DESCRIPTION
FLAVIVIRUS HOST RANGE MUTATIONS AND USES THEREOF
PRIORITY CLAIM
[00011 This application claims priority to U.S. Application No. 61/317,103, filed March 24,
2010 and U.S. Application Serial No. 61/393,151 filed on October 14, 2010, the entire
contents of which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The present invention relates generally to virology and disease control. Specifically,
the present invention relates to mutated arthropod vectored viruses and their uses as vaccines.
n particular aspects, the present invention relates to improved flavivirus constructs for use in
preparing vaccines.
2. Description of Related Art
(00031 Arthropod vectored viruses (Arboviruses) are viral agents which are transmitted in
nature by blood sucking insects. Arboviruses include members of the Alpha-, Flavi- and
Bunyaviridae. The family of flaviviruses includes approximately 60 enveloped, positive
strand RNA viruses, most of which are transmitted by an insect vector. Many members of
this family cause significant public health problems in different regions of the world
(Monath, 1986). The genome of all flaviviruses sequenced thus far has the same gene order:
5'-C-preM-E-NSl-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3' in which the first three genes
code for the structural proteins the capsid (C), the pre-membrane protein (preM) and the
envelope protein (E).
[0004] By their very nature, flaviviruses, like other Arboviruses, must be able to replicate in
the tissues of both the invertebrate insect and the mammalian host (Brown and Condreay,
1986, Bowers et al, 1995). Differences in the genetic and biochemical environment of these
two host cell systems provide a basis for the production of host range mutant viruses which
can replicate in one host but not the other.
[0005] Dengue virus is a positive-sense RNA virus belonging to the Flavivirus genus of the
family Flaviviridae. Dengue virus is widely distributed throughout the tropical and
semitropical regions of the world and is transmitted to humans by mosquito vectors. Dengue
virus is a leading cause of hospitalization and death in children in at least eight tropical Asian
countries (WHO, 1997). Currently, Dengue Fever and other flaviviruses are in resurgence in
the United States. The U.S. Army and other government agencies have been trying to make
vaccines against these viruses since the 1960's with little success. Thus, there is a need to
develop flavivirus vaccines for humans.
SUMMARY OF THE INVENTION
[0006] Viruses that are transmitted in nature by blood sucking insects are a major source of
disease in man and domestic animals. Many of these viruses have lipid membrane bilayers
with associated integral membrane proteins as part of their three dimensional structure. These
viruses are hybrid structures in which the proteins are provided by the genetic information of
the virus and the membrane is the product of the host cell in which the virus is grown.
Differences in the composition of the membranes of the mammalian and insect host are
exploited in aspects of the present invention to produce virus mutants containing deletions in
the membrane spanning domains of the virus membrane proteins. Some of the mutants are
capable of replicating and assembling normally in the insect host cell but assemble poorly in
the mammalian host cell. These host range mutants could produce immunity to wild-type
virus infection when used as a vaccine and represent a novel strategy for the production of
vaccines against arthropod vectored, membrane containing viruses like flaviviruses.
[0007] In certain aspects of the invention, there is provided an engineered nucleic acid
comprising a sequence encoding a modified viral transmembrane protein comprising a
mutation, wherein the mutation inhibits the production or infectivity of a virus comprising the
modified viral transmembrane protein in mammalian cells. The term nucleic acid sequence as
used herein comprises both RNA and DNA sequences, consistent with its usage in the art.
The modified transmembrane protein may be able to .span or correctly integrate into the
membrane of insect cells but not that of mammalian cells due to mutation of one or more
amino acids in the viral transmembrane protein, in particular, a transmembrane domain
thereof. The virus comprising the modified viral transmembrane protein may be capable of
infecting and producing progeny virus in insect cells. The virus also may or may not be
capable of infecting; however, the virus may have reduced ability to produce progeny in
mammalian cells.
[0008] Thus, in accordance with the present invention, the mutation will preferably reside in
a transmembrane protein of a flavivirus, for example, the envelope (E) protein of a flavivirus,
particularly the E protein's transmembrane domain, and more particularly, the N-terminal
transmembrane domain (E-Tl domain).
[0009] A linear sequence of a transmembrane domain has a central amino acid defined as that
amino acid residue that resides essentially at the center of the membrane spanning amino
acids. Thus, in the case of the flavivirus E-Tl domain, the central amino acid will most often
be the amino acid closest to the center of the 16 amino acid transmembrane domain, i.e., the
8 h or 9th amino acid used here refers to the central amino acid, which is Glycine (Gly or G) in
most of the more common Flaviviruses (see, e.g., Table 1). Such flavivirus E-Tl
transmembrane domains include predicted transmembrane domains based on primary
sequences.
[0010] Alignment of representative Flaviviruses from each of the main groups which are
main human pathogens are shown in Table 1. The alignment of the representative
Flaviviruses as compared to Dengue virus serotype 2 (DV2) was performed by DNASTAR
Lasergene software, MegAlign program, using the Clustal W method. The E-Tl domain in
Dengue virus serotype 2 (DV2),served as the basis for E-Tl sequence alignment is predicted
as amino acids 452 to 467 of the E protein (Zhang et al, 2003). Other flavivirus E-Tl
sequences not shown herein can be determined by optimal sequence alignment to the E-Tl
sequences of any of the representative Flaviviruses, for example, by the Bestfit method, and
the "central" amino acid determined accordingly.
Table 1 Flavivirus E-Tl Sequences (bold: central amino acid)
Yellow Fever Virus X03700 45 1NWITKVIMGAVLIWVG 66 3
Zika Virus AY632535
57SWFSQILIGTLLVWLG472 4
[001 ] For the practice of such preferred aspects of the present invention, amino acids of the
transmembrane domain are numbered by relative positions based on the central amino acid of
a unmodified or unmutated domain, which is numbered as position 0 (for example, G460 in
Dengue 2 virus), wherein amino acids proceeding toward the N terminus from the central
amino acid are numbered -1, -2, etc., and amino acids proceeding toward the C-terminus
from the central amino acid are numbered +1, +2, etc. For the purposes of such preferred
aspects, the mutation may comprise a deletion of at least two, three, four, five or more amino
acids comprising the central amino acids. Representative examples of such a mutation may
comprise or consist of a deletion of amino acids at positions of 0 to +3, 0 to +4, -2 to 0, -3 to
0, or - l to +1.
[0012] Non-limiting examples of flavivirus include Dengue viruses (DV), West Nile virus
(WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis
virus (TBE virus), Murray Valley encephalitis virus (MVEV), Saint Louis encephalitis virus
(SLEV), and Powassan virus (PV). The engineered nucleic acid sequences comprising a
modified transmembrane protein, especially a modified transmembrane domain, from each of
these viruses is included as part of the present invention.
[0013] The Dengue virus group may include dengue virus types 1, 2, 3, and 4. In specific
particularly preferred embodiments, the mutation of Dengue virus type 2 may comprise a
deletion at amino acids 458 to 460 (deletion of L458, 1459, and G460, i.e., deletion of
positions -2 through 0) or at amino acids 460 to 463 (deletion of G460, V461, 1462, and 1463,
i.e., deletion of positions 0 through +3). The virus may also be Dengue virus type 1 and the
mutation may comprise a deletion at amino acids 458 to 460, amino acids 460 to 463, amino
acids 457 to 460, amino acids 460 to 464, or amino acids 459 to 461. The virus may also be
Dengue virus type 3 and the mutation may comprise a deletion at amino acids 456 to 458,
amino acids 458 to 461, amino acids 455 to 458, amino acids 458 to 462, or amino acids 457
to 459. The virus may also be Dengue virus type 4 and the mutation may comprise a deletion
at amino acids 458 to 460, amino acids 460 to 463, amino acids 457 to 460, amino acids 460
to 464, or amino acids 459 to 461. The virus may also be West Nile virus and the mutation
may comprise a deletion at amino acids 463 to 465, amino acids 465 to 468, amino acids 462
to 465, amino acids 465 to 469, or amino acids 464 to 466. See examples in Table 2.
Table 2. Dengue and West Nile virus E-Tl Transmembrane Domain Mutants
[0014] The virus comprising the modified transmembrane protein such as flavivirus E protein
may have an ability to produce at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900,
1000, 5000, 104 fold (or any range derivable therein) more progeny virus when infecting
insect cells than when infecting mammalian cells. In certain aspects, the mammalian cells are
human cells. In additional aspects, the insect cells may be mosquito cells.
|0015] In a further aspect, there may be provided a modified flavivirus envelope protein
encoded by an engineered nucleic acid in accordance with aspects of the present invention. In
a still further aspect, there may be provided a genetically engineered flavivirus comprising an
engineered nucleic acid in accordance with aspects of the present invention. In certain
aspects, an immunogenic composition comprising an engineered nucleic acid or a genetically
engineered flavivirus in accordance with aspects of the present invention may be also
provided. In certain aspects of the immunogenic composition, the engineered nucleic acid
may be comprised in a virus particle. Such an immunogenic composition may be further
defined as a vaccine composition in some aspects. Furthermore, the immunogenic
composition may comprise an adjuvant, a preservative, or two or more viruses or nucleic
acids, which are engineered in accordance with aspects of the invention or native. For
example, the vaccine composition may comprise one or more of the genetically engineered
Dengue virus 1, 2, 3, and 4. In a particular aspect, the vaccine composition may comprise the
genetically engineered Dengue virus type 2. For long lasting protection against all Dengue
virus serotypes, the vaccine composition may comprise the genetically engineered Dengue
virus of all sero types, particularly, a tetravalent vaccine composition.
[0016] Aspects of the invention may further include a method of producing a viral vaccine
from the genetically engineered flavivirus for vaccination of mammals, comprising
introducing the genetically engineered virus to insect cells to produce a viral vaccine. There
may also be provided a method of inducing an immune response in a mammal, comprising
administering the immunogenic composition to the mammal.
[0017] In a further embodiment there is provided a vaccine composition comprising one or
more mutant flaviviruses, according to aspects of the invention, and pharmaceutically
acceptable excipient. Thus, it will be understood that the vaccine composition may comprise
any of the mutant flaviviruses described herein. In further specific embodiments, a vaccine
composition may comprise sequences from two or more viruses according to the current
invention. For example, the vaccine composition may comprise engineered sequences from
four Dengue virus serotypes. In some embodiments, the mutant flavivirus is defective in
assembly or infectivity in mammalian cells but competent to assemble in or infect insect cells
due to mutations in the transmembrane domain. In other embodiments the viruses may be
further inactivated. For example in some specific cases the viruses according to the invention
may be inactivated by irradiation, or chemical treatment, such as formalin treatment. In
further embodiments, vaccine compositions according to aspects of the invention may further
comprise additional elements such as an adjuvant, an immunomodulator and/or a
preservative.
[0018] In some further aspects of the invention, there is provided a method of vaccinating an
animal comprising administering the vaccine composition to a mammal. The mammal may
be a human or a primate, such as a monkey. For example, in some specific embodiments the
vaccine composition is administered to a human, however the method may also be used to
vaccinate livestock, wild and domesticated birds, cats, and dogs. In certain cases, the vaccine
composition may be administered, orally, intravenously, intramuscularly, intraperitoneally,
intradermally or subcutaneously. In some cases the vaccine composition is administered
multiple times, and in certain cases each administration is separated by a period of days,
weeks, months or years.
[0019] Embodiments discussed in the context of methods and/or compositions of the
invention may be employed with respect to any other method or composition described
herein. Thus, an embodiment pertaining to one method or composition may be applied to
other methods and compositions of the invention as well.
[0020] As used herein the terms "encode" or "encoding" with reference to a nucleic acid are
used to make the invention readily understandable by the skilled artisan; however, these
terms may be used interchangeably with "comprise" or "comprising" respectively.
