagg
1680gagccacggt tgatgagagc tttgttgtag gtggaccagt tggtgatttt
gaacttttgc 1740tttgccacgg aacggtctgc gttgtcggga agatgcgtga
tctgatcctt caactcagca 1800aaagttcgat ttattcaaca aagccgccgt
cccgtcaagt cagcgtaatg ctctgccagt 1860gttacaacca attaaccaat
tctgattaga aaaactcatc gagcatcaaa tgaaactgca 1920atttattcat
atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
1980gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
tctgcgattc 2040cgactcgtcc aacatcaata caacctatta atttcccctc
gtcaaaaata aggttatcaa 2100gtgagaaatc accatgagtg acgactgaat
ccggtgagaa tggcaaaagc ttatgcattt 2160ctttccagac ttgttcaaca
ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa 2220ccaaaccgtt
attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
2280aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
agcgcatcaa 2340caatattttc acctgaatca ggatattctt ctaatacctg
gaatgctgtt ttcccgggga 2400tcgcagtggt gagtaaccat gcatcatcag
gagtacggat aaaatgcttg atggtcggaa 2460gaggcataaa ttccgtcagc
cagtttagtc tgaccatctc atctgtaaca tcattggcaa 2520cgctaccttt
gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
2580agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
tataaatcag 2640catccatgtt ggaatttaat cgcggcctcg agcaagacgt
ttcccgttga atatggctca 2700taacacccct tgtattactg tttatgtaag
cagacagttt tattgttcat gatgatatat 2760ttttatcttg tgcaatgtaa
catcagagat tttgagacac aacgtggctt tccccccccc 2820cccattattg
aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
2880tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
ccacctgacg 2940tctaagaaac cattattatc atgacattaa cctataaaaa
taggcgtatc acgaggccct 3000ttcgtctcgc gcgtttcggt gatgacggtg
aaaacctctg acacatgcag ctcccggaga 3060cggtcacagc ttgtctgtaa
gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag 3120cgggtgttgg
cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
3180gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
taccgcatca 3240gattggctat tggccattgc atacgttgta tccatatcat
aatatgtaca tttatattgg 3300ctcatgtcca acattaccgc catgttgaca
ttgattattg actagttatt aatagtaatc 3360aattacgggg tcattagttc
atagcccata tatggagttc cgcgttacat aacttacggt 3420aaatggcccg
cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
3480tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
agtatttacg 3540gtaaactgcc cacttggcag tacatcaagt gtatcatatg
ccaagtacgc cccctattga 3600cgtcaatgac ggtaaatggc ccgcctggca
ttatgcccag tacatgacct tatgggactt 3660tcctacttgg cagtacatct
acgtattagt catcgctatt accatggtga tgcggttttg 3720gcagtacatc
aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
3780cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
caaaatgtcg 3840taacaactcc gccccattga cgcaaatggg cggtaggcgt
gtacggtggg aggtctatat 3900aagcagagct cgtttagtga accgtcagat
cgcctggaga cgccatccac gctgttttga 3960cctccataga agacaccggg
accgatccag cctccgcggc cgggaacggt gcattggaac 4020gcggattccc
cgtgccaaga gtgacgtaag taccgcctat agactctata ggcacacccc
4080tttggctctt atgcatgcta tactgttttt ggcttggggc ctatacaccc
ccgcttcctt 4140atgctatagg tgatggtata gcttagccta taggtgtggg
ttattgacca ttattgacca 4200ctcccctatt ggtgacgata ctttccatta
ctaatccata acatggctct ttgccacaac 4260tatctctatt ggctatatgc
caatactctg tccttcagag actgacacgg actctgtatt 4320tttacaggat
ggggtcccat ttattattta caaattcaca tatacaacaa cgccgtcccc
