NOVEL SEQUENCES AND DNA VACCINES AGAINST AVIAN FLU

BACKGROUND OF THE INVENTION

This application claims priority to U.S. Provisional Patent Application 60/921,581 filed April 3, 2007, which is hereby incorporated by reference in its entirety.

The present invention relates generally to the fields of vaccine development, molecular biology, physiology, immunology and disease control. More specifically, the invention relates to the use of novel genetic sequences, preferably DNA, and their use either as traditional DNA-based vaccines where the active DNA component is in the form of plasmid, virus and other similar vectors, or use in the form of DNA produced using a cell-free biosynthetic process for making high quality nucleic acid in a cell-free system, synDNA™, as described in WO2006/063355, WO2007/018744, and US Patent Application 12/012615 (filed 2/4/08) which are hereby incorporated by reference in their entireties.

Avian flu (or avian influenza, pandemic influenza, is commonly known as bird flu) is an influenza type A virus that appears in many different sub-types classified according to the nature of two of the components that make up the virus - hemagglutinin (HA) and neuraminidase (NA).

In humans, influenza is an acute viral disease of the respiratory tract that affects millions of people each year. Apart from seasonal influenza epidemics, influenza pandemics occurs when a virus strain with a novel HA subtype (with or without a novel NA subtype) appears and spreads in the human population. To date, the deadly avian strain H5N1 has posed the greatest risk for a new pandemic. Since its identification in the 1990s, H5N1 viruses have killed millions of domestic fowl in Asia and over 300 human cases of infection were reported to the WHO, 60% of which were fatal. Fortunately, this virus has not yet mutated to a form that spreads easily between people. The socio-economic costs of influenza are attributed to both loss of productivity and related medical expenses as well as the establishment of preventative measures to curtail the spread of the infection. In the United States, seasonal influenza outbreaks are responsible for a total cost of over $US 10 billion per year, In the event of a pandemic, studies based on the 1918 Spanish flu pandemic have predicted that a 30% sickness rate and a 3 -week length of illness would decrease the gross domestic product by 5% with an cost of ~$US 700 billion.

Currently, the major form of human influenza vaccines is the traditional egg-based trivalent virus-based preparation that incorporates three circulating strains. However, each year new batches of "flu" vaccines have to be prepared since influenza strains change frequently. To further complicate matters, the potential pandemic strains of influenza (H5N1 and closely related strains) grow poorly or kill eggs, and inactivated vaccines appear to be poorly immunogenic. There is a need to develop more efficient vaccines utilizing new technology such as high-growth viral reassortants, viral-like particles, recombinant protein subunits and viral vector expressing antigenic proteins; some of which are currently being developed. Although more advanced, these approaches lack the ability to rapidly produce enough vaccine in emergency situations. This has prompted the establishment of guidelines for planning geographical containment, first responders immunization and vaccine stockpiling; all of which are difficult to implement, especially in developing countries where the pandemic threat is at its greatest.

In poultry, the form of avian flu that is currently the subject of concern is known as "Asian" H5N1 and falls into the category of HPAI avian flu because this virus is very contagious among birds and carries a high mortality rate. However, H5N1 is not a single virus; there are over 700 varieties of H5N1 viruses. Governments and the media fall short of making an understandable distinction between the naturally-occurring, harmless virus strains that are only found in wild birds ("North American" H5N1) and which cause only minor disease in birds, and the more virulent deadly strains found in Asian Poultry Markets (HPAI

H5N1).

There are a number of ways that highly pathogenic H5N1 could potentially reach the United States: wild bird migration, illegal smuggling of birds or poultry products, travel by infected people, or people traveling with virus-contaminated articles from regions where HPAI H5N1 already exists. The very rare highly pathogenic avian influenzas, such as the HPAI H5N1 circulate in parts of Asia, Europe and Africa. To date, HPAI H5N1 has not been recorded in the U.S., although outbreaks of related avian influenza viruses lethal to domestic fowl have occurred in Pennsylvania (1983); Texas (2004; Chang- Won Lee, 2005) as well as Michigan, Maryland, Pennsylvania, and Montana (2006; H5N1). Although avian influenza A viruses usually do not infect humans, rare cases of human infection with avian influenza A viruses have been reported. Since November 2003, more than 330 confirmed cases of human infection with highly pathogenic avian influenza A (H5N1) viruses have been reported from 12 countries.

Most human cases of H5N1 virus infection are thought to have occurred during direct contact with sick or dead infected poultry. Human clinical illness from infection with avian influenza A viruses has ranged from eye infections (conjunctivitis) to severe respiratory disease (pneumonia) to death. Because of concerns about the potential for more widespread infection in the human population, public health authorities closely monitor outbreaks of human illness associated with avian influenza. The spread of avian influenza A viruses from one ill person to another has been reported very rarely, and has been limited, inefficient and unsustained.

Once avian influenza is established in domestic poultry, it is a highly contagious disease and wild birds are no longer an essential ingredient for spread. Infected birds excrete virus in high concentration in their feces and also in nasal and ocular discharges. Once introduced into a flock, the virus is spread from flock to flock through the movement of infected birds, contaminated equipment, egg flats, feed trucks, and service crews, to mention a few. The disease generally spreads rapidly in a flock by direct contact. The only available mode of action to prevent the spread of influenza from flock to flock during an outbreak of highly pathogenic avian flu is mass euthanasia with the disposal of infected birds and strict biosecurity measures (current policy of health officials).

Although such eradication efforts may help to protect human health, they can result in significant costs. During the 1983-84 AI epidemic in Pennsylvania poultry, the H5N2 virus initially caused low mortality, but within 6 months became highly pathogenic, with a mortality index approaching 90 percent. Control of this outbreak required destruction of more than 17 million birds at a cost of nearly $65 million dollars. In 2004 evidence of a low and high pathogenic avian influenza virus was detected on a broiler-breeder farm in Canada. Although only 3 million birds were destroyed in this epidemic compared to the 17 million destroyed in Pennsylvania, it has been estimated that the outbreak in Canada cost farmers approximately $340 million to rebuild the industry. In addition to the negative economic impact to the poultry industry, the cost of an outbreak also includes depopulation and disposal of bird carcasses, labor, cleaning, disinfection, and premises or business downtime. As of mid-2006, it was estimated that at least 200 million domestic birds (out of a total world population of 10 billion) had either died or been culled as a result of HPAI H5N1.

For avian flu, inactivated influenza virus vaccines are effective in reducing mortality, preventing disease, or both, in chickens and turkeys. These vaccines, however, may not prevent infection in some individual birds, and if infected could shed virulent virus, albeit

considerably less than that of non-vaccinated and infected birds. Since there is no cross- protection amongst the 15 known HA subtypes, knowledge of the prevailing epidemiological situation is critical.