[0021] As used herein the specification, "a" or "an" may mean one or more. As used herein
in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an"
may mean one or more than one.
[0022] The use of the term "or" in the claims is used to mean "and/or" unless explicitly
indicated to refer to alternatives only or the alternatives are mutually exclusive, although the
disclosure supports a definition that refers to only alternatives and "and/or." As used herein
"another" may mean at least a second or more.
[0023] Throughout this application, the term "about" is used to indicate that a value includes
the inherent variation of error for the device, the method being employed to determine the
value, or the variation that exists among the study subjects.
[0024] Other objects, features and advantages of the present invention will become apparent
from the following detailed description. t should be understood, however, that the detailed
description and the specific examples, while indicating preferred embodiments of the
invention, are given by way of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The following drawings form part of the present specification and are included to
further demonstrate certain aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in combination with the detailed
description of specific embodiments presented herein.
[0026] FIG. 1. Schematic representation of the organization of Dengue virus.
Representation of DV protein structure illustrating the predicted orientation across the
endoplasmic reticulum (ER). Cylinders represent transmembrane (T) helices. prM, membrane
protein precursor; E, envelope protein; NSl, non-structural protein. The minimal predicted
sequences of the E protein Tl and M protein Tl are shown. Underlined residues indicate the
amino acids targeted for deletion. The TM domain of the capsid protein (anchored C) is
cleaved during processing and is not present in the membrane of the assembled virus.
[0027] FIG. 2. Host-range phenotype of DV mutants. Titers for the DV2 WT and mutant
viruses are shown. All virus strains were grown in Vero (mammalian) and the C6/36 (insect)
cells to measure host-range phenotype. The titers of the mutant viruses and WT viruses were
measured in the Vero cell line by the fluorescent focus assay.
[0028] FIGS. 3A-3F. Electron microscopy of WT DV, ALIG, AGVII viruses in C6/36
and Vero cells. Shown in FIGs. 3A-3F are thin sections of the DV strains studied. In FIG.
3A are C6/36 and in FIG. 3B, Vero cells infected with the wild type DV virus. Virus
particles are seen in large paracrystalline structures within the mosquito cell (FIG. 3Aarrows)
and associated with the Vero plasma cell membrane (FIG. 3B-arrows). In FIG. 3C,
are shown mosquito cells infected with the AGVII mutant which displayed similar amounts
of virus particles to that seen in the wt virus infected mosquito cells (arrows). However, in
mammalian cells infected with the same mutant (FIG. 3D AGVII) only the presence of
nucleocapsids in the cytoplasm could be detected in the thin sections (arrowheads). In cells
infected with the mutant ALIG similar results were observed to that of the AGVII mutant.
The presence of virus particles could be observed in the cytoplasm of mosquito cells (FIG.
3E - arrowheads) while only nucleocapsids were detected in the cytoplasm of the Vero cells
(FIG. 3F- arrowheads). Bars are 500 mM.
[0029] FIG. 4. Mouse Neutralizing Antibody (Nab) data. The neutralizing antibody titers
were measured by the focus reduction neutralization assay using WT DV2 and sera from each
individual mouse infected with the mutant virus DV2ALIG or DV2AGVII. The assay was
done on Vero cells. Individual mouse serum from five mice in each group was analyzed and
the titers are shown above as bars. Each bar represents the reciprocal neutralization antibody
titer at 50% neutralization for each mouse.
[0030] FIG. 5. Assessment of pre-challenge viremia in African green monkeys. . Viremia
analysis of serum samples from study day 1 through 14 was done by infectious center assay.
The mock injected animals had no detectable viremia. The titers were plotted as average
viremia titers for 4 monkeys/group.
[0031] FIG. 6. Pre challenge NAb titers. The Nab titers were measured by Plaque
Reduction Neutralization Test assay (PRNT) using the serum from each monkey per
experimental group for Days 0, 5, 7, 14 and 30 post vaccination. Each data point represents
the average of neutralizing antibody titers for all 4 monkeys per group.
[0032] FIG. 7. Assessment of post-challenge viremia in African green monkeys.
Monkeys were initially treated with a negative control, vaccine strain SI6803 variant LAV,
or the experimental DV2ALIG or DV2AGVII vaccine. On day 57, the monkeys were
challenged with live DEN-2 challenge virus (strain SI6803 wild type; 4-5 loglO PFU per
animal). The day 1 data point represents the first day post-challenge, which correlates with
day 58 of the study. Each experimental group contained 4 monkeys, and the data points
shown represent the average of the viremia measurements in genome equivalents/mL
observed for all monkeys in each group.
J0033J FIG. 8. Post-challenge Nab titers. The post-challenge Nab titers were mearured by
PRNT using the serum from each monkey per experimental group for Days 57, 59, 61, 62,
63, 64, 7 1 and 142 post vaccination. Each data point represents the average of neutralizing
antibody titers for all 4 monkeys per group.
[0034] FIG. 9. Total IgG pre and post challenge. Total IgG was measured by Dengue IgG
ELISA kit. Each bar represents the average Antibody Index of 4 monkey serum samples per
experimental group. The measurments were done on the days represented on X-axis.
[0035] FIG. 10. Total IgM post challenge. Total IgM was measured by Dengue IgM ELISA
kit. Each bar represents the average Antibody Index of 4 monkey serum samples per
experimental group. The measurements were done on the days represented on X-axis.
[0036] FIG. 11. Changes in body temperature following vaccine administration. Body
temperature was determined using a rectal thermometer as part of clinical observations. Data
points indicate the mean body temperature with vertical bars representing the standard error,
SEM (n=4). The body temperatures were measured on the days 0, 1, 2, 3 and 30 after
vaccination.
[0037] FIG. 12. Changes in body temperature following viral challenge. Body
temperature was determined using a rectal thermometer as part of clinical observations. Data
points indicate the mean body temperatures with vertical bars representing the standard error,
SEM (n+4). The body temperatures were measured on the day 57 through 65 and on day 7 1
post vaccination.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
I. Introduction
[0038] There are over 700 known arboviruses and at least 80 immunologically distinct types
that cause disease in humans. Arboviruses are transmitted among vertebrates by biting
insects, chiefly mosquitoes and ticks. These viruses are widely distributed throughout the
world, depending on the presence of appropriate hosts (birds, horses, domestic animals,
humans) and vectors. Mosquito-borne arboviruses present some of the most important
examples of emerging' and resurgent diseases of global significance. A strategy has been
developed herein by which host range mutants of flavivirus such as Dengue virus can be
constructed by generating deletions in the transmembrane domain of envelope (E)
glycoprotein. The host range mutants produced are restricted to growth in the insect hosts and
elicited antibody production in preliminary mouse trials. This method of producing vaccine
strains of arboviruses like flaviviruses is novel, simple and inexpensive compared to other
non-infectious vaccine platforms.
[0039] As used herein, the term "membrane-bound virus" refers to a virus which contains a
lipid membrane bilayer as part of its protective exterior coat.
[0040] As used herein the term "viral envelope" refers to the lipid membrane component of
the membrane containing virus and its associated proteins.
[0041] As used herein, the terms "arthropod vectored virus" or "Arbovirus" refer to viral
agents which replicate and produce progeny virus in arthropod (insect) or mammalian cells.
This includes Togaviruses, Flaviviruses and Bunyaviruses. As used herein, the term
"Togavirus" refers to a general classification of membrane containing viruses which include
the Alphaviruses.
[0042] As used herein, the term "membrane bilayer" refers to a structure consisting of
opposed amphipathic phospholipids. The bilayer is organized in cross section from polar
head groups to non-polar carbon chains to nonpolar carbon chains to polar head groups.
[0043] As used herein, the term "transmembrane domain" refers to the amino acid sequence
of the region of a membrane-integrated protein which spans the membrane bilayer.
[0044] As used herein, the term "viral vaccine" refers to a strain of virus or virus mutant or a
combination of such viruses or virus mutants which has the antigenic properties of the virus
but cannot produce disease or a combination of such viruses or virus mutants.
[0045] As used herein the term "immune surveillance" refers to a process by which blood
lymphocytes survey the cells and tissues of a mammal to determine the presence of foreign
(virus) proteins and stimulates the production of lymphocytes capable of targeting cells
producing the foreign protein for destruction. This process also leads to the production of
circulating antibodies against the foreign protein.
[0046] As used herein, the term "infectious virus particles" refers to viruses which are
capable of entering a cell and producing virus protein, whether or not they are capable of
producing progeny virus.
[0047] As used herein, the term "non-infectious virus particles" refers to viruses which are
not capable of infecting or entering a cell.
[0048] As used herein, the term "vertebrate cells" refers to any mammalian cell, such as
human or monkey cells.
[0049] As used herein, the term "invertebrate cells" refers to any insect cell, such as
mosquito cells.
[0050] Antigen may refer to foreign protein recognized by the immune system
[0051] "Attenuated" may refer to impaired in the ability to produce infectious virus particles.
[0052] CBC, or complete blood count, may be measured in a blood test to determine the
health of an individual.
|0053] Challenge virus may refer to a highly infectious strain of virus which is administered
to the host animals after the initial vaccination. It is given after a period during which the
immune system should have responded to the virus. A good vaccine will not allow the
challenge virus to produce significant viremia.
[0054] Efficacy may refer to the ability of a drug to produce the desired therapeutic effect or
the ability of a vaccine to protect against an infection.
[0055] ELISA, or enzyme linked immunosorbent assay, refers to a colorimetric assay used to
detect antibodies (Ab) against specific antigens (proteins).
[0056] Erythema refers to redness of the skin due to inflammation.
[0057] FFU, focus forming units, may be the concentration of foci of infection of any given
virus/ml. It may be used in a test to measure the number of infectious virus particles in a
sample. A focus is a localized infection of cells in a cell culture. It may be viewed by
staining with specific Abs for a substrate that may give a colored or fluorescent product. The
number of infectious virus may be referred to as the virus titer.
[0058] FRNT, or focus reduction neutralization test, refers to an assay for Ab which
neutralizes virus specifically and as a result cause reduction in the number of foci. PRNT, or
Plaque reduction neutralization test, differs from a focus-based test by the way the infections
are visualized. Plaques can be seen with the naked eye.
[0059] Hematocrit refers to proportion of red blood cells in the blood.
[0060] IC refers to infectious centers. Foci of infection that are grown as spherical foci
involving a larger volume of infected cells than a typical flat focus.
[0061] Nab, or neutralizing Ab, may refer to antibodies (Ab) produced by the immune
system which will inactivate virus specifically. IgG is the most desired Ab to induce when an
infection is in the blood stream. It is used as one measure of the immunogenicity of a vaccine.
IgM and IgG are different types of Ab produced at different times during the immune
response to antigen.
[0062] Phenotype may refer to any measurable physical or biochemical characteristics of an
organism, as determined by both genetics and environment.
[0063] Real time PCR, or qPCR, refers to a biochemical measurement of the virus genetic
material or RNA. It is a measure of how much virus has replicated.
[0064] Viremia may refer to the presence of virus in the bloodstream.
[0065] Wild-type virus may refer to original, parental genetic viral sequence containing no
mutations.
II. Flaviviruses
[0066] In certain aspects of the invention, there may be provided compositions and methods
related to modification of flavivirus proteins for generating mutations that affect host range
phenotype. Therefore, flavivirus vaccine may be provided.