4380cgtgcccgca gtttttatta aacatagcgt gggatctcca cgcgaatctc
gggtacgtgt 4440tccggacatg ggctcttctc cggtagcggc ggagcttcca
catccgagcc ctggtcccat 4500gcctccagcg gctcatggtc gctcggcagc
tccttgctcc taacagtgga ggccagactt 4560aggcacagca caatgcccac
caccaccagt gtgccgcaca aggccgtggc ggtagggtat 4620gtgtctgaaa
atgagcgtgg agattgggct cgcacggctg acgcagatgg aagacttaag
4680gcagcggcag aagaagatgc aggcagctga gttgttgtat tctgataaga
gtcagaggta 4740actcccgttg cggtgctgtt aacggtggag ggcagtgtag
tctgagcagt actcgttgct 4800gccgcgcgcg ccaccagaca taatagctga
cagactaaca gactgttcct ttccatgggt 4860cttttctgca gtcaccgtcg
tcgacacgtg tgatcagata tcccaccatg tctgtgagag 4920gccacgctgt
gagaagaaga agggctagca ccagaagcca cgcccctagc gcccacagag
4980ccgatagccc tgtggaggat gagcctgagg gcggaggagg gggcctgatg
ggctacctga 5040gggccgtgtt taacgtggac gacgatagcg aagtggaagc
cgccggagag atggcctctg 5100aggagccccc tcctagaagg agaagagagg
ccagaggcca ccccggctct aggagagcct 5160ctgaggccag agccgccgcc
ccacctagaa gagccagctt ccctagacct agaagcgtga 5220ccgccagaag
ccagtctgtg cgcggcagga gggatagcgc tatcaccaga gcccctagag
5280gcggctacct gggccctatg gaccctcgcg acgtgctggg cagagtgggc
ggctctaggg 5340tggtgcctag ccccctgttc ctggatgagc tgagctacga
ggaggacgac taccctgccg 5400ccgtggccca cgacgacgga gccggagcca
gaccccctgc caccgtggag atcctggccg 5460gcagagtgag cggacctgag
ctgcaagccg ccttccccct ggatcggctg acccctcggg 5520tggccgcctg
ggatgagtct gtgaggagcg ccctggccct gggccaccct gccggcttct
5580acccctgccc tgattccgcc ttcggcctga gcagagtggg agtgatgcac
ttcgccagcc 5640ctgccgaccc taaagtgttc ttccggcaaa cactgcagca
gggcgaggcc ctcgcatggt 5700acatcaccgg cgacgccatc ctggatctga
ccgatagacg ggccaagacc agccctagca 5760gagctatggg ctttctggtg
gacgctattg tgagagtggc tattaacggc tgggtgtgcg 5820gcaccagact
gcacaccgag ggcagaggct ctgagctgga tgatagagcc gccgagctga
5880ggagacagtt cgccagcctg accgccctga gacctgtggg cgccgctgcc
gtgcccctgc 5940tgagcgccgg aggagccgcc cctccccacc ctggccctga
cgccgccgtg tttcggtcta 6000gcctgggcag cctgctgtac tggcccggag
tgagagccct gctgggcagg gactgtagag 6060tggctgccag atacgccggc
aggatgacct acatcgccac cggcgccctg ctggccagat 6120ttaaccctgg
cgccgtgaaa tgcgtgctgc ctagagaagc cgccttcgcc ggaagagtgc
6180tggacgtcct ggccgtgctg gctgagcaga ccgtgcagtg gctgagcgtg
gttgtgggcg 6240ccaggctgca ccctcacagc gcccaccctg ccttcgccga
cgtggagcag gaggccctgt 6300ttagagccct gcctctgggc agccctggcg
tggtggccgc cgagcacgaa gccctgggcg 6360acaccgctgc caggagactg
ctcgccacaa gcggcctgaa cgccgtgctg ggagccgccg 6420tgtacgccct
gcacaccgcc ctggccaccg tgaccctgaa atacgccctg gcctgcggcg
6480acgcccgcag acgccgcgac gacgccgctg cagccagagc cgtcctggcc
accggcctga 6540tcctgcagag gctgctgggc ctggccgaca ccgtggtggc
ctgcgtggcc ctggccgcct 6600ttgacggcgg cagcaccgcc cctgaagtgg
gcacctacac ccctctgaga tacgcctgcg 6660tgctgagagc cacccagcct
ctgtacgcca gaaccacccc tgccaagttc tgggccgatg 6720tgagggccgc
cgccgaacac gtggacctga gacccgcctc tagcgcccca agggcccctg
6780tgagcggcac cgccgacccc gccttcctgc tggaggatct ggccgctttc
cctcccgccc 6840ctctgaatag cgagagcgtg ctggggccta gagtgagagt
ggtggatatt atggcccagt 6900ttagaaagct gctgatgggc gacgaggaaa
cagccgccct gagggcccac gtgtctggca 6960gaagagccac aggcctgggc
ggacctccta gacctcctag gtgat 70057721PRTherpes simplex virus 2 UL46
7Met Gln Arg Arg Ala Arg Gly Ala Ser Ser Leu Arg Leu Ala Arg Cys 1