Currently, the most common production method for influenza vaccines is by growing LPAI flu viruses in embryonated chicken eggs. However, this method cannot be applied for HPAI strains. Indeed, the highly virulent viruses are difficult to grow and amplify in eggs, as they are lethal to the developing embryo. Moreover, the possibility of human contamination seriously hinders and complicates the manufacturing process with needs for stringent bio- containment. Therefore in most HPAI cases, recombinant viruses must be prepared using a method termed reverse genetics where an individual viral component of the circulating HPAI strain is inserted into a LPAI virus which is then grown in eggs. In some cases, related LPAI strains are used. The supply time for LPAI vaccines depends on the availability of product at the time of ordering. If stock is not available, the supply time can be 4 to 8 months from the start of the production process. Potential supply problems are caused by a sudden unexpected and substantial rise in demand. It is precisely in this scenario that DNA vaccines will prove useful.

Nucleic acid immunization is the most recent approach in vaccine development. Genetic vaccines have important advantages over other vaccines and vaccine production methods: 1) they harbor genes made artificially and can be easier to purify than any vaccine made directly from pathogens; 2) they may trigger both the humoral and cellular immune responses thus having the potential to provide long-lived immune responses; 3) several different genes can be mixed and injected simultaneously, making it possible to vaccinate against myltiple variants of a pathogen, or against several different pathogens, at the same time; 4) all DNA vaccines can be produced using similar techniques; 5) they consist of only one (or few) of the many genes necessary for the pathogen to be virulent i.e. enough for the immune system to recognize and mount an immune response, but not enough to pose any adverse effects to the host; 6) the expression of the encoded antigens by the host cells mimics natural infection whereby antigen presentation and processing induces both MHC and class I and class II restricted cellular and humoral immune responses; and 7) they are extremely stable and unlike many conventional vaccines that must be held at a constant temperature.

DNA vaccines can be stored under a vast array of conditions either dried or in solution. This eliminates the need for refrigeration and greatly improves the delivery of vaccines in developing countries. Clearly, DNA vaccines offer significant advantages. The

efficacy of DNA vaccines to provide protection against pathogenic challenges has now been demonstrated in multiple animal models and a handful of different viral pathogens. Positive results have also led to the approval of veterinary DNA vaccines to protect horses, fish and dogs. More importantly, several clinical trials are currently underway including the first human trial of a plasmid DNA vaccine designed to prevent H5N1 infection.

DNA-based vaccines usually consist of a purified plasmid (pDNA) comprising a sequence encoding for expression of the antigen of interest under the control of a eukaryotic promoter. Production of pDNA is traditionally done using bacteria fermentation and requires rigorous purification steps to remove unwanted contaminants derived from bacterial debris (such as genomic DNA, RNA, endotoxins, etc..) and culture medium. Such impurities not only minimize the efficiency of DNA vaccines, they can also lead to dose-related toxicity. pDNA vaccines contain two critical moieties. One constitutes the plasmid backbone which is required for maintenance and replication of the plasmid in the host bacteria. The second moiety consists of the eukaryotic expression cassette (EC) containing the heterologous gene to be expressed. In most instances, the plasmid backbone represents a significant inactive portion of the therapeutic dose. In essence, it is not required for proper ectopic gene expression in mammalian cells and may even be detrimental.

Although immunogenic, CpG dinucleotides present in the backbone sequences have been shown to contribute to episomal gene silencing. In addition, a number of elements from prokaryotic plasmids have been shown to negatively affect gene expression in eukaryotic cells or bind eukaryotic transcription factors. The backbones also harbor sequences, e.g., antibiotic resistance genes, and replication origin sequences, that could later lead to adverse effects. Leading scientists still disagree on the ramifications of producing DNA vaccines in bacteria both regarding potential health hazards and vaccine efficacy. Nonetheless, efforts to develop better methods for producing DNA vaccines devoid of the plasmid backbone and bacterial contaminants are needed.

DNA can be made with polymerase chain reaction (PCR), but this process has multiple limitations. Since its introduction in 1985, PCR has become an indispensable tool in molecular genetic analysis and DNA cloning. More recently, PCR has been used to produce linear DNA expression cassettes for the expression of proteins both in cultured vertebrate cells and in animal models, and has been used to evaluate PCR-derived DNA vaccines with promising results. However, PCR is not always a straightforward method for reliable DNA synthesis. The thermostable polymerase used is very sensitive to various ions, salts and

inhibitory contaminants. Moreover, the conditions for each particular DNA template must be precisely worked out and primer design is extremely important for effective amplification. Finally, although modifications to the PCR buffer and discovery/engineer of novel polymerases have led to the successful amplification of large-size DNA (up to 35 kb), the modifications have been limited to small analytical scale reactions. Most efficient PCR amplification have been used to make DNA in the 4-5 kbp size range.

The effectiveness of cell-free genetic vaccines is rapidly being established. Many groups are currently working on fine-tuning the actual EC to maximize efficacy with dose sparing, efficient delivery methods and inclusion of immunostimulant boosters. Unfortunately, effective vaccination campaigns will still require large and cost effective production capacity to immunize the general population and not just a selected few select, especially in developing countries.

SUMMARY OF THE INVENTION

The current invention addresses the concerns regarding production capacity of DNA by customizing an isothermal DNA amplification process to meet large scale production needs in a small facility with minimal specialized equipment. This cell-free process is based on the naturally occurring replication of circular DNA molecules such as plasmids or viruses.

An important factor for the success of this method in vitro is the unique nature of one of the

DNA polymerases used in the amplification cocktail. Phi29 DNA polymerase is a single subunit, proofreading DNA polymerase isolated from the B. subtilis phage φ29. Without the need for accessory proteins, this polymerase can perform ~10 ⁴ polymerization cycles without dissociating from the template, incorporating on average -70,000 nucleotides per DNA binding event. Its high processivity and robust strand displacement activity, enables the process to easily reach amplification over 1000-fold in 1 hr at constant temperature in vitro. This polymerase has been shown to be very stable, with linear reaction kinetics at 30 ⁰ C for over 12 h.

Although template specific primers can be designed for amplification of each construct, a cocktail of random primers can also be used to amplify virtually any circular template, thus making this a "one-for-all" amplification method. In related patent publications (WO2006/ 063355 and WO2007/018744) and US patent applications (11/792,800 and 12/012,615, herein incorporated by reference in their entireties), we streamlined a cell-free amplification process to synthesize gram quantities of DNA; the process has the capacity to produce hundreds of grams of DNA without significant effort or

costly equipment investments (synDNA™ technology). The process only requires minimal temperature control so that DNA can be manufactured in small facilities having a small footprint. One gram of DNA per liter of amplification cocktail is easily attainable.