[0067] Flaviviruses are small, enveloped, positive-strand RNA viruses, several of which pose
current or potential threats to global public health. Yellow fever virus, for example, has been
the cause of epidemics in certain jungle locations of sub-Saharan Africa, as well as in some
parts of South America. Although many yellow fever infections are mild, the disease can also
cause severe, life-threatening illness. The disease state has two phases. The initial or acute
phase is normally characterized by high fever, chills, headache, backache, muscle aches, loss
of appetite, nausea, and vomiting. After three to four days, these symptoms disappear. In
some patients, symptoms then reappear, as the disease enters its so-called toxic phase. During
this phase, high fever reappears and can lead to shock, bleeding (e.g., bleeding from the
mouth, nose, eyes, and/or stomach), kidney failure, and liver failure. Indeed, liver failure
causes jaundice, which is yellowing of the skin and the whites of the eyes, and thus gives
"yellow fever" its name. About half of the patients who enter the toxic phase die within 10 to
1 days. However, persons that recover from yellow fever have lifelong immunity against
reinfection. The number of people infected with yellow fever virus over the last two decades
has been increasing, with there now about 200,000 yellow fever cases reported, with about
30,000 deaths, each year. The re-emergence of yellow fever virus thus presents a serious
public health concern.
[0068] Fully processed, mature virions of flaviviruses contain three structural proteins:
capsid (C), membrane (M), and envelope (E). The infection also produces seven non¬
structural proteins. Immature flavivirions found in infected cells contain pre-membrane (prM)
protein, which is a precursor to the M protein. The flavivirus proteins are produced by
translation of a single, long open reading frame to generate a polyprotein, followed by a
complex series of post-translational proteolytic cleavages of the polyprotein, to generate
mature viral proteins (Amberg, 1999; Rice, 1995). The virus structural proteins are arranged
in the polyprotein in the order C-prM-E.
[0069] Dengue Virus (DV), the most prevalent arbovirus, is in the family Flaviviridae and
has four distinct serotypes which cause an acute disease of sudden onset with headache,
fever, prostration, severe joint and muscle pain, lymphadenopathy, and rash (Martina et al,
2009; WHO, 2009). DV is transmitted by mosquitoes and as distribution and density of these
has expanded, a considerable increase in Dengue virus transmission in tropical and
subtropical areas throughout the world has been observed, with about 50 million cases of
Dengue Fever and 500,000 cases of the more severe Dengue Hemorrhagic Fever (DHF).
Over 20,000 deaths each year can be attributed to DHF, ranking Dengue with tuberculosis,
STDs (including HIV), childhood diseases or malaria in costs of care and economic impact.
DV is also the only known arbovirus that has fully adapted to the human host and has lost the
need of an enzootic cycle for maintenance The lack of prophylactics, vaccines or antivirals
against DV alone leaves 2 billion people at risk yearly to contract this disease (WHO, 2009).
|0070] DV is an enveloped virus of 40 to 50 nm diameter with an icosahedral capsid that
contains a single-stranded, positive sense RNA genome (Zhang et al, 2003). The envelope of
DV is composed of hetero-dimers of the (E) glycoprotein and the membrane (M) protein that
are embedded in a host-derived lipid bilayer (FIG. 1). The envelope surrounds the capsid
composed entirely of the capsid (C) protein which encapsulates the RNA genome. The E
glycoprotein is important for cell receptor attachment, and infection of the target cell
membrane, and bears the neutralization epitopes (Mukhopadhyay et al., 2005). DV has, as
have all arboviruses evolved to replicate in the unique biochemical environments of both
vertebrate and invertebrate hosts (Condreay and Brown, 1986). The mature viruses are
hybrids which derive their lipid bilayers from the host cell. Insect cell membranes do not
contain cholesterol and are thinner in cross-section (Bretscher and Munro, 1993); therefore,
the membrane-spanning domains (transmembrane domains; TMD) of proteins integrated into
insect cell membranes have evolved to accommodate both host membranes. The TMD of the
virus grown in the insect hosts has been shown in the alphavirus Sindbis to tolerate large
deletions and thus was shown not to require the same spanning length as those integrated into
mammalian membranes (Hernandez et al., 2003). This host-derived TMD difference was
used to develop a method for production of viral mutants with truncated TMD that are
capable of efficient growth in invertebrate cells but incapable of efficient productive
replication in vertebrate cells (Hernandez et al., 2003).
[0071 1As demonstrated by studies herein, a targeted and rational method of deleting amino
acids in the TMD of the envelope glycoproteins was used to create DV serotype 2 (DV2)
mutants. Deleting amino acids in the TMD of the E or M proteins of the virus will make them
shorter such that they are capable of spanning an insect but not the mammalian cell
membrane and as a result will show reduced infectivity in mammalian hosts but will retain
efficient growth in insect hosts, producing a host-range phenotype. This method of generating
a host range phenotype has been demonstrated for Sindbis, a structurally similar but distantly
related arbovirus (Hernandez et al., 2003). Deletions in the TMD of Sindbis virus (SV), the
prototypical arbovirus, resulted in virus with altered infectivity and host range (Hernandez et
al., 2003). Both E and M proteins of DV have a TMD that can be targeted for deletion
mutation analysis using the SV TMD deletion strategy. In the Example, mutant DV2 viruses
were identified as having a host range phenotype restricted to growth in insect cells. Studies
herein demonstrate that truncations of 3 to 4 amino acids in the TMD of the E domain at
amino acid positions (DV2 16681 numbering) between 458 to 463 resulted in virus with
attenuated virulence in mammalian cells that retained the ability to grow in mosquito cells
while larger deletions resulted in either no or very low levels of virus production and
infectivity.
[0072] Additional flaviviruses that can be used in the invention include other mosquito-borne
flaviviruses, such as Japanese encephalitis, Murray Valley encephalitis, St. Louis
encephalitis, West Nile, unj in, Rocio encephalitis, and Ilheus viruses; tick-borne
flaviviruses, such as Central European encephalitis, Siberian encephalitis, Russian Spring-
Summer encephalitis, Kyasanur Forest Disease, Omsk Hemorrhagic fever, Louping ill,
Powassan, Negishi, Absettarov, Hansalova, Apoi, and Hypr viruses.
[0073] In addition to the viruses listed above, as well as other flaviviruses, chimeric
flaviviruses that include one or more mutations that decrease replication in mammalian cells
are also included as a type of flavivirus in the invention. These chimeras can consist of a
flavivirus (i.e., a backbone flavivirus) in which a structural protein (or proteins) has been
replaced with a corresponding structural protein (or proteins) of a second virus (i.e., a test or
a predetermined virus, such as a flavivirus). For example, the chimeras can consist of a
backbone flavivirus (e.g., a yellow fever virus) in which the prM and E proteins of the
flavivirus have been replaced with the prM and E proteins of the second, test virus (e.g., a
dengue virus (serotypes 1-4), Japanese encephalitis virus, West Nile virus, or another virus,
such as any of those mentioned herein). The chimeric viruses can be made from any
combination of viruses. Preferably, the virus against which immunity is sought is the source
of the inserted structural protein(s).
III. Transmembrane domain mutations
|0074] The vaccines of certain aspects of the present invention are based on deletion
mutations in the transmembrane domains of membrane glycoproteins of membraneenveloped
viruses, especially E-Tl domain of flaviviruses. Many membrane-coated viruses
have membrane glycoproteins on their surface which are responsible for identifying and
infecting target cells (Schlesinger and Schlesinger, 1990). These membrane glycoproteins
have hydrophobic membrane-spanning domains which anchor the proteins in the membrane
bilayer (Rice et al, 1982).
[0075] The membrane-spanning domains of these transmembrane proteins need to be long
enough to reach from one side of the bilayer to the other in order to hold or anchor the
proteins in the membrane. Experiments have shown that if the domains are shortened by the
deletion of amino acids within the domain, the proteins do not appropriately associate with
the membrane and fall out (Adams and Rose. 1985). Unlike mammalian cell membranes, the
membranes of insect cells contain no cholesterol (Clayton 1964, Mitsuhashi et a , 1983).
Because insects have no cholesterol in their membranes, the insect-generated viral membrane
will be thinner in cross section than the viral membranes generated from mammals.
Consequently, the membrane-spanning domains of proteins integrated into insect membranes
do not need to be as long as those integrated into the membranes of mammals. It is possible,
therefore, to produce deletions in engineered viruses which remove amino acids from the
transmembrane domain of the viral glycoprotein. This results in a glycoprotein which can
integrate normally into the membrane of a virus replicating in an insect cell, but not into the
membrane of a virus replicating, infecting, or assembling normally in a mammalian cell.
Thus, the mutated virus can replicate and be produced in insect cells as well as the parent
wild-type virus. On the other hand, the mutant virus can infect mammalian cells and produce
viral proteins; however, since the mutated virus glycoprotein cannot span and be anchored in
the mammalian membrane, progeny virus cannot be produced in mammalian cells to wildtype
levels. An advantage to the approach of the present invention is that the mutants are
engineered as deletion mutants, and preferably deletion of at least two or three amino acids,
hence there is little chance for reversion to wild-type phenotype, a common problem with
virus vaccines.
[0076] The methods and compositions described by the present invention may work for any
virus which replicates in insects and mammals and has integral membrane proteins as part of
its structure, namely, Togaviruses, Flaviviruses and Bunya viruses and all other enveloped
viruses which can replicate naturally in both mammalian and insect cells, as well as
enveloped viruses which can be made to replicate in mammalian and insect cells by genetic
engineering of either the virus or the cell.
[0077] Vaccines may be made against any membrane-containing virus by removing amino
acids from the membrane-spanning domain of a protein in the viral envelope. This is
preferably done by removing nucleotides from a cDNA clone of the virus as described below.
RNA transcribed from the altered clone may be then transfected into insect cells. The viruses
produced are amplified by repeated growth in insect cells until large quantities of mutant
viruses are obtained. These viruses are tested for the ability to infect and produce progeny in
mammalian cells. Viruses which produce little to no progeny in mammalian cells are tested
for ability to produce immunity in laboratory animals. Those viruses which do produce
immunity are candidates for production of human and animal vaccines by procedures known
in the art. Non-limiting examples of Flavivirus mutants are shown below in Table 2. Glycine
(G) in the center of E-Tl transmembrane domain (amino acid (aa) G460 in Dengue serotypes
1, 2, and 4; aa G458 in Dengue 3; and aa G465 in West Nile virus) is designated as position
zero in accordance with aspects of the present invention.
|0078| In certain embodiments mutant viruses according to the current invention may
comprise two or more host range mutations or additionally comprise other mutations such as
attenuating mutations, mutations to increase immunogenicity or viral stability or any
mutations that may be used for vaccine production and that are current known in the art.
IV. Viral Vaccine
[0079] Certain aspects of the present invention are drawn to a method of producing a viral
vaccine from the genetically engineered membrane-enveloped virus disclosed herein for
vaccination of mammals, comprising the steps of introducing the engineered virus into insect
cells and allowing the virus to replicate in the insect cells to produce a viral vaccine.
Representative examples of the engineered viruses are Dengue virus E-Tl mutants (for
example deletion of GVII or LIG).
[0080] Both DV host range mutations identified produced a significant numbers of non¬
infectious virions. This was a phenotype also associated with mutants of Sindbis virus, a
member of alphavirus family. A significant difference in the assembly of alpha and
flaviviruses is the association of the glycoprotein-modified viral membrane with the
nucleocapsid. Alphaviruses are characterized by the strong association of the E2 tail with the
nucleocapsid which is required for assembly and infectivity (West et al., 2006). The
flaviviruses do not directly interact with the nucleocapsid and the mechanism by which virus
budding occurs in association with the core is not known (Murray et al, 2008; Samsa et al.,
2009). Additionally, flaviviruses produce empty particles (Lobigs and Lee, 2004; Lobigs et
ai, 2004; Murray et al, 2008) which increase toward late stages of infection suggesting that
some component (viral or host) is depleted as the infection progresses. These specific
differences in the details of virus assembly in the alpha and flavivirus systems underscore the
importance of the membrane in the host range phenotype. It is for this reason that it is
expected that this technology can be applied to other flaviviruses and arboviruses.