5 10 15 Leu Thr Pro Ala Asn Leu Ile Arg Gly Ala Asn Ala Gly Val Pro
Glu 20 25 30 Arg Arg Ile Phe Ala Gly Cys Leu Leu Pro Thr Pro Glu
Gly Leu Leu 35 40 45 Ser Ala Ala Val Gly Val Leu Arg Gln Arg Ala
Asp Asp Leu Gln Pro 50 55 60 Ala Phe Leu Thr Gly Ala Asp Arg Ser
Val Arg Leu Ala Ala Arg His 65 70 75 80 His Asn Thr Val Pro Glu Ser
Leu Ile Val Asp Gly Leu Ala Ser Asp 85 90 95 Pro His Tyr Asp Tyr
Ile Arg His Tyr Ala Ser Ala Ala Lys Gln Ala 100 105 110 Leu Gly Glu
Val Glu Leu Ser Gly Gly Gln Leu Ser Arg Ala Ile Leu 115 120 125 Ala
Gln Tyr Trp Lys Tyr Leu Gln Thr Val Val Pro Ser Gly Leu Asp 130 135
140 Ile Pro Asp Asp Pro Ala Gly Asp Cys Asp Pro Ser Leu His Val Leu
145 150 155 160 Leu Arg Pro Thr Leu Leu Pro Lys Leu Leu Val Arg Ala
Pro Phe Lys 165 170 175 Ser Gly Ala Ala Ala Ala Lys Tyr Ala Ala Ala
Val Ala Gly Leu Arg 180 185 190 Asp Ala Ala His Arg Leu Gln Gln Tyr
Met Phe Phe Met Arg Pro Ala 195 200 205 Asp Pro Ser Arg Pro Ser Thr
Asp Thr Ala Leu Arg Leu Ser Glu Leu 210 215 220 Leu Ala Tyr Val Ser
Val Leu Tyr His Trp Ala Ser Trp Met Leu Trp 225 230 235 240 Thr Ala
Asp Lys Tyr Val Cys Arg Arg Leu Gly Pro Ala Asp Arg Arg 245 250 255
Phe Val Ala Leu Ser Gly Ser Leu Glu Ala Pro Ala Glu Thr Phe Ala 260
265 270 Arg His Leu Asp Arg Gly Pro Ser Gly Thr Thr Gly Ser Met Gln
Cys 275 280 285 Met Ala Leu Arg Ala Ala Val Ser Asp Val Leu Gly His
Leu Thr Arg 290 295 300 Leu Ala His Leu Trp Glu Thr Gly Lys Arg Ser
Gly Gly Thr Tyr Gly 305 310 315 320 Ile Val Asp Ala Ile Val Ser Thr
Val Glu Val Leu Ser Ile Val His 325 330 335 His His Ala Gln Tyr Ile
Ile Asn Ala Thr Leu Thr Gly Tyr Val Val 340 345 350 Trp Ala Ser Asp
Ser Leu Asn Asn Glu Tyr Leu Arg Ala Ala Val Asp 355 360 365 Ser Gln
Glu Arg Phe Cys Arg Thr Ala Ala Pro Leu Phe Pro Thr Met 370 375 380
Thr Ala Pro Ser Trp Ala Arg Met Glu Leu Ser Ile Lys Ser Trp Phe 385
390 395 400 Gly Ala Ala Leu Ala Pro Asp Leu Leu Arg Ser Gly Thr Pro
Ser Pro 405 410 415 His Tyr Glu Ser Ile Leu Arg Leu Ala Ala Ser Gly
Pro Pro Gly Gly 420 425 430 Arg Gly Ala Val Gly Gly Ser Cys Arg Asp
Lys Ile Gln Arg Thr Arg 435 440 445 Arg Asp Asn Ala Pro Pro Pro Leu
Pro Arg Ala Arg Pro His Ser Thr 450 455 460 Pro Ala Ala Pro Arg Arg
Phe Arg Arg His Arg Glu Asp Leu Pro Glu 465 470 475 480 Pro Pro His
Val Asp Ala Ala Asp Arg Gly Pro Glu Pro Cys Ala Gly 485 490 495 Arg
Pro Ala Thr Tyr Tyr Thr His Met Ala Gly Ala Pro Pro Arg Leu 500 505
510 Pro Pro Arg Asn Pro Ala Pro Pro Glu Gln Arg Pro Ala Ala Ala Ala
515 520 525 Arg Pro Leu Ala Ala Gln Arg Glu Ala Ala Gly Val Tyr Asp
Ala Val 530 535 540 Arg Thr Trp Gly Pro Asp Ala Glu Ala Glu Pro Asp
Gln Met Glu Asn 545 550 555 560 Thr Tyr Leu Leu Pro Asp Asp Asp Ala
Ala Met Pro Ala Gly Val Gly 565 570 575 Leu Gly Ala Thr Pro Ala Ala
Asp Thr Thr Ala Ala Ala Trp Pro Ala 580 585 590 Lys
Ser His Ala Pro Arg Ala Pro Ser Glu Asp Ala Asp Ser Ile Tyr 595 600
605 Glu Ser Val Ser Glu Asp Gly Gly Arg Val Tyr Glu Glu Ile Pro Trp
610 615 620 Val Arg Val Tyr Glu Asn Ile Cys Leu Arg Arg Gln Asp Ala
Gly Gly 625 630 635 640 Ala Ala Pro Pro Gly Asp Ala Pro Asp Ser Pro
Tyr Ile Glu Ala Glu 645 650 655 Asn Pro Leu Tyr Asp Trp Gly Gly Ser
Ala Leu Phe Ser Pro Pro Gly 660 665 670 Ala Thr Arg Ala Pro Asp Pro