We have also streamlined a purification regimen to purify milligram quantities of an expression cassette away from the pDNA backbone and with minimal handling. A circular template is added to an enzyme/buffer cocktail containing phi29 DNA polymerase. The reaction is left to proceed for a few hours at a set temperature and the product rendered to single EC copies by treatment with selected restriction endonucleases (RE) specific for the EC and starting material. This process yields large amounts of DNA in a single run, which can be purified in a streamlined method that easily separates the desired therapeutic DNA from other amplification by-products and reagents. The method is capable of handling gram quantities of raw material in a single day. Quality Control tests can then be performed for assurance of identity, fidelity and purity. In most cases, it would take less than seven weeks to transform a newly identified viral strain into large quantities of therapeutic quality DNA and useful vaccines.

Most existing avian influenza infections require the induction of the immune system and the production of antibodies to the highly variable surface viral proteins, HA and NA. Accordingly, current vaccine preparations work mainly by triggering strong humoral responses to these two proteins. Unfortunately, each time a new viral variant emerges, new vaccines need to be produced which can take up to 9 months. Some of these DNA vaccines have been shown to induce a protective immune response against lethal influenza viral infections in mice, ferrets and chickens; however, production of these vaccines requires large facilities and often implicates growth in a host microorganism whose by-products need to be thoroughly removed. Existing DNA vaccine production technology does not readily allow for the production of large DNA constructs expressing more than one large protein. If a genetic vaccine preparation targeting multiple viral proteins is to be formulated, several plasmid vectors generally need to be mixed in defined ratios. Although feasible, the use of DNA vaccine cocktails does not take into account the variability in cellular uptake and expression levels from one construct to another. From a manufacturing standpoint, the production of DNA vaccine mixes requires stringent control, and validation must take into account every individual component within the DNA cocktail. The need for a large scale production process that addresses these concerns is great.

The current invention combines the synDNA™ process with some novel H5 and Nl sequences for the production of effective DNA vaccines. This combination is capable of meeting the challenge of a quickly occurring epidemic. Not only can large quantities (grams- kilograms) of DNA be produced in a small production facility (on average less than 500 sq feet), the therapeutic DNA so produced requires only minimal downstream processing because there are no live biological agents involved. These attributes reflect on both product cost and availability. This combination can easily accommodate possible clade or sub-clade specific mutations in the influenza viral genome with potential turn around times of less than four weeks to enable meeting the demand in an emergency scenario. Modern poultry farming has resulted in the development of high-density poultry areas. The efficacy of a vaccination requires proper vaccine administration, a challenging feat when thousands of birds need to be vaccinated at one time. A wide range of methods of administration of poultry vaccines is available for use both in hatcheries and on farms: drinking water, intramuscular injection (im), subcutaneous injection (sc), coarse/fine spray, eye-drop and in-ovo (egg) injections.

The present invention includes a DNA-based vaccine having novel sequences designed to optimally express the HA and NA sequences of the H5N1 virus. The sequences were designed using codon-optimization strategies, were made using the cell-free synDNA™ process described above, and have been tested independently and together in animal challenge experiments (mouse model) using a lethal viral load to challenge the vaccinated or the untreated mice. The sequences were co don-optimized for the HA and NA genes of avian influenza viral strain A/Vietnam/ 1203/04 (Figures 1 and 2). Challenge experiments were done using each of the HA and NA optimized sequences, alone, as well as together. The H5 construct (alone and in tandem with Nl) of the current invention protected mice against a lethal challenge with the Vietnam virus. The vaccine DNA was produced using our synDNA™ technology as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

The H5 and Nl sequences of the current invention as shown in the sequence listing and used in the examples are codon-optimized for improved expression in mammalian cells. FIGURE 1 : (A) MAP of the tandem H5N1 expression cassette (SEQ ID NO: 1), having a codon-optimized H5 and Nl sequence (SEQ ID NOs: 3 and 5), each under the control of two individual CMV promoters, two individual B-globin like introns, two

individual IG signal peptides, and two individual polyA sequences; (B) Map of the single H5 expression cassette (SEQ ID NO: 2) having the codon-optimized H5 (SEQ ID NO: 3) under the control of the CMV promoter, with a bGH polyA tail, a β-Globin like intron, and an individual Ig signal peptide; (C) Map of the single Nl expression cassette (SEQ ID NO: 4) having the codon-optimized Nl sequence (SEQ ID NO: 5) under the control of the CMV promoter with a bGH poly A tail.

FIGURE 2: Percent SURVIVAL of vaccinated mice following lethal challenge with influenza A/H5N1. (A) and (B) represent two independent challenge trials showing post- challenge percent survival over the 21-22 day study period. Mice were vaccinated via intramuscular (i.m.) with the indicated DNAs (50 μg per mouse) or with a control (saline or control DNA) on days -42, -28 and -14 relative to day 0 (H5N1 challenge). Challenge was done on day 0, two weeks after the last DNA injection, using intranasal (i.n.) administration. (A) Trial 1 : 15 mice/group; challenged with a lethal dose of virulent H5N1 virus (6.8 x 10 TCID ₅₀ per mouse) (positive control mice surviving a prior i.n. infection with a low dose of H5N1 convalescent). (B) Trial 2: 19 mice/group; challenged with a lethal dose of virulent H5N1 virus (5.3 x 10 ³ TCID ₅₀ per mouse). H5N1 synDNA™, H5 synDNA™, Nl synDNA™, control luciferase synDNA™, saline, and convalescent groups.

FIGURE 3: Percent change in BODY WEIGHT following challenge with influenza

A/H5N1. (A) and (B) represent the two independent trials as in Fig. 2. Data is represented as percent change in body weight relative to baseline (day 0). Each data point represents the average of 15 mice (A) or 19 mice (B). H5N1 synDNA™, H5 synDNA™, Nl synDNA™, control luciferase synDNA™ and saline groups.

FIGURE 4: Percent change in BODY TEMPERATURE of vaccinated mice following challenge with influenza A/H5N1. Data collected from the same two trials as in Figs 2 and 3. (A) and (B) represent the results of the two independent trials. The percent change in body temperature relative to baseline (day 0) was calculated for individual mice and the percent change averaged by group. Each data point represents the average for H5N1 synDNA™, H5 synDNA™, Nl synDNA™, control synDNA™ and saline groups.

FIGURE 5: Serological analysis of H5 ANTIBODIES in vaccinated mice. Sera from H5N1, H5 and control synDNA™ immunized mice were collected prior to challenge (on day

0, two weeks following third vaccination dose) and analyzed for the presence of anti-H5 antibodies by ELISA (OD ₄₅ Q readings). (A) Anti-H5 antibody titers in mice that survived the

lethal challenge with H5N1 virus are represented by solid bars for H5N1 (black) and H5 (grey). Hatched bars depict antibody titers in H5N1 and H5 immunized mice that did not survive the challenge (black and grey, respectively). Anti-H5 antibody titers in mice immunized with placebo are represented by white solid bars. (B) Comparative analysis of anti-H5 antibody titers in the pre-challenge sera of mice that survived the challenge without detectable infection symptoms (normal) and in morbid mice that recovered by the end of the trial (sick).