[0081] It is contemplated in certain aspects of the invention that one, two, three, four or more
of these types of mutations can be combined, for example, to use constructs of DV1-4 in
order to formulate a tetravalent vaccine. This is a novel approach which only requires
molecular biology methods until the mutants are identified. This combination of host range
mutants for DV1-4 would constitute a live virus vaccine grown in insect cells. The DV host
range mutants were shown not to revert to wild type after multiple passages displaying
genetic stability in the host used for production. The reasons for this characteristic are not
known but alphavirus complementation does not occur in insect cells implying component
sequestration (Condreay and Brown. 1986; Renz and Brown. 1976). These data provide
further evidence that sequence elements which define host range are expressed throughout the
arbovirus genome. This host range mutant methodology could continue to be applied to other
pathogenic flaviviruses and alphaviruses to produce vaccine strains.
[0082) Furthermore, certain aspects of the present invention provide a method of producing a
viral vaccine to a disease spread by a wild mosquito population to a mammal; comprising the
steps of genetically engineering a deletion of one or more amino acids in a flavivirus E
protein such as the E-Tl domain to produce an engineered virus, wherein the transmembrane
protein is able to span the membrane envelope when the virus replicates in mosquito cells,
but is unable to efficiently span the membrane envelope when the virus replicates in
mammalian cells, and wherein the virus remains capable of replicating in mosquito cells;
introducing the engineered virus into a wild mosquito population; and allowing the virus to
replicate in cells of the wild mosquito population to produce a population of mosquitoes
which excludes the wild type pathogenic virus and harbors the vaccine strain of the virus
such that the mosquito bite delivers the vaccine to a mammal bitten.
[0083] In addition, certain aspects of the present invention provide a method of vaccinating
an individual in need of such treatment, comprising the steps of introducing the viral vaccine
of the present invention into the individual and allowing the vaccine to produce viral proteins
for immune surveillance and stimulate the immune system for antibody production in the
individual.
A. Vaccine preparations
[0084] In any case, a vaccine component (e.g., an antigenic peptide, polypeptide, nucleic acid
encoding a proteinaceous composition or virus particle) may be isolated and/or purified from
the chemical synthesis reagents, cell or cellular components. In a method of producing the
vaccine component, purification is accomplished by any appropriate technique that is
described herein or well known to those of skill in the art (e.g., Sambrook et al., 1987).
Although preferred for use in certain embodiments, there is no general requirement that an
antigenic composition of the present invention or other vaccine component always be
provided in their most purified state. Indeed, it is contemplated that less substantially purified
vaccine component, which is nonetheless enriched in the desired compound, relative to the
natural state, will have utility in certain embodiments, such as, for example, total recovery of
protein product, or in maintaining the activity of an expressed protein. However, it is
contemplated that inactive products also have utility in certain embodiments, such as, e.g., in
determining antigenicity via antibody generation.
[0085] Certain aspects of the present invention also provide purified, and in preferred
embodiments, substantially purified vaccines or vaccine components. The term "purified
vaccine component" as used herein, is intended to refer to at least one vaccine component
(e.g., a proteinaceous composition, isolatable from cells), wherein the component is purified
to any degree relative to its naturally obtainable state, e.g., relative to its purity within a
cellular extract or reagents of chemical synthesis. In certain aspects wherein the vaccine
component is a proteinaceous composition, a purified vaccine component also refers to a wild
type or mutant protein, polypeptide, or peptide free from the environment in which it
naturally occurs.
[0086] Where the term "substantially purified" is used, this will refer to a composition in
which the specific compound (e.g., a protein, polypeptide, or peptide) forms the major
component of the composition, such as constituting about 50% of the compounds in the
composition or more. In preferred embodiments, a substantially purified vaccine component
will constitute more than about 60%, about 70%, about 80%, about 90%, about 95%, about
99% or even more of the compounds in the composition.
[0087] In certain embodiments, a vaccine component may be purified to homogeneity. As
applied to the present invention, "purified to homogeneity," means that the vaccine
component has a level of purity where the compound is substantially free from other
chemicals, biomolecules or cells. For example, a purified peptide, polypeptide or protein will
often be sufficiently free of other protein components so that degradative sequencing may be
performed successfully. Various methods for quantifying the degree of purification of a
vaccine component will be known to those of skill in the art in light of the present disclosure.
These include, for example, determining the specific protein activity of a fraction (e.g.,
antigenicity), or assessing the number of polypeptides within a fraction by gel
electrophoresis.
[0088] It is contemplated that an antigenic composition of the invention may be combined
with one or more additional components to form a more effective vaccine. Non-limiting
examples of additional components include, for example, one or more additional antigens-,
immunomodulators or adjuvants to stimulate an immune response to an antigenic
composition of the present invention and/or the additional component(s). For example, it is
contemplated that immunomodulators can be included in the vaccine to augment a cell or a
patient's (e.g., an animal's) response. Immunomodulators can be included as purified proteins,
nucleic acids encoding immunomodulators, and/or cells that express immunomodulators in
the vaccine composition.
[0089] Immunization protocols have used adjuvants to stimulate responses for many years,
and as such adjuvants are well known to one of ordinary skill in the art. Some adjuvants
affect the way in which antigens are presented. For example, the immune response is
increased when protein antigens are precipitated by alum. Emulsification of antigens also
prolongs the duration of antigen presentation.
[0090] Optionally, adjuvants that are known to those skilled in the art can be used in the
' administration of the viruses of the invention. Adjuvants that can be used to enhance the
immunogenicity of the viruses include, for example, liposomal formulations, synthetic
adjuvants, such as (e.g., QS21), muramyl dipeptide, monophosphoryl lipid A, or
polyphosphazine. Although these adjuvants are typically used to enhance immune responses
to inactivated vaccines, they can also be used with live vaccines. In the case of a virus
delivered via a mucosal route, for example, orally, mucosal adjuvants such as the heat-labile
toxin of E. coli (LT) or mutant derivations of LT can be used as adjuvants. In addition, genes
encoding cytokines that have adjuvant activities can be inserted into the viruses. Thus, genes
encoding cytokines, such as GM-CSF, IL-2, IL-12, IL-13, or IL-5, can be inserted together
with foreign antigen genes to produce a vaccine that results in enhanced immune responses,
or to modulate immunity directed more specifically towards cellular, humoral, or mucosal
responses.
[0091] An immunologic composition of the present invention may be mixed with one or
more additional components (e.g., excipients, salts, etc.) which are pharmaceutically
acceptable and compatible with at least one active ingredient (e.g., antigen). Suitable
excipients are, for example, water, saline, dextrose, glycerol, ethanol and combinations
thereof.
[0092] An immunologic composition of the present invention may be formulated into the
vaccine as a neutral or salt form. A pharmaceutically acceptable salt, includes the acid
addition salts (formed with the free amino groups of the peptide) and those which are formed
with inorganic acids such as, for example, hydrochloric or phosphoric acid, or such organic
acids as acetic, oxalic, tartaric, mandelic, and the like. A salt formed with a free carboxyl
group also may be derived from an inorganic base such as, for example, sodium, potassium,
ammonium, calcium, or ferric hydroxide, and such organic bases as isopropylamine,
trimethylamine, 2 ethylamino ethanol, histidine, procaine, and combinations thereof.
[0093] In addition, if desired, an immunologic composition may comprise minor amounts of
one or more auxiliary substances such as for example wetting or emulsifying agents, pH
buffering agents, etc. which enhance the effectiveness of the antigenic composition or
vaccine.
B. Vaccine administration
[0094] The viruses of the invention can be administered as primary prophylactic agents in
adults or children at risk of infection, or can be used as secondary agents for treating infected
patients. For example, in the case of yellow fever/dengue chimeras, the vaccines can be used
in adults or children at risk of Dengue infection, or can be used as secondary agents for
treating Dengue- infected patients. Examples of patients who can be treated using the denguerelated
vaccines and methods of the invention include (i) children in areas in which Dengue is
endemic, such as Asia, Latin America, and the Caribbean, (ii) foreign travelers, (iii) military
personnel, and (iv) patients in areas of a Dengue virus epidemic. Moreover, inhabitants of
regions into which the disease has been observed to be expanding (e.g., Argentina, Chile,
Australia, parts of Africa, southern Europe, the Middle East, and the southern United States),
or regions in which it may be observed to expand in the future (e.g., regions infested with
Aedes aegypti, or Aedes albopictus), can be treated according to the invention.
[0095] Formulation of the viruses of the invention can be carried out using methods that are
standard in the art. Numerous pharmaceutically acceptable solutions for use in vaccine
preparation are well known and can readily be adapted for use in the present invention by
those of skill in this art. (See, e.g., Remington's Pharmaceutical Sciences, 18th Ed., 1990) In
two specific examples, the viruses are formulated in Minimum Essential Medium Earle's Salt
(MEME) containing 7.5% lactose and 2.5% human serum albumin or MEME containing 10%
sorbitol. However, the viruses can simply be diluted in a physiologically acceptable solution,
such as sterile saline or sterile buffered saline. In another example, the viruses can be
administered and formulated, for example, in the same manner as the yellow fever 17D
vaccine, e.g., as a clarified suspension of infected chicken embryo tissue, or a fluid harvested
from cell cultures infected with the chimeric yellow fever virus. Preferably, virus can be
prepared or administered in FDA-approved insect cells.
[0096] The vaccines of the invention can be administered using methods that are well known
in the art, and appropriate amounts of the vaccines administered can be readily be determined
by those of skill in the art. For example, the viruses of the invention can be formulated as
sterile aqueous solutions containing between 10 and 10 infectious units (e.g., plaque-forming
units or tissue culture infectious doses) in a dose volume of 0.1 to 1.0 ml, to be administered
by, for example, intramuscular, subcutaneous, or intradermal routes. In addition, because
flaviviruses may be capable of infecting the human host via the mucosal routes, such as the
oral route (Gresikova et al., 1988), the viruses can be administered by mucosal routes as well.
Further, the vaccines of the invention can be administered in a single dose or, optionally,
administration can involve the use of a priming dose followed by a booster dose that is
administered, e.g., 2-6 months later, as determined to be appropriate by those of skill in the
art.
[0097] The manner of administration of a vaccine may be varied widely. Any of the
conventional methods for administration of a vaccine are applicable. For example, a vaccine
may be conventionally administered intravenously, intradermally, intraarterial ly,
intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly,
intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intratumorally,
intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally,
intrapericardially, orally, rectally, nasally, topically, in eye drops, locally, using aerosol,
injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a
catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method
or any combination of the forgoing as would be known to one of ordinary skill in the art (see,
for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by
reference).
[0098| A vaccination schedule and dosages may be varied on a patient by patient basis,
taking into account, for example, factors such as the weight and age of the patient, the type of
disease being treated, the severity of the disease condition, previous or concurrent therapeutic
interventions, the manner of administration and the like, which can be readily determined by
one of ordinary skill in the art.
