A vaccine for West Nile virus that protects a subject against West Nile infection comprising an a pharmaceutically acceptable carrier and a therapeutically effective dose of an infectious agent selected from the group consisting of: a live attenuated infectious (+) RNA virus designated as WN1415, a vector comprising infectious DNA encoding an infectious (+) RNA molecule encoding the West Nile virus, and the West Nile (+) RNA virus designated as WN956D117B3 (GenBank #M12294).

Description of the Invention

BACKGROUND OF THE INVENTION

Flaviviruses are (+) RNA viruses that cause such diseases as West Nile fever ("WN"), dengue fever ("DEN"), yellow fever ("YF"), St. Louis encephalitis, Japanese encephalitis ("JE"), and tick-borne encephalitis ("TBE"). West Nile virus was first isolated over 60 years ago from the blood of a febrile patient (Smithburn et al., 1940), and is one of the most widespread flaviviruses worldwide. The virions of the West Nile fever virus are spherical particles with a diameter of 50 nm constituted by a lipoproteic envelope surrounding an icosahedric nucleocapsid containing a positive polarity, single-strand RNA. A single open reading frame ("ORF") encodes all the viral proteins in the form of a polyprotein. The cleaving and maturation of this polyprotein leads to the production of about ten different viral proteins. The structural proteins are encoded by the 5' part of the genome and correspond to the nucleocapsid designated C (14 kDa), the envelope glycoprotein designated E (50 kDa), the pre-membrane protein designated prM (23 kDa), the membrane protein designated M (7 kDa). The non-structural proteins are encoded by the 3' part of the genome and correspond to the proteins NS1 (40 kDa), NS2A (19 kDa), NS2B (14 kDa), NS3 (74 kDa), NS4A (15 kDa), NS4B (29 kDa), NS5 (97 kDa).

The West Nile virus is endemic to Africa and has been repeatedly registered in Europe and Asia for decades causing self-limiting epidemics and epizootics (Murgue et al., 2001; Savage et al., 1999). Recent introduction of the virus into the naive environment of the North American continent (Lanciotti et al., 1999) had disastrous consequences both for wildlife and human population (Roehrig et al., 2002) and in a few years has developed into a nationwide epidemiological problem. In addition to hundreds of human mortality cases reported to the Centers for Disease Control (CDC, 2004), the virus imposes a substantial economical burden, especially on the equine industry (Anonymous, 2003).

Cell-mediated immune response plays an important role in virus clearance and in protection from the disease (Diamond et al., 2003; Shrestha and Diamond, 2004). Since flavivirus nonstructural proteins supply the majority of dominant T-cell peptide determinants (Co et al., 2002), cell-mediated response induced by chimeric flaviviruses are mostly to vector proteins. However, development of a live attenuated West Nile vaccine, which may have a better capability to elicit balanced humoral and cell-mediated immune responses, is hindered by the high virulence and pathogenicity of the NY99 strain circulating in the U.S. (Beasley et al., 2002; Roehrig et al., 2002).

Based on serological data and genetic characterization, West Nile viruses were subdivided into two distinct lineages (Berthet et al., 1997; Price and O'Leary, 1967). Viruses of lineage 1, which includes the highly virulent NY99 strain, are most widespread and often were found in association with epidemics or epizootics (Roehrig et al., 2002). Although a few strains with a high virulence were also found among lineage 2 representatives (Beasley et al., 2002), viruses of this lineage have not been associated with disease outbreaks (Lanciotti et al., 1999). For this reason, lineage 2 viruses may be more attractive for development of live attenuated West Nile vaccine.

Modern (+) RNA virus studies increasingly rely on the infectious clone methodology, which allows for targeted manipulation of viral genomes. In the "classical approach", (+) RNA viruses are recovered from cells transfected with synthetic RNA made by in vitro transcription of infectious clone cDNA templates. In a layered DNA/RNA approach, also known as "infectious DNA," the infectious (+) RNA viruses are recovered directly after transfection of plasmids carrying viral genome cDNA into susceptible cells. Unfortunately, difficulties are often encountered in the design of flavivirus infectious DNA. Few studies have reported on the use of such infectious DNA construct as a vaccine.