FIGURE 6: H5N1 virus level (viral load) in the brain and lungs of vaccinated and challenged mice. Mice were vaccinated and challenged as in Figs 2-5. At 5 days post- challenge, five mice per vaccination group were euthanized and organs collected for viral titration. Viral titer in brain (solid) and lung (open) tissues for individual mice in each H5N1 (square), H5 (triangle) and control (circle) synDNA™ immunized group are shown. Y axis represents H5N1 TCID ₅₀ per g of given tissue. Marked points (+) refer to mice that already presented signs of morbidity at day 5 post-challenge. The dotted line at TCID ₅ 0 : 1E+04 represents the limit of detection for our assay.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "nucleic acid" refers to: DNA; RNA; DNA mimetics having chemically altered backbones; oligodeoxynucleotides (oligos) which may be chemically modified with morpholino groups, phosphorothioates, radioisotopes, fi orescent, magnetic or other markers; and DNA-RNA hybrid structures. Such nucleic acid molecules may be single- stranded, double-stranded or triplex structures, and may exist as hairpin-like or clover-like structures.

As used herein, the term "promoter" shall be used to describe a nucleic acid having a sequence that functions to initiate binding of necessary proteins for initiation of mRNA synthesis using a DNA-like structure as a template, and includes variations of commonly used promoters comprising functional sequence changes. Common eukaryotic promoters include the Cytomegalovirus (CMV) and Rous Sarcoma virus (RSV) promoters.

As used herein, the term "termination sequence" shall refer to a nucleic acid sequence that facilitates the termination of mRNA transcription by signaling the RNA polymerase to stop, detach or skip over the template.

As used herein, the term "expression cassette" is a nucleic acid sequence comprising the necessary sequences for synthesis of a RNA from the nucleic acid template.

As used herein, the term "vector" is a self-contained nucleic acid structure that aids in the transfer, delivery, replication or transcription of a useful nucleic acid sequence or expression cassette. A vector includes, but is not limited to, viral particles (DNA and RNA), plasmids, linear expression cassettes, minicircles and other related forms of nucleic acid. Medicament, as used herein, is a therapeutic, vaccine, prophylactic or similar compound that either prevents, halts progress, ameliorates or cures a disease process in an organism.

Derivative - a derivative effectuates protection of a host organism when expressed in the context of a DNA-based vaccine and the host is challenged with avian flu or a related virus.

Mimetic - a nucleic acid comprising a backbone other than a phosphodiester backbone, including PNA, LNA, and other chemically modified backbones that confer biological activity similar to phosphodiester based nucleic acids.

Although the use of inactivated or attenuated viruses has been the traditional standby in the preparation of vaccines, this method has limited effectiveness. For example, commonly used inactivated vaccines provide very short-term and highly specific humoral immunity due to frequent antigenic variations in the influenza virion and because this type of vaccine fails to induce sufficient protective immunity. Stronger, more effective, and longer lasting vaccines are needed for both humans and poultry. Recombinant DNA vaccines not only offer the advantage of "targeting" a specific virulence factor but are known to be highly effective inducers of both humoral and cellular immunity; they show great promise as alternatives for protection against viral infections. The current invention includes codon-optimized sequences of the H5 and Nl antigenic proteins of the H5N1 influenza virus, expression vectors used herein to demonstrate the effectiveness of the sequences in viral challenge experiments in mice, and proposed medicaments containing nucleic acids having the codon- optimized sequences.

When a foreign microorganism invades the bird's body, antibodies are produced and specific cells whose purpose is to engulf and destroy foreign pathogens (also called antigens) are activated. This defense mechanism constitutes the basis of the humoral and cellular immune response, respectively. Once a bird's immune system has responded to an antigen and survived the infection, if it is exposed again to that microbe, it will respond very quickly due to the innate ability of the immune system to "remember" the microbe (Cutler, 2002).

The quick response of the immune system prevents the disease from happening or shortens its duration and severity. For most poultry diseases the natural progression is the same: infection, development of immunity and recovery (Cutler, 2002). However in the case of highly virulent microbes, recovery is either slow or all together absent. In order to trick the immune system into responding to a pathogen without exposing the birds to virulent disease- causing microbes, modern science has developed vaccines. The main objective of vaccination is to increase the specific immunity to infections to which the vaccinated poultry are likely to be exposed so that, when challenged, they either do not suffer the disease, or suffer to a much lesser extent than if they had not been vaccinated. The associated economic objective is to ensure that, on average, the cost of the vaccines purchased, their application, and any loss of productivity caused by their application, is less than the cost of the disease if vaccines are not used. Vaccines are categorized into, Live, Inactivated, Recombinant and Nucleic Acid.

Live vaccines contain live viruses, bacteria or parasites. They are nearly always weakened (or 'attenuated') in some way to ensure that they do not induce significant disease when administered. They can sometimes be found as naturally weak strains in poultry populations. Sometimes a related pathogen, even from another species, may be used to vaccinate. However, more commonly they are grown through multiple generations in an artificial culture system (such as cell cultures, embryos, or artificial media) so that they become poorly adapted to grow in the target host. Live vaccines cause infection with living organisms, which then, to a greater or lesser extent, multiply in the host and the resulting infection induces an immune response. This ability to multiply in the host means that effective live vaccines can contain a very low dose of the agent, making them less expensive to produce than some other vaccines. Some live vaccines are capable of lateral, bird to bird, spread and can, thus achieve some protection even in those birds which do not receive an adequate dose initially. Only live vaccines can currently be administered by by drinking water, as aerosols or sprays.

Inactivated vaccines are often described as 'dead' vaccines and as their name implies, do not contain live organisms. To manufacture these, the pathogen must be grown in large amounts in the laboratory then inactivated, usually by a chemical treatment. Because they contain no live organisms, they do not multiply in vaccinated birds or spread between birds in the flock. They therefore must be applied to each individual bird by injection. They nearly always contain something to stimulate the immune system locally at the site of injection. These compounds are called 'adjuvants' and the two most common types are mineral oils and

aluminium hydroxide. Oil-based inactivated vaccines are usually formulated as an emulsion (either oil-in- water or water-in-oil). Because of the need to have a high content of the antigen, inactivated vaccines tend to be expensive. Uniformity of application (both in terms of % of birds injected and volume injected in each) is critical to a successful outcome, because they do not spread between birds.