Gly Leu Ser Leu Ser Pro Met Pro Ala 675 680 685 Arg Pro Arg Thr Asn
Ala Leu Ala Asn Asp Gly Pro Thr Asn Val Ala 690 695 700 Ala Leu Ser
Ala Leu Leu Thr Lys Leu Lys Arg Gly Arg His Gln Ser 705 710 715 720
His 87080DNAherpes simplex virus 2 UL46 8atcccaccat gcagcggaga
gccagaggcg cctctagcct gagactggcc cggtgcctga 60cccccgccaa tctgattaga
ggcgccaacg ccggcgtgcc tgagagaaga atcttcgccg 120gctgcctgct
gcctacccct gagggcctgc tgagcgccgc tgtgggcgtg ctgagacaga
180gggccgatga cctgcagccc gccttcctga ccggcgccga tagatctgtg
aggctggccg 240ccagacacca caacaccgtg cctgagtccc tgatcgtgga
cggcctggcc tctgaccccc 300actacgacta cattaggcac tacgccagcg
ccgccaagca ggccctgggc gaagtggagc 360tgagcggcgg acagctgagc
agagccatcc tggcccagta ctggaagtac ctgcagaccg 420tggtgcctag
cggcctggac atccctgatg atcctgccgg cgactgtgac cctagcctgc
480acgtgctgct gagacccaca ctgctgccta agctgcttgt gagggccccc
tttaagagcg 540gcgctgccgc cgccaaatac gccgccgccg tggccggcct
gagggacgcc gcccacagac 600tgcagcagta tatgttcttt atgagacccg
ccgaccctag cagacctagc accgacaccg 660ccctgagact gagcgagctg
ctggcctatg tgagcgtgct gtaccactgg gccagctgga 720tgctgtggac
cgccgataaa tacgtgtgta ggcggctggg ccctgccgat agaagattcg
780tggccctgag cggcagcctg gaggcccctg ccgagacctt tgcccggcac
ctggatagag 840gccctagcgg caccaccggc tctatgcagt gtatggccct
gagagccgcc gtgtctgacg 900tgctgggcca cctgaccaga ctggcccacc
tgtgggagac cggcaagaga agcggcggca 960cctacggcat cgtggacgcc
attgtgagca ccgtggaagt gctgagcatc gtgcaccacc 1020acgcccagta
catcattaac gccaccctga ccggctacgt tgtgtgggcc tctgatagcc
1080tgaataatga gtacctgagg gctgccgtcg atagccagga gcggttctgt
agaacagccg 1140cccctctgtt ccccaccatg accgcccctt cctgggccag
aatggaactg agcattaaga 1200gctggttcgg agccgccctg gcccctgacc
tgctgagaag cggcacccct agccctcact 1260acgagagcat cctgcgcctg
gctgccagcg gccctcctgg cggcagagga gctgtgggcg 1320gcagctgtag
ggataagatc cagcggaccc ggagagataa cgcccctccc cccctgcctc
1380gggccagacc ccacagcacc cctgctgccc ctcggcggtt tagacggcac
agagaggacc 1440tgcctgagcc tccccacgtg gacgccgccg ataggggccc
tgagccctgc gccggcagac 1500ccgccaccta ctacacccac atggccggag
ccccccctcg gctgccccct cggaaccctg 1560cccctcctga gcagagacct
gccgccgctg cccggcctct ggccgcccag agagaagccg 1620ccggagtgta
tgacgctgtg agaacctggg gccctgacgc cgaggccgag cctgatcaga
1680tggagaacac ctacctgctg cctgacgacg acgccgccat gcctgccgga
gtgggcctgg 1740gcgccacccc agccgccgat accacagccg ccgcctggcc
cgccaagagc cacgccccta 1800gagcccctag cgaggacgcc gatagcatct
acgaaagcgt gtctgaggac ggcggcagag 1860tgtatgagga gatcccctgg
gtgcgggtgt acgaaaacat ctgcctgagg agacaggacg 1920ccggaggagc
cgccccaccc ggcgacgccc ctgatagccc ttacattgag gccgagaacc
1980ccctgtacga ctggggcggc agcgccctgt ttagcccccc tggcgccacc
agagcccctg 2040accccggcct gagcctgagc cccatgcccg ccagacctag
aaccaacgcc ctggccaatg 2100acggccccac caacgtggcc gccctgagcg
ccctgctgac caagctgaag aggggcagac 2160accagagcca ccctaggtaa
tctagaccag gccctggatc cagatctgct gtgccttcta 2220gttgccagcc
atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
2280ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
agtaggtgtc 2340attctattct ggggggtggg gtggggcagg acagcaaggg
ggaggattgg gaagacaata 2400gcaggcatgc tggggatgcg gtgggctcta
tgggtaccca ggtgctgaag aattgacccg 2460gttcctcctg ggccagaaag
aagcaggcac atccccttct ctgtgacaca ccctgtccac 2520gcccctggtt