[0099] A vaccine is administered in a manner compatible with the dosage formulation, and in
such amount as will be therapeutically effective and immunogenic. For example, the
intramuscular route may be preferred in the case of toxins with short half lives in vivo. The
quantity to be administered depends on the subject to be treated, including, e.g., the capacity
of the individual's immune system to synthesize antibodies, and the degree of protection
desired. The dosage of the vaccine will depend on the route of administration and will vary
according to the size of the host. Precise amounts of an active ingredient required to be
administered depend on the judgment of the practitioner. In certain embodiments,
pharmaceutical compositions may comprise, for example, at least about 0.1% of an active
compound. In other embodiments, the active compound may comprise between about 2% to
about 75% of the weight of the unit, or between about 25% to about 60%, for example, and
any range derivable therein. However, a suitable dosage range may be, for example, of the
order of several hundred micrograms active ingredient per vaccination. In other non-limiting
examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5
microgram/kg/body weight, about 10 microgram/kg/body weight, about 50
microgram/kg/body weight, about 100 microgram/kg/body weight, about 200
microgram/kg/body weight, about 350 microgram/kg/body weight, about 500
microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body
weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100
milligram/kg/body weight, about 200 milligram/kg/body weight, about 350
milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body
weight or more per vaccination, and any range derivable therein. In non-limiting examples of
a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to
about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500
milligram/kg/body weight, etc., can be administered, based on the numbers described above.
A suitable regime for initial administration and booster administrations (e.g., inoculations)
are also variable, but are typified by an initial administration followed by subsequent
inoculation(s) or other administration(s).
[00100] In many instances, it will be desirable to have multiple administrations of the
vaccine, usually not exceeding six vaccinations, more usually not exceeding four
vaccinations and preferably one or more, usually at least about three vaccinations. The
vaccinations will normally be at from two to twelve week intervals, more usually from three
to five week intervals. Periodic boosters at intervals of 1.5 years, usually three years, will be
desirable to maintain protective levels of the antibodies.
[00101] The course of the immunization may be followed by assays for antibodies for
the supernatant antigens. The assays may be performed by labeling with conventional labels,
such as radionuclides, enzymes, fluorescents, and the like. These techniques are well known
and may be found in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384
and 3,949,064, as illustrative of these types of assays. Other immune assays can be performed
and assays of protection from challenge with the flavivirus can be performed, following
immunization.
[00102] Certain aspects of the present invention include a method of enhancing the
immune response in a subject comprising the steps of contacting one or more lymphocytes
with a flavivirus immunogenic composition, wherein the antigen comprises as part of its
sequence a sequence nucleic acid or amino acid sequence encoding mutant E protein,
according to the invention, or a immunologically functional equivalent thereof. In certain
embodiments the one or more lymphocytes is comprised in an animal, such as a human. In
other embodiments, the lymphocyte(s) may be isolated from an animal or from a tissue (e.g.,
blood) of the animal. In certain preferred embodiments, the lymphocyte(s) are peripheral
blood lymphocyte(s). In certain embodiments, the one or more lymphocytes comprise a Tlymphocyte
or a B-lymphocyte. In a particularly preferred facet, the T-lymphocyte is a
cytotoxic T-lymphocyte.
|001 03] The enhanced immune response may be an active or a passive immune
response. Alternatively, the response may be part of an adoptive immunotherapy approach in
which lymphocyte(s) are obtained with from an animal (e.g., a patient), then pulsed with
composition comprising an antigenic composition. In a preferred embodiment, the
lymphocyte(s) may be administered to the same or different animal (e.g., same or different
donors).
V. Pharmaceutical compositions
[00104] It is contemplated that pharmaceutical compositions may be prepared using
the novel mutated viruses of certain aspects of the present invention. In such a case, the
pharmaceutical composition comprises the novel virus and a pharmaceutically acceptable
carrier. A person having ordinary skill in this art readily would be able to determine, without
undue experimentation, the appropriate dosages and routes of administration of this viral
vaccination compound. When used in vivo for therapy, the vaccine of certain aspects of the
present invention is administered to the patient or an animal in therapeutically effective
amounts, i.e., amounts that immunize the individual being treated from the disease associated
with the particular virus. It may be administered parenterally, preferably intravenously or
subcutaneously, but other routes of administration could be used as appropriate. The amount
of vaccine administered may be in the range of about 10 to about 106 pfu/kg of patient
weight. The schedule will be continued to optimize effectiveness while balancing negative
effects of treatment. See Remington's Pharmaceutical Science, 17th Ed., (1990); and
Klaassen In: Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed.
( 1990); which are incorporated herein by reference. For parenteral administration, the vaccine
may be formulated in a unit dosage injectable form (solution, suspension, emulsion) in
association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are
preferably non-toxic and non-therapeutic. Examples of such vehicles are water, saline,
Ringer's solution, dextrose solution, and 5% human serum albumin.
VI. Examples
[00105] The following examples are included to demonstrate preferred embodiments
of the invention. It should be appreciated by those of skill in the art that the techniques
disclosed in the examples which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be considered to constitute
preferred modes for its practice. However, those of skill in the art should, in light of the
present disclosure, appreciate that many changes can be made in the specific embodiments
which are disclosed and still obtain a like or similar result without departing from the spirit
and scope of the invention.
Example 1 Host range mutants of DV2
[00106] Deletion mutants constructed around a specific TMD position/region
exhibit host range phenotype. Two mutants DV2ALIG and DV2AGVII showed host range
phenotype restricted to preferential growth in the insect cells (FIG. 2). Mutant viruses with
preferential growth in the C6/36 mosquito cell line and attenuated growth in Vero cells as
defined by at least 2 orders of magnitude less virus production are considered to have host
range phenotype. The deletion mutants infected both Vero and C6/36 cells at a known MOI
with a titer > 104 FFU/ml. The Vero and C6/36 cells were infected with the mutants at MOI ~
0.03 FFU/cell. The mutant viruses were grown in each cell line, harvested on day 7 and
titered on Vero cells, and showed the host range phenotype. The WT DV2 routinely
generates titers of 106 ffu/ml in Vero cells and 107 ffu/ml in C6/36 cells. Titers were in the
range of 103- 04 ffu/ml for both DV2AGVII and DV2ALIG mutants grown in C6/36 cells
(FIG. 2).
|00107] Expression and processing of DV2 host-range mutants. The two DV2 hostrange
mutants DV2AGVII and DV2ALIG were found to produce infectious virus, as
observed in the focus assay. In order to determine if all viral proteins were indeed produced
and processed as WT DV2, a western blot analysis was performed. Virus grown in mosquito
cells was harvested from the cell supernatant at day 7 post-infection and examined by SDSPAGE.
To confirm the presence of specific DV proteins, the gel was transferred to substrate
and blotted with an anti-DV antibody. Visualization of the protein bands revealed a similar
banding pattern to that of WT DV2, verifying the correct production of virus proteins by the
mutants.
[00108] Electron micrographs of the mutants (AGVII and ALIG) suggest
impaired assembly. In order to analyze the differences in virus production between
mammalian and mosquito cells, thin sections of cells infected with wild type and mutant
viruses were prepared and evaluated. In FIG. 3A, C6/36 and in FIG. 3B, Vero cells were
infected with the wild type virus respectively. Virus particles are seen in large paracrystalline
structures within the mosquito cell (FIG. 3A- arrows) and associated with the mammalian
plasma cell membrane (FIG. 3B-arrows). In FIG. 3C, are shown mosquito cells infected
with the AGV mutant which exhibited similar amounts of virus production (arrows).
However, in mammalian cells infected with the same mutant FIG. 3D only the presence of
nucleocapsids in the cytoplasm could be detected in the thin sections (arrowheads) suggesting
a defect in budding. In cells infected with the mutant ALIG similar results were observed.
The presence of virus particles could be observed in the cytoplasm of mosquito cells (FIG.
3E - arrowheads) and nucleocapsids were detected in the cytoplasm of mammalian cells
(FIG. 3F- arrowheads). The specific defect in the assembly pathway of these mutants in Vero
cells is not known. However these results clearly demonstrate differences in virus production
between these two cell types as expected for host range mutants.
100109] Particle to FFU ratios of AGVII and ALIG from C6/36 cells. The
ultrastructural analysis of the host range mutant infected cells and the large quantities of
protein produced from the insect cell cultures indicated that these mutations were producing
large numbers of non-infectious virus. To determine if this was indeed the case, the
particle/ffu ratios were determined for each of the DV2 strains. The wild type DV strains
reproducibly displays a particle to ffu ratio of 103 particles/ffu. The particle to ffu ratios for
the mutants DV2AGVII and DV2ALIG were assayed at 5.5 X 107 and 1.4 X 107 particles/ffu
respectively. Thus, for each strain, infectious titer added to the particle to ffu ratio gives a
total particle/ml quantity of ~10'° virus particles.
[00110] Mutants with host range phenotype generate neutralizing antibody
response in BALB/cJ mice. Mutants showing an insect cell preferential host range
phenotype were analyzed for the ability to generate neutralizing antibodies in BALB/cJ mice.
First, a mouse trial was conducted with WT DV2. BALB/cJ mice (n=5) injected
subcutaneously (SC) with 106 ffu/mouse and 105 ffu/mouse of WT DV2, followed by a
booster dose on Day 14, generated antibody titers at 50% neutralization of virus (ND50; the
serum dilution of antibody needed to inactivate 50% of virus) in the range of 8-256 ND50 and
32-256 ND50 respectively. These experiments demonstrated that for WT DV2 an initial dose
of 105 to 106 ffu/ml followed by a booster dose on Day 14 is required to generate an immune
response in mice. This experiment was repeated with the host range mutants DV2AGVII and
DV2ALIG (Table 3).
J0 111] As shown in Table 3, the mouse trial was conducted to determine if the host
range mutants were able to generate an immune response in mice. Five groups (n=5) of 8
week old BALB/cJ mice were inoculated subcutaneously with 29 g (~10 -10 ffu/mouse) of
the WT DV2, DV2ALIG, DV2AGVII, iodixanol buffer alone and mock control. The mice
were boosted with an equal dose on Day 14 and the serum samples were collected from all
groups on Day 28.
Table 3. Mouse trial design
Buffer (Mock) 5 -
[001 12] For the mouse trials with the host range mutants the inoculum was given as
protein units. For each of the groups, 29 mg of protein were injected. Both mutants were able
to generate reciprocal neutralizing antibody titers in the range of 10-80 ND5o. Mice
inoculated with DV2AGVII gave a higher neutralizing antibody response than from
DV2ALIG and WT DV2. Four out of 5 mice in the DV2AGVII group gave an ND 0 between
the range of 20-80 while 2 mice were responders in the DV2ALIG group and gave an ND50 of
~20 (FIG. 4). Mice injected with buffer alone exhibited no neutralizing antibody response.
[00113] Mutant viruses grown in C6/36 cells are stable and do not revert to wild
type virus. The host-range mutant viruses identified in this study propagate two orders of
magnitude less virus than the WT virus in insect cells. These host range mutants, however,
still produce two to three orders of magnitude less infectious virus from the Vero cells. As a
result, there would seem to be some selective pressure on the mutant viruses to revert to the
WT virus in the insect system. To confirm whether or not this was the case, reversion of the
mutant viruses to WT in the C6/36 cells was evaluated over five serial passages. The cell
culture supernatant was collected from the infection after each round of infection and the
sequence of viral RNA was analyzed by RT-PCR as described above. It was found that the
mutant viruses retained the deletions after 5 sequential passages and there was no reversion to
the WT DV2 sequence. The same experiment was applied to virus grown in Vero cells.
After four serial passages in the Vero cells, no WT virus RNA was recovered.
Methods
[001 14] Cell Culture . C6/36 cells (Aedes albopictus, American Type Culture
Collection [ATCC] # CRL-1660, Manassas, VA) were maintained in minimal essential
medium (MEM) containing Earl's salts supplemented with 10% fetal bovine serum (FBS),
5% tryptose phosphate broth (TPB) and 2 mM L-glutamine. Vero cells (African Green
monkey kidney, ATCC #CCL-81) were maintained in IX MEM supplemented with 10%
FBS, 5% TPB, 2 mM glutamine, 10 mM Hepes pH 7.4 and IX MEM nonessential amino
acids (NEAA) (1:100 dilution of NEAA from Gibco # 11140, Carlsbad, CA).