Recombinant vaccines are a sub-set of the category live vaccine. They are created as a mix of two different organisms by artificial means. Nucleic acid from one organism is "grafted" into the nucleic acid of another in such a way that, when the carrier organism multiplies in the body it also expresses the protein to induce immunity to the second one (without inducing an infection of the second organism). Development of this type of vaccine is highly complex as it is necessary to ensure that the modification does not damage the ability of the carrier organism to infect and multiply. In addition the chosen antigen for the second organism must be the correct protein (in structure and conformation) to achieve protection. For some infections it is necessary to provide immunity to multiple antigens for full immune efficacy to be achieved. Recombinant vaccines share the same features as other live vaccines - they can contain small numbers of organisms, they can be spread bird-to-bird and can be applied by mass routes. However the features of a particular recombinant vaccine will depend upon the nature of the carrier organism. To date, the more common carriers for viral recombinants have been fowlpox virus and Marek's disease herpevirus (or HVT). These are not yet widely used.

Nucleic acid vaccines are a relatively new approach whereby the naked nucleic acid (usually DNA) of a pathogen is injected into the target bird, egg or host. This has been an area of active research for the past decade and, to date, three veterinary DNA vaccines have been approved for use in horses and dogs, as well as for farm-raised trout and salmon destined for human consumption. While such products need to be individually administered, there is a need to develop techniques to rapidly produce large amounts of DNA in a consistent fashion. Because only the nucleic acid is injected, the vaccine is not infectious and does not spread between vaccinated birds.

The current invention combines the use of our synDNA™ process (US Patent App: 12/012615) with novel sequences and a robust vaccination regimen. The current invention comprises a novel sequence H5N1 DNA vaccine which can be made quickly, in large amounts using our cell-free production process. This allows for rapid testing for efficacy in

chickens, eggs or other animal hosts, and provides a way to develop an economical preventative inoculation process for use in large scale vaccination campaigns.

It is commonly believed that the prevention of influenza infection requires induction of antibodies mainly to the most variable surface viral proteins HA and NA. Accordingly, current vaccine preparations work mainly by triggering a strong humoral response to these two proteins. Recent DNA vaccines expressing the influenza proteins HA and NA have shown to be reliable in generating a protective antibody-mediated immune response against lethal influenza virus infections in mice, ferrets and chickens, by restricting viral replication.

There are reports demonstrating that some influenza A DNA vaccines expressing viral proteins HA- and NA- in conjunction with some other proteins, can confer a cross-protective effect in animals.

Unfortunately, the current DNA vaccine production technology does not readily allow the production of large DNA constructs expressing more than one large protein. If a genetic vaccine preparation targeting multiple viral proteins is desired, several plasmid vectors need to be mixed in defined ratios. Although feasible, the use of DNA vaccine cocktails does not take into account the variability in cellular uptake and expression levels from one construct to another. From a manufacturing standpoint, the production of DNA vaccine mixes requires stringent control, and validation must take into account every individual component within the DNA cocktail. Successful vaccines should induce strong immune responses which are long-lasting and in most cases capable of providing protection against different strains of the same pathogen. The current invention focuses on a single combination of novel H 5 and Nl sequences that has been tested in challenge assays in mice with promising results. It is feasible to extrapolate that this protective effect would also be found in humans, chickens and eggs. The current construct once tested in chickens should be a useful agent in inducing protective immunological responses in a susceptible host. Combination with the cell-free biosynthesis of DNA should enable the immunization of large poultry flocks (and if needed, humans) in a short amount of time in response to a potential threat.

Current sub-unit vaccines predominantly induce strong antibody responses and weak cellular immunity. DNA vaccines in animal models can induce both strong humoral and cellular mediated responses. With the threat of an HPAI H5N1 at our door step, with no reliable conventional vaccine available, the reported success of DNA vaccines against

influenza in animal models cannot be ignored. As the nature of these vaccines is being further refined, our simple, robust and economical production process (synDNA™) offers a way to take on the need for rapid mass production. Significant advances have recently been made in identifying influenza virus proteins capable of triggering both an antibody and cellular response - essential for long term protection. Moreover, DNA vaccines using some of these polypeptides have shown to induce cross protection. The current invention provides novel sequences for the expression of the hemaglutinin (HA) and neuraminidase (NA) proteins and a way to manufacture these sequences quickly and with minimal handling. These sequences have been tested in vivo using synDNA™ to effectively protect against challenges with the influenza A/Vietnam/I 203/04 H5N1 virus.

EXAMPLES

The current invention addresses the use of novel codon-optϊmized H5 and Nl sequences designed for convenient downstream cloning, cloned into optimally designed expression cassettes, together with our own proprietary and biologically active synDNA™ processing, to create DNA-based vaccines which confer protection against an H 5Nl viral challenge in mice.

EXAMPLE 1 : Codon Optimization. The H5 and Nl viral gene sequences were codon-optimized for mammalian cell expression using standard techniques as provided by computerized optimization techniques (GeneArt, Inc.). Sequences were additionally designed to have specific Kpnl and Xbal sites for streamlined cloning of the codon-optimized sequences.

EXAMPLE 2: DNA Vaccination. The codon-optimized open reading frame sequences (SEQ ID NOs: 3 and 5) were cloned into expression cassettes (SEQ ID NOs: 1, 2, and 4) having CMV promoters upsteam of each optimized sequence and polyA tails downstream of each optimized sequence. Intervening introns, signaling sequences and RNA transport sequences were included, as diagrammed in Fig. 1. The expression cassettes were configured into circular double-stranded DNA templates and used to replicate the template in the cell-free synDNA™ process described above. Naked DNA was injected without any carrier. Following synthesis, the DNA material was purified using a chromatography-based process to >90% as judged by densitometry-coupled DNA electrophoresis. The vaccines were recovered in physiological citrate buffer and used directly (naked linear dsDNA) in

immunization experiments. A conservative immunization protocol was used which consisted of 3 intramuscular leg injections in 6-8 week old Balb/C mice, carried out at 2 week intervals, using 50 μg of DNA for each injection. The mice were challenged intranasally with 6x10 ⁴ EIDso/dose (egg infectious dose) of H5N1 influenza A/Vietnam/ 1203 /04, two weeks after the last booster. As positive control, survivors of previous H5N1 -challenge (convalescent) were infected with H5N1 using half the virus dose of the other groups. Clinical observations, paralysis / mortality, body temperature and weight were all monitored for 21 days postinfection. Animals were checked daily for survival, morbidity and mortality as indicated in Table 1.