cttagttcca gccccactca taggacactc atagctcagg agggctccgc
2580cttcaatccc acccgctaaa gtacttggag cggtctctcc ctccctcatc
agcccaccaa 2640accaaaccta gcctccaaga gtgggaagaa attaaagcaa
gataggctat taagtgcaga 2700gggagagaaa atgcctccaa catgtgagga
agtaatgaga gaaatcatag aatttcttcc 2760gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 2820cactcaaagg
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
2880tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
ggcgtttttc 2940cataggctcc gcccccctga cgagcatcac aaaaatcgac
gctcaagtca gaggtggcga 3000aacccgacag gactataaag ataccaggcg
tttccccctg gaagctccct cgtgcgctct 3060cctgttccga ccctgccgct
taccggatac ctgtccgcct ttctcccttc gggaagcgtg 3120gcgctttctc
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
3180ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
cggtaactat 3240cgtcttgagt ccaacccggt aagacacgac ttatcgccac
tggcagcagc cactggtaac 3300aggattagca gagcgaggta tgtaggcggt
gctacagagt tcttgaagtg gtggcctaac 3360tacggctaca ctagaagaac
agtatttggt atctgcgctc tgctgaagcc agttaccttc 3420ggaaaaagag
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
3480tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
tcctttgatc 3540ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
gttaagggat tttggtcatg 3600agattatcaa aaaggatctt cacctagatc
cttttaaatt aaaaatgaag ttttaaatca 3660atctaaagta tatatgagta
aacttggtct gacagttacc aatgcttaat cagtgaggca 3720cctatctcag
cgatctgtct atttcgttca tccatagttg cctgactcgg gggggggggg
3780cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg
aatcgcccca 3840tcatccagcc agaaagtgag ggagccacgg ttgatgagag
ctttgttgta ggtggaccag 3900ttggtgattt tgaacttttg ctttgccacg
gaacggtctg cgttgtcggg aagatgcgtg 3960atctgatcct tcaactcagc
aaaagttcga tttattcaac aaagccgccg tcccgtcaag 4020tcagcgtaat
gctctgccag tgttacaacc aattaaccaa ttctgattag aaaaactcat
4080cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
tatttttgaa 4140aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
gttccatagg atggcaagat 4200cctggtatcg gtctgcgatt ccgactcgtc
caacatcaat acaacctatt aatttcccct 4260cgtcaaaaat aaggttatca
agtgagaaat caccatgagt gacgactgaa tccggtgaga 4320atggcaaaag
cttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
4380catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
tgagcgagac 4440gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
aatcgaatgc aaccggcgca 4500ggaacactgc cagcgcatca acaatatttt
cacctgaatc aggatattct tctaatacct 4560ggaatgctgt tttcccgggg
atcgcagtgg tgagtaacca tgcatcatca ggagtacgga 4620taaaatgctt
gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
4680catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
tctggcgcat 4740cgggcttccc atacaatcga tagattgtcg cacctgattg
cccgacatta tcgcgagccc 4800atttataccc atataaatca gcatccatgt
tggaatttaa tcgcggcctc gagcaagacg 4860tttcccgttg aatatggctc
ataacacccc ttgtattact gtttatgtaa gcagacagtt 4920ttattgttca
tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca
4980caacgtggct ttcccccccc ccccattatt gaagcattta tcagggttat
tgtctcatga 5040gcggatacat atttgaatgt atttagaaaa ataaacaaat
aggggttccg cgcacatttc 5100cccgaaaagt gccacctgac gtctaagaaa
ccattattat catgacatta acctataaaa 5160ataggcgtat cacgaggccc
tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 5220gacacatgca
gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
5280aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
aactatgcgg 5340catcagagca gattgtactg agagtgcacc atatgcggtg
tgaaataccg cacagatgcg 5400taaggagaaa ataccgcatc agattggcta
ttggccattg catacgttgt atccatatca 5460taatatgtac atttatattg
gctcatgtcc aacattaccg ccatgttgac attgattatt 5520gactagttat
taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
5580ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
acccccgccc 5640attgacgtca ataatgacgt atgttcccat agtaacgcca
atagggactt tccattgacg 5700tcaatgggtg gagtatttac ggtaaactgc
ccacttggca gtacatcaag tgtatcatat 5760gccaagtacg ccccctattg
acgtcaatga cggtaaatgg cccgcctggc attatgccca 5820gtacatgacc
ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
5880taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
ttgactcacg 5940gggatttcca agtctccacc ccattgacgt caatgggagt
ttgttttggc accaaaatca 6000acgggacttt ccaaaatgtc gtaacaactc
cgccccattg acgcaaatgg gcggtaggcg 6060tgtacggtgg gaggtctata
taagcagagc tcgtttagtg aaccgtcaga tcgcctggag 6120acgccatcca
cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccgcgg
6180ccgggaacgg tgcattggaa cgcggattcc ccgtgccaag agtgacgtaa
gtaccgccta 6240tagactctat aggcacaccc ctttggctct tatgcatgct
atactgtttt tggcttgggg 6300cctatacacc cccgcttcct tatgctatag
gtgatggtat agcttagcct ataggtgtgg 6360gttattgacc attattgacc
actcccctat tggtgacgat actttccatt actaatccat 6420aacatggctc
tttgccacaa ctatctctat tggctatatg ccaatactct gtccttcaga
6480gactgacacg gactctgtat ttttacagga tggggtccca tttattattt
acaaattcac 6540atatacaaca acgccgtccc ccgtgcccgc agtttttatt
aaacatagcg tgggatctcc 6600acgcgaatct cgggtacgtg ttccggacat
gggctcttct ccggtagcgg cggagcttcc 6660acatccgagc cctggtccca
tgcctccagc ggctcatggt cgctcggcag ctccttgctc 6720ctaacagtgg
aggccagact taggcacagc acaatgccca ccaccaccag tgtgccgcac
6780aaggccgtgg cggtagggta tgtgtctgaa aatgagcgtg gagattgggc
tcgcacggct 6840gacgcagatg gaagacttaa ggcagcggca gaagaagatg
caggcagctg agttgttgta 6900ttctgataag agtcagaggt aactcccgtt
gcggtgctgt taacggtgga gggcagtgta 6960gtctgagcag tactcgttgc
tgccgcgcgc gccaccagac ataatagctg acagactaac 7020agactgttcc
tttccatggg tcttttctgc agtcaccgtc gtcgacacgt gtgatcagat
708091023DNAherpes simplex virus 2 gD 9atgggcagac tgactagcgg
agtgggcaca gccgccctgc tcgtggtggc tgtgggcctg 60agagtggtgt gtgctaagta
cgccctggct gaccctagcc tgaagatggc tgatcctaat 120aggtttaggg
gcaagaacct gcccgtgctg gaccagctga ctgacccccc tggcgtgaag
180agagtgtacc acatccagcc tagcctggag gaccccttcc agccccctag
catccctatc 240accgtgtact acgccgtgct ggagagagcc tgtagaagcg
tgctgctgca cgcccctagt 300gaggcccctc agattgtgag aggcgctagt
gacgaggcta ggaagcacac ctataacctg 360accatcgcct ggtataggat
gggcgataac tgcgccatcc ccatcacagt gatggagtac 420actgagtgcc
cctataataa gagcctgggc gtgtgtccca ttaggaccca gcctaggtgg
480agctactacg atagctttag cgccgtgagt gaggataacc tgggcttcct
gatgcacgcc 540ccagcctttg agaccgccgg cacctacctg agactggtga
agattaacga ctggactgag 600atcacccagt tcatcctgga gcatagggct
agggctagct gtaaatacgc cctgcccctg 660agaatccccc ctgccgcctg
cctgactagt aaggcctacc agcaaggcgt gaccgtggat 720agcatcggca
tgctgcctag attcatccct gagaaccaga gaaccgtggc cctgtatagc
780ctgaaaatcg ccggctggca cggccctaag cctccttaca ctagcaccct