The present invention is directed to the isolation of a virus useful for development of a West Nile vaccine. Isolate 956D117B3 (earlier also referred to as WN-Nigeria or WN-Wengler (Berthet et al., 1997; Lanciotti et al., 1999)) is a descendant of the West Nile virus prototype B956 (Smithburn et al., 1940), and is one of the first flaviviruses for which the complete nucleotide sequence has been determined (Castle et al., 1986; Castle et al., 1985; Wengler et al., 1985; GenBank #M12294). Earlier, the first West Nile infectious clone designed on the basis of the isolate 956D117B3 (Yamshchikov et al., 2001) was reported. While the virus was produced, the viral population was not characterized. In the present invention, it was demonstrated for the first time that the virus recovered from the molecular clone is highly attenuated, induces vigorous and balanced immune response and even at low doses protects mice against the virulent NY99 strain. Combined with its stable genotype and excellent growth characteristics in tissue culture, the recovered virus is well-suited for the development of veterinary and human live West Nile vaccines.

BRIEF SUMMARY OF THE INVENTION

An object of the present invention is to provide a (+) RNA viral material formed by infectious DNA.

Still another object of the present invention is to provide plasmid DNA encoding for an (+) RNA viral genome, which can be amplified in E. coli and easily prepared in large amounts.

Another object of the present invention is to use DNA immunization methodology for direct vaccination using infectious DNA, which will provide a stable and safe vaccine for an (+) RNA virus with increased shelf life due to a higher stability of the purified DNA.

Yet another object of the present invention is to provide a West Nile type 2 viral strain 956D117B3 and its WN1415 isolate that is immunogenic to type 1 viruses, such as the highly virulent NY99 strain.

Another object of the present invention is to provide a flavivirus infectious DNA that is dichotomous nature such that it is partially protective against its own infectivity. Although in cell culture higher inputs of infectious DNA correspond to higher infection rate, this results in decreased mortality even for a highly virulent virus.

Additional aspects of the invention, together with the advantages and novel features appurtenant thereto, will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned from the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a composition for eliciting an immune response or a protective immunity against West Nile virus. According to a related aspect, the present invention relates to a vaccine for preventing and/or treating a West Nile virus associated disease. As used herein, the term "treating" refers to a process by which the symptoms of a West Nile associated disease are ameliorated or completely eliminated. As used herein, the term "preventing" refers to a process by which a West Nile associated disease is obstructed or delayed. The compositions and vaccines of the invention comprises a live attenuated virus or an infectious DNA capable of producing a live attenuated virus. Most preferably, the live attenuated virus is produced in vivo using an "infectious DNA" approach.

As used herein, the term "immune response" refers to a T cells response or increased serum levels of antibodies to an antigen, or to the presence of neutralizing antibodies to an antigen, such as a West Nile polypeptide.

The term "protection" or "protective immunity" refers herein to the ability of the serum antibodies or T cell response induced during immunization to protect (partially or totally) against disease or death caused by the West Nile virus.

The methods of the invention comprise administering a composition having a therapeutically effective amount of a live attenuated West Nile virus or the infectious DNA encoding for the attenuated virus in an acceptable pharmaceutical carrier to the subject in need.

The "subject" or "patient" of the present invention is preferably an animal, e.g., such as mice, cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.

The term "therapeutically effective dose" or "therapeutically effective amount" means a dose or amount that produces the desired effect for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the attenuated virus or infectious DNA is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

Thus, as used herein, the term "pharmaceutically acceptable carrier" means, but is not limited to, a vehicle for containing the DNA plasmid that can be injected into a mammalian host without adverse effects. Suitable pharmaceutically acceptable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions. Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.