TABLE 1 : Effect of lethal IAV/H5N1 viral challenge on immunized mice

Vaccine Asymptomatic Sick Survivors Dead

Trial 1 H5N1 9 (60%) 4(26%) 13 (87%) 2(13%) n=15 H5 4 (26.5%) 5(33%) 9 (60%) 6 (40%) Nl 15(100%) 15(100%) Control 15(100%) 15(100%)

Trial 2 H5N1 13 (90%) 1 (5%) 14 (95%) 1 (5%) n=19 ^a) H5 6 (42%) 6(42%) 12 (84%) 3 (16%) N1 15(100%) 15(100%) Control 15(100%) 15(100%)

EXAMPLE 3: Challenge with H5N1 virus. DNA vaccines for flu have show promising results in several animal models. DNA constructs expressing HA and NA have previously been shown to be the most immunogenic viral proteins for inducing an immune response. We have previously shown that DNA made with our synDNA™ process is a bona fide alternative to plasmid vaccines produced from bacteria culture (US Patents 11/792,800 and 12/012,615). Here, we designed and tested the avian flu DNA vaccine prototypes of the current invention against a pandemic influenza A/1203/04 H5N1 virus (made using the synDNA ™ process). The current constructs were designed without a plasmid backbone and the DNA amplified in our cell-free system. The expression cassettes contained sequences encoding for a codon optimized H 5 and/or a co don-optimized Nl gene from the H5N1 isolate. Three linear prototype vaccines were prepared (Figure 1): (i) one comprising a codon- optimized H5 sequence; (ii) one comprising a codon- optimized Nl sequence; and (iii) one comprising both expression cassettes cloned in tandem. The firefly luciferase gene was used as a control.

EXAMPLE 4: Poultry and Egg Inoculation a) Evaluation of influenza synDNA™ vaccines in vivo. Once the constructs are made, their immunogenic activity can be tested in chickens. Initially, the synDNA™ vaccines will be injected into chickens using a conservative intramuscular delivery method. Various amounts of synDNA™ and several numbers of injections will be tested. We will conduct quantitative and qualitative analysis of the antibody and cellular immune response along with constant monitoring of the animals after each vaccination and booster shot. b) Confirmation study. Once an optimal vaccination regimen is determined, the test can be done using a larger group of animals using a lethal challenge with a virulent HPAI H5N1 virus strain using Biosafety Level-4 containment to assess the extent of the protection. All animals will be humanely euthanized following the challenge to analuze the viral loads in various target organs such as gut, lung and brain. c) Evaluation of in-ovo immunization. In poultry, vaccines are generally administered as aerosols, oculo-nasal drops, through drinking water or by injection. An in ovo vaccination technique can be used that offers several advantages over the abovementioned methods, including neonatal resistance and better protection, administration of vaccine in eggs en masse (up to 40,000 eggs per hour) resulting in reduced labor costs and handling. In ovo vaccines are administered in embryonated eggs with the help of an in-egg vaccine delivery system (available for testing through Embrex, Inc). Soon after hatching, the birds are protected against the disease. In ovo vaccination has been proved to be effective against other avian diseases. d) Optimization of in-ovo immunizations. An effective procedure will be optimized for:

(i) The site of injection - usually between the amnion and the shell, the allantois functions as a sort of waste bag, storing waste products that are formed during the embryo's growth, including metabolic water whereby vaccines must be injected deep enough into the egg to be delivered into the amnionic fluid, where it can be absorbed by the embryo as it takes up nutrients. If the vaccine is injected too deep, the needle will hit the developing embryo directly, and although the embryo will be vaccinated, there is a risk of damage to the developing chick. If the vaccine is not injected deeply enough, it will be delivered into the allantois, or waste fluid. This vaccine will not be utilised by the embryo and the vaccination will be ineffective.

(ii) Position of the embryo - the needle is usually inserted into the egg at a fixed depth under the shell, positioned for each individual egg. If the embryo is in the correct position, the egg will be injected correctly and the embryo successfully vaccinated. However, the position of the embryo depends on its stage of development. If the embryo is too small, it will not sit high enough in the egg, the needle will not penetrate through the allantoic and the vaccine will be delivered into the allantoic fluid and wasted. If the embryo is too big, the needle will go through the amnion and hit the embryo directly.

(iii) Size of the embryo — which depends mainly on the speed of its development and time since incubation started. To inject the embryos at the optimum size, vaccination must be done at the proper stage of development. If the embryo is not adequately developed, it is simply a matter of waiting slightly longer before it is vaccinated. The stage and speed of development depends on the embryonic temperature inside the egg, which varies with the incubator, breed, and egg size. To help identify optimum timing, the eggs can be injected with a dye periodically during incubation, to check where the dye is delivered. The timing of vaccination can also be adjusted by measuring the length of the embryo at 18 days of incubation.

(iv) Temperature fluctuations - embryonic temperature (inside the egg) is not equal at every spot in a given incubator. This is dependent on a number of factors, including air temperature, air velocity and the volume and path of water spray. This will influence the rate of development and if not standardized or monitored, a percentage of the embryos will not be vaccinated correctly and will not be effectively immunized, especially when injected relatively early. To achieve optimum results with in-ovo vaccination, it is therefore, more important to monitor the size of the embryos closely, rather than time elapsed during incubation. To achieve uniform protection, all embryos must be as close to the same stage of development as possible, and of the right size to deliver the vaccine into the amnion.

(v) Confirmation and challenge - Once the optimal injection protocol is determined, a range of DNA amounts will be used for vaccinating chicks in-ovo. Once hatched, analysis of the antibody and cellular responses over time will be conducted for each group of animals, to determine the optimal vaccination regimen. The analysis will be conducted over a period of 24 to 44 weeks matching

EXAMPLE 5: Fragments and Sequence Derivatives

Although the data presented here focuses on the novel H5 and Nl sequences as intact sequences, both alone and in the context of an expression cassette, one skilled in the art would recognize that smaller fragments of larger genes can be shown to be equally as effective, and may be more desireable if multiple antigens are to be produced simultaneously. As such, the current invention also includes any fragments or closely related derivatives of the sequences herein presented, which would be able to induce an immunological response either alone orin conjunction with another fragment or derivative of any of the sequences presented here as SEQ ID NOS: 1-5. For example, a smaller peptide of possibly twenty amino acids of the H5 codon-optimized sequence could be inserted into an expression cassette and cloned in tandem on the same vector with a similar expression cassette encoding for a smaller fragment of possibly twenty amino acids of the Nl codon-optimized sequence presented here.

From the data presented here, it is likely that the H5 which works well alone, but better with the Nl sequence, could work as well as the H5N1 tandem cassette if the entire H5 sequence was co-expressed with a not yet identified 'active' fragment of the Nl codon- optimized sequence. Likewise, there may simply be a small localized area on the expressed H5 expressed peptide that interacts with a small localized area on the expressed Nl peptide, that can more efficiently induce the host to trigger an immune response. As such, the current invention would include smaller sequence fragments of the H5 and Nl codon-optimized sequences presented here as small as ten amino acids (or thirty nucleotides) which when expressed and presented in the host would help to effectuate an immune response in the host, as detected by serological assay or by challenge protection.

Derivatives would include minor modifications that would be used to streamline cloning or to incorporate stabilizing bases into the nucleic acid (such as phosphorothioate, morpholino groups, PNA, LNA or other similar modifications) that would strengthen the immune response upon introducing the nucleic acid into the host.