gctgccccct 840gagctgagtg ataccactaa cgccacccag cctgagctgg
tgcctgagga ccctgaggat 900agcgctctgc tggaagatcc tgccggcacc
gtgagtagcc agatcccccc taactggcac 960atccctagca ttcaggacgt
ggccccccac cacgcccctg ccgctcctag taaccctggc 1020tga
102310900DNAherpes simplex virus 2 UL49 10atgacctcta ggcggagcgt
gaagagctgc cctagagagg cccctagagg cacccacgag 60gagctgtact acggccctgt
gtcccctgcc gaccctgaga gccctagaga tgactttaga 120cggggagccg
gccctatgag agccagacct agaggcgaag tgagattcct gcactacgac
180gaggccggct acgccctgta tcgggatagc agctctgacg acgacgagtc
tagggatacc 240gccaggccta gaagaagcgc cagcgtggcc ggcagccacg
gccctggccc tgccagagcc 300ccccctcctc ctggcggccc tgtgggagcc
ggcggaagaa gccacgcccc tcccgcccgg 360acccctaaga tgaccagagg
cgcccctaag gccagcgcca cccccgccac cgatcccgcc 420agaggcagga
gacccgccca ggccgatagc gccgtgctgc tggacgcccc tgcccccacc
480gcctccggca gaaccaagac ccctgcccag ggcctggcca agaagctgca
ctttagcacc 540gcccctcctt cccccaccgc cccctggacc cctagagtgg
ccggctttaa taagcgcgtg 600ttctgtgccg ctgtgggcag actggccgcc
acccacgcca ggctggccgc cgtgcagctg 660tgggatatga gcagacccca
caccgacgag gacctgaacg agctgctgga cctgaccaca 720attagagtga
ccgtgtgtga gggcaagaac ctgctgcaga gggccaacga gctggtgaac
780cctgacgccg cccaggacgt ggacgccacc gccgccgcca ggggcagacc
tgccggcaga 840gccgccgcca cagccagggc ccctgccaga agcgcctcta
ggccaagacg gcccctggag 900112088DNAherpes simplex virus 2 UL47
11atgtctgtga gaggccacgc tgtgagaaga agaagggcta gcaccagaag ccacgcccct
60agcgcccaca gagccgatag ccctgtggag gatgagcctg agggcggagg agggggcctg
120atgggctacc tgagggccgt gtttaacgtg gacgacgata gcgaagtgga
agccgccgga 180gagatggcct ctgaggagcc ccctcctaga aggagaagag
aggccagagg ccaccccggc 240tctaggagag cctctgaggc cagagccgcc
gccccaccta gaagagccag cttccctaga 300cctagaagcg tgaccgccag
aagccagtct gtgcgcggca ggagggatag cgctatcacc 360agagccccta
gaggcggcta cctgggccct atggaccctc gcgacgtgct gggcagagtg
420ggcggctcta gggtggtgcc tagccccctg ttcctggatg agctgagcta
cgaggaggac 480gactaccctg ccgccgtggc ccacgacgac ggagccggag
ccagaccccc tgccaccgtg 540gagatcctgg ccggcagagt gagcggacct
gagctgcaag ccgccttccc cctggatcgg 600ctgacccctc gggtggccgc
ctgggatgag tctgtgagga gcgccctggc cctgggccac 660cctgccggct
tctacccctg ccctgattcc gccttcggcc tgagcagagt gggagtgatg
720cacttcgcca gccctgccga ccctaaagtg ttcttccggc aaacactgca
gcagggcgag 780gccctcgcat ggtacatcac cggcgacgcc atcctggatc
tgaccgatag acgggccaag 840accagcccta gcagagctat gggctttctg
gtggacgcta ttgtgagagt ggctattaac 900ggctgggtgt gcggcaccag
actgcacacc gagggcagag gctctgagct ggatgataga 960gccgccgagc
tgaggagaca gttcgccagc ctgaccgccc tgagacctgt gggcgccgct
1020gccgtgcccc tgctgagcgc cggaggagcc gcccctcccc accctggccc
tgacgccgcc 1080gtgtttcggt ctagcctggg cagcctgctg tactggcccg
gagtgagagc cctgctgggc 1140agggactgta gagtggctgc cagatacgcc
ggcaggatga cctacatcgc caccggcgcc 1200ctgctggcca gatttaaccc
tggcgccgtg aaatgcgtgc tgcctagaga agccgccttc 1260gccggaagag
tgctggacgt cctggccgtg ctggctgagc agaccgtgca gtggctgagc
1320gtggttgtgg gcgccaggct gcaccctcac agcgcccacc ctgccttcgc
cgacgtggag 1380caggaggccc tgtttagagc cctgcctctg ggcagccctg
gcgtggtggc cgccgagcac 1440gaagccctgg gcgacaccgc tgccaggaga
ctgctcgcca caagcggcct gaacgccgtg 1500ctgggagccg ccgtgtacgc
cctgcacacc gccctggcca ccgtgaccct gaaatacgcc 1560ctggcctgcg
gcgacgcccg cagacgccgc gacgacgccg ctgcagccag agccgtcctg