[00115] Construction of DV2 deletion mutants. A full-length cDNA clone of Dengue
serotype 2 (DV2; Thai strain 16681, GenBank U8741 1) in pGEM3z+ was obtained from
the Walter Reed Army Institute of Research for these studies (Irie et al., 1989). The clone
produces full-length DV2 RNAs when transcribed in vitro with T7 RNA polymerase and
after transfection of the transcripts into mammalian or insect cells, infectious virions are
generated.
[00116] Deletions in the TMD of the DV2 E and M proteins were produced by
polymerase chain reaction (PCR)-based site-directed mutagenesis, using Pfu Turbo® DNA
polymerase AD (Stratagene, La Jolla, CA). Primers were designed to create sets of single,
double, triple, quadruple, and quintuple amino acid (aa) deletions within the Tl domain of the
E or M protein of DEN2. PCR conditions were as follows: 25 ng DV2 DNA, IX or 1.5X Pfu
Turbo Buffer, 0.4 h M/m dNTPs (New England Biolabs, Ipswich, MA), 5 each
primeri and 0.1 I\ Pfu Turbo DNA polymerase AD. Reactions were run with and without
DMSO (4% final concentration). PCR cycles were as follows: 95°C for 2 min, then 25 cycles
of 95°C for 5 sec, 45 sec of annealing (TA = Primer T -5°C for each set of primers), 68°C
for 24 min. Extension was performed for 28 min at 68°C; samples were held at 4°C until
analysis by gel electrophoresis. Following mutagenesis of the WT DV2 clone, the PCR
products were digested with Dpnl (New England Biolabs) and transformed into SURE®2
Supercompetent E.coli cells (Stratagene) as per manufacturer's instructions with a few
alterations. Following heat shock and recovery on ice, room temperature NZY* broth
(Teknova, Hollister, CA) was added and incubation was performed at 30°C for 1 to 2 hours
with shaking. After plating on Luria Broth (LB) agar containing 50 g/mL carbenicillin
(Teknova) incubation was performed at 30°C for 32 to 48 hours. A colony PCR screen was
then used to quickly identify the presence of the mutations in the resulting bacterial colonies
prior to culture. Growth of all DV2 clones in SURE®2 cells was conducted in LB containing
50 mg/mL carbenicillin at 28 to 30°C for approximately 24 to 48 hours with shaking. DV2
plasmid DNA was recovered using the Wizard® Plus Minipreps DNA Purification System
(Promega, Madison, WI) following manufacturer's instructions. All DV2 deletion mutant
clones were confirmed by sequence analysis (Eurofins MWG Operon, Huntsville, AL).
[00117] In vitro transcription and RNA transfection . Transcripts were generated for
each DV2 mutant clone using the RiboMAX™ Large Scale RNA Product Systems for T7
RNA Polymerase (Promega) following manufacturer's instructions, with the addition of RNA
cap analog 7mg (ppp)G (NEB # S1404S). The RNA transcripts were transfected into Vero
and C6/36 cells as follows: Cells were pelleted and washed in RNase free electroporation
buffer (PBS-D for Vero and MOPS for C6/36) and resuspended in their respective buffers at
a concentration of lxlO 7 to 5x1 07 cells/ml. RNA transcripts were added to 400 mÀ of cells and
electroporated at 1.0 KV, 50 m and ¥ resistance using the BioRad Gene Pulsar P (Bio-Rad
Laboratories, Hercules, CA). The transfected cells were then plated at different
concentrations in three different 24 well plates with 1.0 ml of the media and incubated at
37°C for Vero cells and 28°C for C6/36 cells for 1 hour with slow rocking. The media was
removed and the plates overlayed with 1.0 ml of 1% carboxymethylcellulose (CMC) in IX
Vero media or IX C6/36 media and incubated for 7, 10 and 14 days. The plates were
developed by focus assay.
[00118] Focus assay . The focus assay may be developed as a colorimetric or
fluorescent assay using antibodies labeled with either HRPO (color substrate) or Alexa Fluor
fluorescent dye. For the color assay, plates with transfected or infected cells were washed
twice with IX PBS and fixed with 80% methanol for 15 minutes at room temperature,
followed by incubation with antibody dilution buffer (5% skim milk in IX PBS-D) for 10
minutes. Primary antibody (a-DV NS1 glycoprotein, Abeam #ab41623, Cambridge, MA)
was added at a dilution of 1:400 in antibody (Ab) dilution buffer and incubated for 1 hour at
37°C with slow rocking. The wells were then washed twice with IX PBS followed by the
addition of secondary antibody conjugated with horse radish peroxidase (HRP) (Sigma #
8924, St. Louis, MO) at a dilution of 1:500 in Ab dilution buffer. Wells were washed again
twice with IX PBS. Foci were visualized by the addition of 150 mÀ TrueBlue™ peroxidase
substrate ( PL# 50-78-02, Gaithersburg, MD) to each well and developing for 15 minutes.
Foci were counted and titer determined in focus forming units/ml (ffu l) of virus. For the
fluorescent assay, the protocol is similar to the color assay with the following exceptions:
Cells are fixed for 20 minutes at room temperature in 100% methanol. A second 10 minute
incubation with 1 X PBS plus 0.05% Tween, followed by 2 washes with 1 X PBS plus 0.2%
BSA. Antibody is diluted in 1 X PBS + 0. 2% BSA. The washes between the primary and
secondary antibodies are performed in 1X PBS + 0.2% BSA. The secondary antibody, Alexa
fluor® 488 F(ab')2 fragment of goat anti-mouse IgG (Invitrogen # A-1 1017, Carlsbad, CA),
incubation is conducted for 45 minutes in darkness. After the final wash, 50 m of water is
added to each well for visualization of the fluorescent foci.
[00119] Infection and purification of selected mutants . The WT and DV2 mutants were
grown in the Aedes albopictus mosquito-derived C6/36 cell line. Cells were split one day
prior to infection at a ratio of 1:3. Subconfluent monolayers of C6/36 cells were infected at an
MOI of -0.03 ffu/cell. Virus was diluted in C6/36 media and each 75 cm3 flask infected with
1.0 ml of diluted virus for 1 hour at room temperature with slow rocking. After the initial
infection, 4.0 ml of fresh media was added to each flask. Flasks were then incubated for 7
days at 28°C. Virus was harvested by centrifugation of the supernatant at 4000 rpm for 10
min. Purification and concentration of WT and mutant DV2 were achieved using isopycnic
ultracentrifugation with iodixanol (Optiprep) gradients (Sigma, St. Louis, MO). Virus was
spun to equilibrium in gradients of 12% to 35% iodixanol and isolated.
[00120] Expression and Processing of DV2 mutants . Production and processing of
DV2 host-range mutants was determined in A. albopictus C7-10 cells. Mosquito cells were
infected with WT, DV2AGVII, and DV2ALIG and incubated at 28°C for 1 week. Infected
mosquito cell supernatants were then harvested and concentrated by tangential flow filtration
(TFF) as per manufacturer's recommendations (PALL, Post Washington, NY). Twenty-five
m of each preparation was loaded onto a 4-12% bis-tris gradient gel (Invitrogen). Proteins
from the gel were transferred to a polyvinylidene difluoride membrane and blotted as
described previously (Hernandez et ai, 2001) with the following modifications: Primary antiwhole
dengue virus mouse monoclonal antibody (Abeam #ab9202) was used for detection; an
anti-mouse-HRP conjugate was used as a secondary antibody; and viral proteins were
visualized by the addition of TrueBlue™ peroxidise substrate (KPL).
[00121] RT-PCR analysis of mutant viruses . To confirm that the desired deletions
remained intact in virus grown in cell culture, RNA was extracted from each mutant virus,
reverse transcribed, and amplified by PCR (RT-PCR). RNA extraction was performed by
two methods. The first method involved extracting RNA from a minimum of 104 ffu of virus
by pelleting the virus at 50,000 rpm in a SW55Ti (Beckman Coulter, Brea, CA) rotor for 1
hour. The pelleted virus was extracted as described previously (Hernandez et ai, 2000). The
RNA pellet was resuspended in 10 mÀ of diethyl pyrocarbonate (DEPC) treated water and
checked on 1% agarose gel. For smaller quantities of virus, a second method of purification
was employed. Viral RNA was harvested from C6/36 cells by RNeasy Mini kit (Qiagen,
Valencia, CA). Infected cells were scraped off flasks on Day 7 post-infection and suspended
in media at a cell density of ~ 1x107 cells/ml. Cells were spun down, resuspended in lysis
buffer, and homogenized. Viral RNA was purified as directed according to manufacturer's
instructions. The resulting RNA was suspended in 30 mÀ of RNase free water and checked on
a 1% agarose gel.
[00122) The extracted RNA was reverse transcribed and amplified by PCR using the
One-Step RT-PCR kit (Qiagen). Primers were designed for use in the RT-PCR reaction by
analysing the folded DV2 RNA structures to optimize RNA binding accessibility (Mathews
et ai, 1999). The products generated in the RT-PCR reaction (~ 640 bp) were
phenol/chloroform extracted, precipitated and sequenced to confirm the identity and presence
of deletions. Some of the RT-PCR products were of insufficient quantity and quality to be
sequenced directly. These products were amplified by nested PCR, subcloned into the pDrive
cloning vector and transformed in QIAGEN EZ Competent cells using the QIAGEN PCR
cloningplus kit (Qiagen). White colonies containing the vector-ligated PCR product were
amplified and the minipreped DNA was sequenced for verification (Eurofins MWG Operon).
[00123] Focus reduction neutralization test (FRNT) . The relative amount of virusneutralizing
antibody present in the mouse sera was determined by a focus reduction assay on
Vero cells, which is similar to the standard Plaque Reduction Neutralization Test (WHO,
2009). The assay was performed as described above for the focus assay, with the addition of a
pre-incubation step to allow serum antibody to bind WT DV2. In short, serum samples were
heat inactivated for 30 minutes at 56°C. A dilution of 1:10 for each sample was prepared
followed by serial 2-fold dilutions in dilution buffer containing 3% FBS. Each dilution was
mixed with an equal volume of virus suspension containing 50 ffu of WT DV2 and incubated
at 37°C for 1 hour. 200 mÀ of the mixture was then added to duplicate wells seeded with Vero
in a 24 well plate. After 1 hour of adsorption at 37°C, the cells were overlayed with IX Vero
media containing 1% CMC. At day 7 post-infection, antibody titers were determined by
developing the plates according to the focus assay protocol. Neutralizing antibody titers
(FRNT50) were reported as the highest dilution of the sera that reduced focus formation by
50%.
[00124] Experimental design for determination of neutralizing antibody titers . To
evaluate DV2 specific neutralizing antibody responses, five groups (n=5) of 8 week old
BALB/cJ mice were inoculated subcutaneously (SC) with 29 g (-102-103 ffu/mouse) of the
purified WT DV2, DV2ALIG, DV2AGVII, and iodixanol buffer alone. Protein estimates
were made using the EZQ® Protein Quantitation Kit (Molecular Probes). Mice were boosted
with an equal dose on Day 14 and serum samples were collected from all groups on Day 28,
at the termination of the study. The relative amount of virus-neutralizing antibody present in
mouse sera was determined by focus reduction neutralization test as described above
(Mathews et ai, 1999).