AVIAN FLU DNA VACCINE SEQUENCES

(1) CMV-H5,CMV-N1 (H5N1 tandem cassette):

1 CGGCCGCACG CGTGGGATCC GAGCTAGTTA TTAATAGTAA TCAATTACGG

51 GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG 101 GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC

151 AATAATGACG TATGTTCCCA TAGTAACGTC AATAGGGACT TTCCATTGAC

201 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA

251 GTGTATCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG

301 GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT 351 GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT

401 TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC

451 AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGC ACCAAAΆTCA

501 ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG ACGCAAATGG

551 GCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC TCGTTTAGTG 601 AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG

651 AAGACACCGG GACCGATCCA GCCTCCGCGG ATACCGCCGA GACCGCGTCC

701 GCCCCGCGAG CACAGAGCCT CGCCTTTGCC GATCCGCCGC CCGTCCACAC

751 CCGCCGCCAG CTCACCTCGA ATCCCGGCCG GGAACGGTGC ATTGGAACGC

801 GGATTCCCCG TGCCAAGAGT GACGTAAGTA CCGCCTATAG AGTCTATAGG 851 CCCACAAAAA ATGCTTTCTT CTTTTAATAT ACTTTTTTGT TTATCTTATT

901 TCTAATACTT TCCCTAATCT CTTTCTTTCA GGGCAATAAT GATACAATGT

951 ATCATGCCTC TTTGCACCAT TCTAAAGAAT AACAGTGATA ATTTCTGGGT

1001 TAAGGCAATA GCAATATTTC TGCATATAAA TATTTCTGCA TATAAATTGT

1051 AACTGATGTA AGAGGTTTCA TATTGCTAAT AGCAGCTACA ATCCAGCTAC 1101 CATTCTGCTT TTATTTTAAG GTTGGGATAA GGCTGGATTA TTCTGAGTCC

1151 AAGCTAGGCC CTTTTGCTAA TCATGTTCAT ACCTCTTATC TTCCTCCCAC

1201 AGCTCCTGGG CAACGTGCTG GTCTGTGTGC TGGCCCATCA CTTTGGCAAA

1251 GAATTGGGAT TCGAACATCG ATTGAATTCG GTACCATGGA TTGGACTTGG

1301 ATCTTATTTT TAGTTGCTGC TGCTACTAGA GTTCATTCTA ACTGGATGGA 1351 AAAGATCGTG CTGCTGTTCG CCATCGTGAG CCTGGTGAAG AGCGACCAGA

1401 TCTGCATCGG CTACCACGCC AACAACAGCA CCGAGCAGGT GGACACCATC

1451 ATGGAAAAAA ACGTGACCGT GACCCACGCC CAGGACATCC TGGAAAAGAA

1501 GCACAACGGC AAGCTGTGCG ACCTGGACGG CGTGAAGCCC CTGATCCTGC

1551 GGGACTGCAG CGTGGCCGGC TGGCTGCTGG GCAACCCCAT GTGCGACGAG 1601 TTCATCAACG TGCCCGAGTG GAGCTACATC GTGGAGAAGG CCAACCCCGT

1651 GAACGACCTG TGCTACCCCG GCGACTTCAA CGACTACGAG GAACTGAAGC

1701 ACCTGCTGTC CCGGATCAAC CACTTCGAGA AGATCCAGAT CATCCCCAAG

1751 AGCAGCTGGT CCAGCCACGA GGCCAGCCTG GGCGTGAGCA GCGCCTGCCC

1801 ATACCAGGGC AAGTCCAGCT TCTTCCGGAA CGTGGTGTGG CTGATCAAGA 1851 AGAACAGCAC CTACCCCACC ATCAAGCGGA GCTACAACAA CACCAACCAG

1901 GAAGATCTGC TGGTCCTGTG GGGCATCCAC CACCCCAACG ACGCCGCCGA

1951 GCAGACCAAG CTGTACCAGA ACCCCACCAC CTACATCAGC GTGGGCACCA

2001 GCACCCTGAA CCAGCGGCTG GTGCCCCGGA TCGCCACCCG GTCCAAGGTG

2051 AACGGCCAGA GCGGCCGGAT GGAATTCTTC TGGACCATCC TGAAGCCCAA 2101 CGATGCCATC AACTTCGAGA GCAACGGCAA CTTCATCGCC CCCGAGTACG

2151 CCTACAAGAT CGTGAAGAAG GGCGACAGCA CCATCATGAA GAGCGAGCTG

2201 GAATACGGCA ACTGCAACAC CAAGTGCCAG ACCCCCATGG GCGCCATCAA

2251 CAGCAGCATG CCCTTCCACA ACATCCACCC CCTGACCATC GGCGAGTGCC

2301 CCAAGTACGT GAAGAGCAAC AGGCTGGTGC TGGCCACCGG CCTGCGGAAC 2351 AGCCCCCAGC GGGAGCGGCG GAGGAAGAAG AGGGGCCTGT TCGGCGCCAT

2401 CGCCGGCTTC ATCGAGGGCG GCTGGCAGGG CATGGTGGAC GGGTGGTACG

2451 GCTACCACCA CAGCAATGAG CAGGGCAGCG GCTACGCCGC CGACAAAGAG

2501 AGCACCCAGA AGGCCATCGA CGGCGTCACC AACAAGGTGA ACAGCATCAT

2551 CGACAAGATG AACACCCAGT TCGAGGCCGT GGGCCGGGAG TTCAAC7VACC

2601 TGGAACGGCG GATCGAGAAC CTGAACAAGA AAATGGAAGA TGGCTTCCTG

2651 GACGTGTGGA CCTACAACGC CGAGCTGCTG GTGCTGATGG AAAACGAGCG

2701 GACCCTGGAC TTCCACGACA GCAACGTGAA GAACCTGTAC GACAAAGTGC

2751 GGCTGCAGCT GCGGGACAAC GCCAAAGAGC TGGGCAACGG CTGCTTCGAG

2801 TTCTACCACA AGTGCGACAA CGAGTGCATG GAAAGCGTGC GGAACGGCAC

2851 CTACGACTAC CCCCAGTACA GCGAGGAAGC CCGGCTGAAG CGGGAGGAAA

2901 TCAGCGGCGT GAAACTGGAA AGCATCGGCA TCTACCAGAT CCTGAGCATC