1620gccaccggcc tgatcctgca gaggctgctg ggcctggccg acaccgtggt
ggcctgcgtg 1680gccctggccg cctttgacgg cggcagcacc gcccctgaag
tgggcaccta cacccctctg 1740agatacgcct gcgtgctgag agccacccag
cctctgtacg ccagaaccac ccctgccaag 1800ttctgggccg atgtgagggc
cgccgccgaa cacgtggacc tgagacccgc ctctagcgcc 1860ccaagggccc
ctgtgagcgg caccgccgac cccgccttcc tgctggagga tctggccgct
1920ttccctcccg cccctctgaa tagcgagagc gtgctggggc ctagagtgag
agtggtggat 1980attatggccc agtttagaaa gctgctgatg ggcgacgagg
aaacagccgc cctgagggcc 2040cacgtgtctg gcagaagagc cacaggcctg
ggcggacctc ctagacct 2088122163DNAherpes simplex virus 2 UL46
12atgcagcgga gagccagagg cgcctctagc ctgagactgg cccggtgcct gacccccgcc
60aatctgatta gaggcgccaa cgccggcgtg cctgagagaa gaatcttcgc cggctgcctg
120ctgcctaccc ctgagggcct gctgagcgcc gctgtgggcg tgctgagaca
gagggccgat 180gacctgcagc ccgccttcct gaccggcgcc gatagatctg
tgaggctggc cgccagacac 240cacaacaccg tgcctgagtc cctgatcgtg
gacggcctgg cctctgaccc ccactacgac 300tacattaggc actacgccag
cgccgccaag caggccctgg gcgaagtgga gctgagcggc 360ggacagctga
gcagagccat cctggcccag tactggaagt acctgcagac cgtggtgcct
420agcggcctgg acatccctga tgatcctgcc ggcgactgtg accctagcct
gcacgtgctg 480ctgagaccca cactgctgcc taagctgctt gtgagggccc
cctttaagag cggcgctgcc 540gccgccaaat acgccgccgc cgtggccggc
ctgagggacg ccgcccacag actgcagcag 600tatatgttct ttatgagacc
cgccgaccct agcagaccta gcaccgacac cgccctgaga 660ctgagcgagc
tgctggccta tgtgagcgtg ctgtaccact gggccagctg gatgctgtgg
720accgccgata aatacgtgtg taggcggctg ggccctgccg atagaagatt
cgtggccctg 780agcggcagcc tggaggcccc tgccgagacc tttgcccggc
acctggatag aggccctagc 840ggcaccaccg gctctatgca gtgtatggcc
ctgagagccg ccgtgtctga cgtgctgggc 900cacctgacca gactggccca
cctgtgggag accggcaaga gaagcggcgg cacctacggc 960atcgtggacg
ccattgtgag caccgtggaa gtgctgagca tcgtgcacca ccacgcccag
1020tacatcatta acgccaccct gaccggctac gttgtgtggg cctctgatag
cctgaataat 1080gagtacctga gggctgccgt cgatagccag gagcggttct
gtagaacagc cgcccctctg 1140ttccccacca tgaccgcccc ttcctgggcc
agaatggaac tgagcattaa gagctggttc 1200ggagccgccc tggcccctga
cctgctgaga agcggcaccc ctagccctca ctacgagagc 1260atcctgcgcc
tggctgccag cggccctcct ggcggcagag gagctgtggg cggcagctgt
1320agggataaga tccagcggac ccggagagat aacgcccctc cccccctgcc
tcgggccaga 1380ccccacagca cccctgctgc ccctcggcgg tttagacggc
acagagagga cctgcctgag 1440cctccccacg tggacgccgc cgataggggc
cctgagccct gcgccggcag acccgccacc 1500tactacaccc acatggccgg
agccccccct cggctgcccc ctcggaaccc tgcccctcct 1560gagcagagac
ctgccgccgc tgcccggcct ctggccgccc agagagaagc cgccggagtg
1620tatgacgctg tgagaacctg gggccctgac gccgaggccg agcctgatca
gatggagaac 1680acctacctgc tgcctgacga cgacgccgcc atgcctgccg
gagtgggcct gggcgccacc 1740ccagccgccg ataccacagc cgccgcctgg
cccgccaaga gccacgcccc tagagcccct 1800agcgaggacg ccgatagcat
ctacgaaagc gtgtctgagg acggcggcag agtgtatgag 1860gagatcccct
gggtgcgggt gtacgaaaac atctgcctga ggagacagga cgccggagga
1920gccgccccac ccggcgacgc ccctgatagc ccttacattg aggccgagaa
ccccctgtac 1980gactggggcg gcagcgccct gtttagcccc cctggcgcca
ccagagcccc tgaccccggc 2040ctgagcctga gccccatgcc cgccagacct
agaaccaacg ccctggccaa tgacggcccc 2100accaacgtgg ccgccctgag
cgccctgctg accaagctga agaggggcag acaccagagc 2160cac 2163
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