[00125] Transmission Electron Microscopy . Vero or C6/36 cells were transfected with
RNA transcribed from WT DV2, DV2ALIG, or DV2AGVII clones. Incubation proceeded at
37°C for 16-18 hours, after which the cell monolayers were scraped from the flasks and
pelleted by low speed centrifugation. Cell pellets were washed twice with PBS and fixed with
3% glutaraldehyde (Ladd Research Industries, Inc., Williston, VT) in 0.1M cacodylic acid
buffer pH 7.4 (Ladd Research Industries). After washing 3 times with 0.1M cacodylic acid,
cells were stained with 2% osmium tetroxide in cacodylic buffer for 1 hour. Cells were then
washed as before and embedded in 2% agarose. Agarose containing the cell sample was then
pre-stained with 1% uranyl acetate (Polaron Instruments Inc., Hatfield, PA) overnight at 4°C.
The samples were washed and carried through sequential dehydration with ethanol.
Infiltration was achieved using SPURR compound (LADD Research Industries). Next blocks
were trimmed on an LKB NOVA Ultrotome (Leica Microsystems, Inc., Deerfield, IL). Ultrathin
sections were obtained and stained with 5% uranyl acetate in distilled water for 60
minutes and in Reynolds lead citrate pH 12 (Mallinkrodt Baker Inc., Paris, KY) for 4
minutes. The samples were examined at 80 kV in a JEOL JEM 100S transmission electron
microscope.
Example 2 Clinical results using host range mutant viruses as a novel Dengue Virus-2
vaccine
[00126] To evaluate the immunogenicity, safety, and efficacy of a vaccine against
DEN2, immunization experiments were performed in African green monkeys. African green
monkeys provide a useful model for the preclinical assessment of novel candidates for
Dengue vaccines (Martin et ai, 2009a; Martin et al., 2009b).
[00127) Study design. In the present study, serum samples were collected and clinical
observations made at baseline and at 1, 3, 5, 7, 14, 30 and 57 days after vaccine
administration (Table 4). After serum collection on day 57 animals received live DEN-2
challenge virus (strain SI6803 wild type; 10 PFU per animal) before continued serum
collection and clinical observations at 58-64, 7 1 and 142 days post-vaccine administration.
All animals were prescreened for the presence of anti-Dengue 1-4 IgM or IgG by ELISA.
Monkeys positive by ELISA were excluded from the study.
Table 4: Study design
Day 1 Animal observation (including injection site) Blood 2 ml aliquot samples (7 ml)
sample for vaccine viremia
Day 2 Animal observation (including injection site) Blood 2 ml aliquot (9 ml)
Sample for vaccine viremia
Day 3 Blood sample for vaccine viremia 2 ml ( 11 ml)
Day 4 Observation -
Day 5 Blood sample for IgM antibody, viremia 2 ml (13 ml)
Day 7 Blood sample for IgM antibody, viremia 2 ml (15 ml)
Day 4 Blood sample for IgG antibody 2 ml (17 ml)
Day 30 Blood sample for IgG antibody 2 ml (19 ml)
PCV/Hematocrit (RxGEN)
Day 57 Blood sample for IgG antibody 2 ml (21 ml)
PCV/Hematocrit (RxGEN)
Virus Challenge
Day 58- Blood samples collected for 7 consecutive days for 20 ml, 2 ml per day (41 ml)
64 WT challenge virus viremia measurement
Day 7 1 Blood sample for IgG antibody 2 ml (43 ml)
Day 142 Blood sample for IgG antibody, study termination 2 ml (45 ml)
Total blood volume drawn (142 Study Days) 45 ml
[00128] It was determined that four monkeys per group (Table 5) would be sufficient
to generate statistically significant data (Fisher's exact test P=0.05). The study design was
based on previous studies done in the African Green Monkey model system (Martin et ai,
2009a; Martin et ai, 2009b) and rhesus monkeys (Halstead et ai, 1973). No virus boost was
incorporated because it was expected that the vaccine strains would generate sufficient
viremia not to require a second dose. Cell supernatant from uninfected mosquito C6/36 cells
was used as the negative control inoculum to monitor for the effect of mosquito antigens on
the animals. The positive control used was the live attenuated (LAV) strain of DV2 SI 6803.
Table 5 : Vaccine and Monkey Groups
[00129] Vaccines and challenge virus. Vaccine strains and the negative controls
were administered in a total volume of 0.5 ml iodixanol solution (33% in PBS-D) after
concentration by tangential flow filtration (TFF) and purification on 12% and 35% step
iodixanol gradients to remove serum albumin and further concentrate the virus. Doses given
of the host range mutant vaccine strains were as follows: DV2ALIG (2.5x10 ffu/monkey),
DV2AGVII (7.5x1 0 ffu/monkey) and DV2G460P (7.5xl0 4 ffu/monkey) (Table 5). The
positive control (DV2 S16803 LAV) was administered in doses of 5.0x10 pfu/monkey
(Tables 5-6). Sequences of the mutagenized viruses and the control TMDs are shown in
Table 6. All monkeys were challenged with DV2 SI6803 wild type virus at a dose of lxl 0
pfu/monkey (Table 6) (Eckels et al, 2003). DV2 SI6803 LAV passaged through PDK cells
(Halstead and Marchette, 2003) and DV2 SI6803 wild type virus were used as the control
and challenge strains respectively because these strains have been extensively studied
(Halstead et al, 1973; Marchette et al, 1973; Putnak et al, 2008). The response of monkey
hosts to these DV2 S I6803 LAV and wild type strains is well documented (Riedel and
Brown, 1977; Sun et al, 2006; Vaughn et al, 1996).
[00130] A single vaccination with no boost was given via subcutaneous injection. The
positive control, derivative LAV (strain 16803) was obtained from Robert Putnak of the
WRAIR (Eckels et al, 2003). Table 6 shows the virus titers used for the wild-type and
mutant viruses. Table 6 includes titers used for the following additional control strains: the
DV2 16681 strain, which is the parent strain that was used to make the mutant ALIG and
AGVII viruses, and the DV2S 16803 strain, which is an attenuated LAV derivative strain that
was also obtained from Robert Putnak of the WRAIR (Eckels et al, 2003).
Table 6 : Virus titers for the DEN-2 WT virus and host range mutant viruses
[00131] Pre challenge viremia. Days 1, 2, 3, 5 and 7 post inoculation were chosen as
the time points to assay for viremia in the monkey hosts. Viremia was found to peak on days
2-3 post injection for all test viruses. Shown in FIG. 5 is the peak viremia titer for the
average of all 4 animals in each monkey group expressed as infectious centers/ml. The raw
data (not presented) demonstrate that each individual monkey responded differently to the
inoculation, although each group followed a notable trend. An assay of infectious centers (IC)
was chosen over plaque assay since this assay is more sensitive by approximately 10 fold
(data not presented) (Edwards and Brown, 1 84; Putnak et al., 2005). The IC assay was also
chosen to facilitate the isolation of individual IC to determine the sequence of the virus after
replication in the primate host. This method of virus isolation made it possible to determine
if reversion of the mutants was occurring during the viremia (see below). No viremia was
detected in the mock infected controls. The amount of viremia detected in the test animals
compares with the test vaccines reported in the literature (Missailidis and Brady, 2004) and
compared to the control vaccine DV2 SI6803 provided by Dr. Robert Putnak [Walter Reed
Army Institute of Research (WRAIR)]. It is important to consider that while the control LAV
injected at 05 pfu/monkey produced a moderate level of viremia, an inoculum of 10 total
virus/500 mÀ in two of the test vaccines (AGVII and ALIG) produced equivalent virus titers in
the host animals. This is significant because vaccine efficacy has been linked to viral load
with lower dosages producing less protection (Simmons et al, 2006). This was not the case
with the host range mutants in this study. Notably, viremia is seen in two distinct peaks, one
at days 2-3 and a second between days 5 and 7. Distinct peaks of viremia have been detected
in monkeys as well as humans. It is hypothesized that the first peak represents amplification
of the virus in the sub-dermal dendritic cells in the epithelium at the site of injection (Libraty
et al, 2001) with the infection progressing to a second site, possibly the draining lymph
nodes (Cassetti et al, 2010). This type of dissemination of the virus has been seen in other
test primates and in humans (Guy et al, 2009; Marchette et al, 1973; Martina et al, 2009).
While the maximum titer of the two peaks of each of the viruses differs in quantity and day of
onset, the total amount of virus for all vaccine strains days 1 through 14 is in the range of 104
total IC/ml/monkey. These data demonstrate that the test vaccine strains did not produce
more viremia in the test animals than the SI6803 LAV control. These data are particularly
important to demonstrate that the test vaccines are not more viremic than the well-established
model DV2 LAV (Heegaard and Kennedy, 2002) and are indeed attenuated for replication in
the animal host as proposed. Evaluation of IC from each virus group by sequence analysis
has confirmed that AGVII virus from all four monkeys at day 2 had no reversions
demonstrating the genetic stability of the largest deletion in the animal host.
[001321 No infectious centers were seen in the assay of serum from the mock infected
animals.
[00133] Neutralizing Ab (NAb) data. Assay for the production of neutralizing IgM
and IgG titers post vaccination began with day 5 post injection samples and included days 7,
14 and 30 (FIG. 6). Three different assays PKNT, FRNT and ELISA were used to test for
Ab production and were performed on each individual sample in each group on the days
reported. The average antibody titers from each group of animals are shown in FIG. 6. The
numbers shown are from a plaque reduction neutralization assay (PR T) and represent the
inverse of the serum dilution in which 50% of the control DV2 virus was inhibited (Scott et
al, 1983). Focus reduction neutralization assay (FRNT) was not as sensitive an assay. The
monkeys were found to test positive for IgM on days 5, 7 and 14, with day 14 also beginning
to show IgG. Only IgG was detected on day 30 which peaked at day 62 correlating well with
the time course of virus neutralization (see FIG. 6 and FIGS. 9-10). These findings follow
the progression for the production of IgM and IgG as seen in other test primates and humans
(Velzing et al, 1999). As with the viremia raw data, the individual monkey Ab titers (not
presented) demonstrate that each individual monkey responded differently to the inoculation,
although each group followed a notable trend. Ab production (pre-challenge) appears to peak
on day 14 for the LAV strain (DV2 SI6803) and the ALIG test vaccine strains. Mutant
G460P peaked on days 5 and 14 while AGVII peaked on day 7. All strains showed
neutralizing Ab titers on day 30 with the peak trailing to 0 at day 57. The control strain DV2
S 6803 LAV produced NAb titers equivalent to those seen previously, -500 PR Tso l
(Putnak et al, 96). Compared to the control PRNT/50 titers, AGVII and G460P produced
higher levels of NAb, in the 600 to 1300 PRNT o ml levels. As can be observed from FIG. 6,
the levels of NAb are similar for all virus strains at day 14 which coincide with the end of the
viremia (refer to FIG. 5).
[00134] Post Challenge viremia. Post challenge viremia and neutralizing Ab titers
were determined for day 57 to 64 for 7 consecutive days. Data showing viremia after
challenge are shown in FIG. 7. Plotted are the averages of the viremias assayed in all four
monkeys of each of the vaccination groups determined by real time qRT-PCR (Bustin and
Mueller, 2005) on days 1-5 post challenge. qRT-PCR was used because detection of virus by
plaque assay cannot be used due to the high levels of NAb in the sera. All virus groups
experienced viremia as measured in genome equivalents/ml with the mock vaccination group
and the SI6803 LAV control group producing the highest viremias. The test virus vaccines
AGVII, G460P and D LIG allowed the least amount of virus replication/viremia of the
challenge virus compared to the SI6803 LAV control.