As discussed more fully below, in the present invention, the genetic material of West Nile virus type 2 strain 956D117B3 was rescued from archival RNA. Like many RNA viruses, a large heterogeneity exists within the viral population of 956D117B3, and the reported genome identifies the dominant quasispecies (GenBank #M11294, which is incorporated by reference). In the present invention, the 956D117B3 virus was used in the design of West Nile plasmid DNA construct. From the plasmid, a molecularly defined clonal variant designated WN1415 was isolated. WN1415 is significantly attenuated but remains highly immunogenic and protective in mice. As discussed more fully below, the immune responses developed upon infection with the substantially attenuated virus WN1415 protects mice against challenge with 100 times the lethal doses of West Nile NY99. The WN1415 clonal variant has LD.sub.50 greater than 10.sup.6 pfu; yet immune responses developed after infection with much smaller doses of WN1415 protect mice against challenge with 100 LD.sub.50 of the highly virulent NY99. Similar protection is achieved upon immunization with infectious DNA consisting of the genome of West Nile virus type 2 strain 956D117B3. While the reported genomes of both the 956D117B3 and the WN1415 isolate are substantially the same and differ only by two silent nucleotides, the heterogeneity of the viral population associated with the 956D117B3 strain makes it more pathogenic and virulent compared to the homogeneous WN1415 viral population as determined by present day sequencing technology.

Further, in the present invention, it was shown that West Nile infectious DNA, which is comprised of cDNA of the West Nile virus type 2 genome placed under transcriptional control of an eukaryotic promoter and inserted into a derivative of the pBR322 plasmid, initiates the flavivirus infectious cycle directly after transfection into susceptible cells or after inoculation in animals in vivo by intramuscular needle or needle-free injection, or by intradermal biolistic delivery. Due to the stability of supercoiled DNA plasmid and a high specific infectivity of the construct, West Nile infectious DNA is capable of initiating flavivirus infection even when used in very small amounts. Further, the protection achieved with the infectious construct is similar to that of WN1415.

Although West Nile infectious DNA as a plasmid carrying the full flavivirus genome controlled by eukaryotic transcription elements resembles a DNA vaccine, it is about 1000 to 10,000-fold more efficient in inducing antiviral protective immunity in mice via different inoculation routes.

It will be appreciated to those skilled in the art that the infectious DNA of the present invention may be formed using any suitable vector. In general, a vector is a nucleic acid molecule (typically DNA or RNA) that serves to transfer a passenger nucleic acid sequence (i.e., DNA or RNA) into a host cell. Three common types of vectors include plasmids, phages and viruses. Preferably, the vector is a plasmid. That is the infectious DNA vaccines of the present invention are comprised of DNA that is produced as a plasmid that can be introduced into animal tissue and therein is expressed by animal cells to produce a messenger ribonucleic acid (mRNA) molecule of the size of the flavivirus genome, which is translated to produce a viral polyprotein, that is processed by cellular machinery to provide a full set of flavivirus proteins that are capable to initiate replication of the above primary RNA transcript and thus initiate the virus replication cycle in animal tissue into which the above DNA plasmid was introduced.

Suitable and exemplary plasmid vectors that have been used in conventional DNA vaccines include, but are not limited to pBR322 (ATCC# 31344); pUC19 (ATCC# 37254); pcDNA3.1 (Invitrogen, Carlsbad Calif. 92008; Cat. NO. V385-20; pNGVL (National Gene Vector Laboratory, University of Michigan, Mich.); p414cyc (ATCC# 87380), p414GALS (ATCC# 87344), pBAD18 (ATCC# 87393), pBLCAT5 (ATCC# 77412), pBluescriptIIKS, (ATCC# 87047), pBSL130 (ATCC# 87145), pCM182 (ATCC# 87656), pCMVtkLUC (ATCC# 87633), pECV25 (ATCC#77187), pGEM-7zf (ATCC# 87048), pGEX-KN (ATCC# 77332), pJC20 (ATCC# 87113, pUB110 (ATCC# 37015), pUB18 (ATCC# 37253).

The infectious DNA of the present invention is also under the control of a suitable promoter. For eukaryotic expression, suitable vectors include the cytomegalovirus ("CMV") early promoter, or alternatively the Rous sarcoma virus ("RSV") LTR promoter, and the SV40 promoter.