[00135] Pre and Post Challenge Neutralizing Ab data. Assay of neutralizing Ab
titers after challenge began at day 57 post inoculation and continued for 7 consecutive days,
sampled again at day 7 1 with a terminal blood draw taken at day 142. Results from the
PRNT tests are shown in FIG. 8. NAb begins to appear again on day 4 post challenge. With
the exception of ALIG, which peaks at day 71, all virus strains show a peak of NAb on day
6 1 or 62 which drops through day 63 and begins to increase again on day 71. Maximal
values of NAb titer, including those seen for ALIG were seen at day 7 1 post vaccination and
are similar in their time course to the NAb response in the initial post vaccination period
(FIG. 5). All vaccine strains tested elicited a neutralizing response above 1:300 PRNT50 titer
by day 7 1 post challenge. The bimodal peaks of neutralization produced after challenge were
unexpected but were reproducible. Additionally the bimodal peaks of IgG Ab were also
detected in the ELISA assay suggesting that there was a real drop in IgG titer on day 63 post
vaccination. Because this peak also occurred in the mock vaccinated monkeys, it is possible
that this response was due to some component of the challenge inoculum. Whatever the
cause, Nab titers returned to high levels at day 7 1 post vaccination. The plaque reduction
neutralization tests completed for the post challenge virus samples demonstrate that the levels
of NAb detected for AGVII and G460P exceeded the Ab titers produced by the control virus
LAV SI6803 based on the quantity of virus injected (see Table 5).
[00136] Total Ab, pre and post virus challenge. The Ab types elicited during the
virus neutralization response were determined by ELISA to be both Ig and IgG. This
suggests that both IgG and IgM may contribute to the neutralizing activity because the levels
of both Abs followed the trends for NAb seen in the PRNT50 assays. Shown in FIG. 9 are the
total IgG indices for each of the virus groups' pre and post challenge. There was no peak of
IgM detected at day 61-62 post challenge, rather IgM peaked at day 7 1 post challenge (FIG.
10) NAb levels which peaked on day 7 1 post vaccination declined at day 142 in the AGVII,
G460P and LAV samples, while ALIG and the mock samples plateaued. At a PRNT50 level
of 600 units, the amount of NAb for ALIG and G460P were still high as was the challenged
mock. Total IgG against all mutant viruses tested peaked at the end of the study at day 142
while IgM levels declined. Comparison of the IgG and IgM levels suggests that IgM levels
were elicited after IgG as has been reported previously to occur after virus challenge
(Bernardo et ai, 2008; WHO, 2006). It was unexpected to find that the total IgG levels
against the LAV control virus were so much higher than the test or control serum samples
through day 142 post vaccination. This suggests that the amount of total IgG against LAV
must be largely non-neutralizing because the NAb titers for the mutant and LAV vaccine
strains were similar or larger in magnitude during the post vaccination period. By day 142 the
levels of neutralization are declining for LAV and AGVII while ALIG, G460P and the
challenge DV2 SI 6803 levels remain high. NAb titers at maintenance levels were not
determined since no additional blood draws were taken past 142 days.
(00137] Safety assessment of Dengue 2 vaccine. Data generated in this Example was
designed to assess efficacy, immunogenicity and safety in African green monkeys to support
preclinical validation of novel candidate dengue vaccines. Safety was assessed by clinical
observations performed from baseline until completion of in vivo studies on study day 7 1
(extended to day 142) as well as determination of complete blood cell counts (CBCs) at
baseline and on study days 30 and 57. No major clinical concerns related to experimental
vaccines were identified as part of performed assessments. No erythema was observed at
injection sites and no fever was observed in the days following experimental vaccine
administration.
[00138] Twenty monkeys were assigned to one of five treatment groups to evaluate
viremia and antibody responses to test vaccine delivery and subsequent challenge with live
virus. Clinical observations were made over the initial 3 days following vaccine delivery and
again after the viral challenges were performed in the same animals. No increases in body
temperatures were observed following subcutaneous delivery of experimental vaccines
(AGVII, G460P or ALIG) or administration of LAV or the negative control (FIG. 11).
Similarly, no major changes in heart rate or respiratory rate were observed as a result of
experimental vaccine administration compared to control groups (data not presented).
[00139] Body temperature was determined using a rectal thermometer as part of
clinical observations. Data points indicate the mean body temperature with vertical bars
representing the standard error, SEM (n=4).
[00140] Clinical observations made after viral challenge at day 57 highlighted modest
but significant differences between treatment groups. Minimal body temperature increases
were observed in the initial 4 days following viral challenge across all treatment groups. On
day 62 (5 days post-viral challenge) a significant spike in temperatures was observed for
animals that had received AGVII and G460P vaccines (FIG. 12). Body temperatures steadily
declined towards baseline levels over the next 2-3 days with no other clinical abnormalities
observed during this time. This spike in temperature was preceded by viremia at day 58 and
followed by the second spike in NAb by one to three days (63 and 64). Analysis of the data
suggests that these small spikes are not significantly different from that seen in the LAV
control at day 7. Minor changes in specific CBC measures were noted but these changes,
which included reduced platelet counts, were not consistent with a vaccine-specific safety
concern as similar findings were observed in control groups.
[00141] Body temperature was determined using a rectal thermometer as part of
clinical observations. Data points indicate the mean body temperature with vertical bars
representing the SEM (n=4).
Conclusions
[00142] Administration of experimental vaccines produced no significant safety
concerns with no evidence of injection site erythema and no evidence of fever following
subcutaneous delivery. No pyrexia or other clinical signs were observed following
administration of experimental vaccines, positive (LAV) or negative controls.
[00143] Dengue infection in African green monkeys demonstrated similar absence of
pyrexia following live dengue serotype-2 infection (Martin et ai, 2009a; Martin et ai,
2009b). Interestingly, following subsequent viral challenge at 57 days post-vaccine delivery
a brief spike in body temperature was observed in animals receiving AGVII and G460P
experimental vaccines and to some extent in LAV but not in any other treatment groups
suggesting differing responses to infection in these treatment groups. It is of particular
interest to assess whether observed body temperature changes following viral challenge
reflect any differences in immunogenicity and efficacy as a result of vaccine treatment.
Indeed, this was not found to be the case because all three test vaccines displayed high
efficacy in suppressing the challenge virus viremia. Of the three host range vaccine strains
tested post challenge virus NAb levels elicited were shown to be GVII> G460P >LIG as
compared to the LAV control. All vaccine candidates gave NAb levels of > 320 PRNT50
values. A minimal sero-conversion PRNT50 value of 10 or greater is generally accepted
which puts these values into the high responding NAb category (Russell et ai, 1967). All
test vaccines were able to protect monkeys from viremia to a greater extent than the LAV
vaccine control.
(00144] Overall, with the endpoints assessed in the present studies no significant safety
concerns were identified that would adversely impact continued preclinical development.
The data support that one or more of the test vaccines would be efficacious and immunogenic
for clinical applications. More detailed analysis of CBC data may provide more information
on the immune response of these monkeys to the vaccine strains tested. In conjunction with
viremia and antibody response results these data have provided great insights into the safety
and efficacy of these test viruses. This initial study provides evidence that all mutants tested
appeared safe, producing no significant side effects. All three vaccine strains tested were
efficacious in reducing the amount of viremia upon WT DV2 16803 challenge. All three
candidates also were found to induce an excellent neutralizing immune response compared to
the LAV control. One important observation is that all three test vaccine strains elicited
much less non neutralizing IgG than the LAV control. This is a significant finding because it
is thought that non neutralizing Ab contributes to the Ab dependent enhancement leading to
the abnormal immune response resulting in hemorrhagic disease (Halstead et ai, 1977;
Huang et ai, 2006; Littaua et ai, 1990). These tests and data support the development of
similar vaccine strains in DV 1, 3 and 4 which will be poised to continue development either
singly or in combination to enter phase 1 clinical trials.
[00145] All of the methods disclosed and claimed herein can be made and executed
without undue experimentation in light of the present disclosure. While the compositions and
methods of this invention have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be applied to the methods and in the
steps or in the sequence of steps of the method described herein without departing from the
concept, spirit and scope of the invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be substituted for the
agents described herein while the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those skilled in the art are deemed to be within the
spirit, scope and concept of the invention as defined by the appended claims.
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WHAT IS CLAIMED IS:
1. A modified flavivirus envelope protein (E) comprising a mutated E protein's Nterminal
transmembrane domain (E-Tl domain), such a mutation comprising a deletion of
amino acids at positions of 0 to +3, 0 to +4, -2 to 0, -3 to 0, or - 1 to + 1 relative to the central
amino acid of the unmodified E-Tl domain (the central amino acid of the unmodified E-Tl
domain numbered position 0, amino acids proceeding toward the N terminus numbered -1, -
2. etc., and amino acids proceeding toward the C-terminus numbered +1, +2, etc.), wherein
the mutation inhibits the replication in mammalian cells of a virus comprising the modified
flavivirus E protein.
2. The modified flavivirus envelope protein of claim 1, wherein the mutation comprises
a deletion of amino acids at positions of 0 to +3 relative to the central amino acid.
3. The modified flavivirus envelope protein of either of claims 1 or 2, wherein the
mutation comprises a deletion of amino acids at positions of -2 to 0 relative to the central
amino acid.
4. The modified flavivirus envelope protein of any of preceding claims, wherein the
modified flavivirus E protein is a modified E protein of Dengue virus (DV), West Nile virus
(WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis
virus (TBE virus), Murray Valley encephalitis virus (MVEV), Saint Louis encephalitis virus
(SLEV), or Powassan virus (PV),.
5. The modified flavivirus envelope protein of any of preceding claims, wherein the
modified flavivirus E protein is a modified E protein of Dengue virus.
6. The modified flavivirus envelope protein of any of preceding claims, wherein the
modified flavivirus E protein is a modified E protein of Dengue virus type 2.
7. The modified flavivirus envelope protein of any of claims 3-6, wherein the mutation
comprises a deletion at amino acids 458 to 460 {i.e., a deletion of L458, 1459 and G460).
8. The modified flavivirus envelope protein of any of claims 2, 4, 5, and 6, wherein the
mutation comprises a deletion at amino acids 460 to 463 {i.e., a deletion of G460, V461, 1462
9. The modified flavivirus envelope protein of any of preceding claims, wherein a virus
comprising the modified flavivirus E protein has an ability to produce at least 100 fold more
progeny viruses when infecting insect cells than when infecting mammalian cells.
10. The modified flavivirus envelope protein of any of preceding claims, wherein a virus
comprising the modified flavivirus E protein has an ability to produce at least 1000 fold more
progeny viruses when infecting insect cells than when infecting mammalian cells.
11. An engineered nucleic acid encoding a modified flavivirus envelop protein of any of
preceding claims.
12. A genetically engineered flavivirus comprising at least an engineered nucleic acid of
claim 11.
13. An immunogenic composition comprising a genetically engineered flavivirus of claim
12.
14. The immunogenic composition of claim 13, further defined as a vaccine composition.
15. The immunogenic composition of claim 14, wherein the vaccine composition
comprises one or more of the genetically engineered Dengue virus types 1, 2, 3, and 4.
16. The immunogenic composition of claim 15, wherein the vaccine composition
comprises the genetically engineered Dengue virus type 2.
17. The immunogenic composition of claim 15, wherein the vaccine composition is a
tetravalent vaccine composition comprising the genetically engineered Dengue virus types 1,
2, 3, and 4.
18. The immunogenic composition of any of claims 13-17, further comprising an
adjuvant or a preservative.
19. A method of producing a viral vaccine for vaccination of mammals, comprising
introducing the genetically engineered flavivirus of claim 12 to insect cells to produce a viral
vaccine.
20. A composition in accordance with any one of claims 13 to 18 for use in vaccinating a
mammal for preventing flaviviral infections.
21. The composition of claim 20, wherein the mammal is a human or a primate.
22. The composition of claims 20 or 21, wherein the composition is to be administered
intravenously, intramuscularly, intraperitonealy or subcutaneously.
23. The composition of any of claims 19-22, wherein the composition is to be
administered as a single-dose.