The amount of plasmid present in the compositions or in the DNA vaccines of the present invention is preferably a therapeutically effective amount. A therapeutically effective amount of plasmid is that amount necessary so that the nucleotide sequence coding for the West Nile polypeptide performs its immunological role without causing overly negative effects in the host to which the composition is administered. The exact amount of plasmid to be used and the composition/vaccine to be administered will vary according to factors such as the strength of the transcriptional and translational promoters used, the type of condition being treated, the mode of administration, as well as the other ingredients in the composition. Preferably, the composition or the vaccine formulation is composed of from about 10 ng to about 1 .mu.g of plasmid.

The immunogenicity of the DNA vaccine and pharmaceutical compositions of the present invention can also be modified by formulating with a pharmaceutically acceptable adjuvants or imunostimulants, such as alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulator factor ("GM-CSF"), macrophage colony stimulator factor ("M-CSF"), interleukin 2 ("IL-2"), interleukin 12 ("IL-12"), and CpG oligonucleotides. For preparing such compositions, methods well known in the art may be used.

Subcutaneous injection, intradermal introduction, impression through the skin, and other modes of administration such as intraperitoneal, intravenous, oral, or inhalation delivery are also suitable. For example, vectors containing the infectious DNA of the present invention can be introduced into the desired host by methods known in the art, for example, transfection, electroporation, microinjection, microparticles, microcapsules, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lyposome fusion), use of a gene gun (particle bombardment), or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

Administration may be single or multiple (i.e. single-dose or including a booster). Such administration may be alone or in combination with other active therapeutic agents against West Nile virus.

In the following examples, virus titers were determined by a microplate method. Serial 10-fold dilutions of virus stocks in DMEM containing 0.5% FCS were prepared in duplicate wells of a 96-well cluster and 50 .mu.l was transferred in parallel to confluent monolayers of Vero cells in 96-well plates using a multichannel pipettor. The plates were incubated for 1 hour at 37.degree. C. in the CO.sub.2 incubator with occasional shaking. The inocula were aspirated, replaced with 100 .mu.l of the growth medium, and plates returned to the incubator for additional 24 hours. Under these conditions, virus multiplication foci consisted of compact clusters of about 5-15 cells, which stained positive for the viral antigen as described below.

PRNT assays were done in a similar microplate format. Two-fold dilutions of immune sera in DMEM plus 0.5% FCS were mixed in duplicate in 96-well plates with equal volume of NY99 virus prediluted in the same medium to 2.times.103 pfu/ml and the plate was kept in CO.sub.2 incubator at 37.degree. C. for 1 hour. Fifty microliters of each mix (containing about 50 pfu) was transferred in parallel into a 96-well plate with confluent Vero monolayers and incubated for another hour as above. The inocula were aspirated, replaced with 100 .mu.l of the growth medium and plates returned to the incubator for 24 hours.

At the end of the incubation period, cells were fixed by addition of 25 .mu.l/well of 10% formalin in PBS and incubation for 30 minutes at room temperature. Foci of viral multiplication were visualized on fixed monolayers with DAB substrate (Vector Laboratories, Burlingame, Calif.) after the following treatment sequence (50 .mu.l/well): 0.5% Thesit (Sigma-Aldrich) for 10 minutes, 1:1000 dilution of WN mouse hyperimmune antiserum for 30 minutes, 1:1000 dilution of biotinylated horse anti-mouse IgG(H+L) (Vector Laboratories) for 30 minutes, 2 .mu.g/ml streptavidin (ICN, Aurora, Ohio) for 30 minutes, 3.5 .mu.g/ml biotinylated peroxidase (ICN) for thirty minutes. PBS supplemented with 1% horse serum was used as a diluent throughout the assay, and plates were washed extensively in tap water between treatments. For PRNT, endpoint serum dilutions providing 50% reduction in the number of foci over control wells that contained no immune serum were counted as positive.
 

Claim 1 of 19 Claims

1. A live attenuated homogeneous viral population comprising the WN 1415 (+) RNA virus.
 

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