2951 TACAGCACCG TGGCCAGCAG CCTGGCCCTG GCCATCATGG TGGCCGGCCT

3001 GAGCCTGTGG ATGTGCAGCA ACGGCAGCCT GCAGTGCCGG ATCTGCATCT

3051 GAGAAATCGA GTTCAGATTG TAGTTAACGA TTCAGAAGAG GCGCCGTATA

3101 CTCTACGCGC GGATCTCTAG AGTCGACCTG CTCGGGGACG GTGAAGGTGA

3151 CAGCAGTCGG TTGGAGCGAG CATCTCTACG GGTGGCATCC CTGTGACCCC

3201 TCCCCAGTGC CTCTCCTGGC CCTGGAAGTT GCCACTCCAG TGCCCACCAG

3251 CCTTGTCCTA ATAAAATTAA GTTGCATCAT TTTGTCTGAC TAGGTGTCCT

3301 TCTATAATAT TATGGGGTGG AGGGGGGTGG TATGGAGCAA GGGGCAAGTT

3351 GGGAAGACAA CCTGTAGGGC CTGCGGGGTC TATTGGGAAC CAAGCTGGAG

3401 TGCAGTGGCA CAATCTTGGC TCACTGCAAT CTCCGCCTCC TGGGTTCAAG

3451 CGATTCTCCT GCCTCAGCCT CCCGAGTTGT TGGGATTCCA GGCATGCATG

3501 ACCAGGCTCA GCTAATTTTT GTTTTTTTGG TAGAGACGGG GTTTCACCAT

3551 ATTGGCCAGG CTGGTCTCCA ACTCCTAATC TCAGGTGATC TACCCACCTT

3601 GGCCTCCCAA ATTGCTGGGA TTACAGGCGT GAACCACTGC TCCCTTCCCT

3651 GTCCTTCTGA TTTTGTAGGT AACCACGATC CGAGCTAGTT ATTAATAGTA

3701 ATCAATTACG GGGTCATTAG TTCATAGCCC ATATATGGAG TTCCGCGTTA

3751 CATAACTTAC GGTAAATGGC CCGCCTGGCT GACCGCCCAA CGACCCCCGC

3801 CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGT CAATAGGGAC

3851 TTTCCATTGA CGTCAATGGG TGGAGTATTT ACGGTAAACT GCCCACTTGG

3901 CAGTACATCA AGTGTATCAT ATGCCAAGTA CGCCCCCTAT TGACGTCAAT

3951 GACGGTAAAT GGCCCGCCTG GCATTATGCC CAGTACATGA CCTTATGGGA

4001 CTTTCCTACT TGGCAGTACA TCTACGTATT AGTCATCGCT ATTACCATGG

4051 TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG GTTTGACTCA

4101 CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA GTTTGTTTTG

4151 CACCAAAATC AACGGGACTT TCCAAAATGT CGTAACAACT CCGCCCCATT

4201 GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG

4251 CTCGTTTAGT GAACCGTCAG ATCGCCTGGA GACGCCATCC ACGCTGTTTT

4301 GACCTCCATA GAAGACACCG GGACCGATCC AGCCTCCGCG GATACCGCCG

4351 AGACCGCGTC CGCCCCGCGA GCACAGAGCC TCGCCTTTGC CGATCCGCCG

4401 CCCGTCCACA CCCGCCGCCA GCTCACCTCG AATCCCGGCC GGGAACGGTG

4451 CATTGGAACG CGGATTCCCC GTGCCAAGAG TGACGTAAGT ACCGCCTATA

4501 GAGTCTATAG GCCCACAAAA AATGCTTTCT TCTTTTAATA TACTTTTTTG

4551 TTTATCTTAT TTCTAATACT TTCCCTAATC TCTTTCTTTC AGGGCAATAA

4601 TGATACAATG TATCATGCCT CTTTGCACCA TTCTAAAGAA TAACAGTGAT

4651 AATTTCTGGG TTAAGGCAAT AGCAATATTT CTGCATATAA ATATTTCTGC

4701 ATATAAATTG TAACTGATGT AAGAGGTTTC ATATTGCTAA TAGCAGCTAC

4751 AATCCAGCTA CCATTCTGCT TTTATTTTAA GGTTGGGATA AGGCTGGATT

4801 ATTCTGAGTC CAAGCTAGGC CCTTTTGCTA ATCATGTTCA TACCTCTTAT

4851 CTTCCTCCCA CAGCTCCTGG GCAACGTGCT GGTCTGTGTG CTGGCCCATC

4901 ACTTTGGCAA AGAATTGGGA TTCGAACATC GATTGAATTC GGTACCATGG

4951 ATTGGACTTG GATCTTATTT TTAGTTGCTG CTGCTACTAG AGTTCATTCT

5001 AACTGGATGA ACCCCAACCA GAAGATCATC ACCATCGGCA GCATCTGCAT

5051 GGTGACCGGC ATCGTGAGCC TGATGCTGCA GATCGGCAAC ATGATCAGCA

5101 TCTGGGTGTC CCACAGCATC CACACCGGCA ACCAGCACCA GAGCGAGCCC

5151 ATCAGCAACA CCAACTTTCT GACCGAGAAG GCCGTGGCCA GCGTGAAGCT

5201 GGCCGGCAAC AGCAGCCTGT GCCCCATCAA CGGCTGGGCC GTGTACAGCA

5251 AGGACAACAG CATCCGGATC GGCAGCAAGG GCGATGTGTT CGTGATCCGG

5301 GAGCCCTTCA TCAGCTGCAG CCACCTGGAA TGCCGGACCT TCTTCCTGAC

5351 CCAGGGGGCC CTGCTGAACG ACAAGCACAG CAACGGCACC GTGAAGGACA 5401 GAAGCCCCCA CCGGACCCTG ATGAGCTGCC CCGTGGGCGA GGCCCCCAGC 5451 CCCTACAACA GCCGGTTCGA GAGCGTGGCC TGGTCCGCCA GCGCCTGCCA 5501 CGACGGCACC AGCTGGCTGA CCATCGGCAT CAGCGGCCCT GACAACGGCG 5551 CCGTGGCCGT GCTGAAGTAC AACGGCATCA TCACCGACAC CATCAAGAGC 5601 TGGCGGAACA ACATCCTGCG GACCCAGGAA AGCGAGTGCG CCTGCGTGAA 5651 CGGCAGCTGC TTCACCGTGA TGACCGACGG CCCCAGCAAC GGCCAGGCCA 5701 GCCACAAGAT CTTCAAGATG GAAAAGGGCA AGGTGGTGAA GAGCGTGGAG 5751 CTGGACGCCC CCAACTACCA CTACGAGGAA TGCAGCTGCT ACCCCAACGC 5801 CGGCGAGATC ACCTGCGTGT GCCGGGACAA CTGGCACGGC AGCAACCGGC 5851 CCTGGGTGTC CTTCAACCAG AACCTGGAAT ACCAGATCGG CTACATCTGC 5901 AGCGGCGTGT TCGGCGACAA CCCCAGGCCC AACGATGGCA CCGGCAGCTG 5951 CGGCCCTGTG AGCAGCAACG GCGCCTACGG CGTGAAGGGC TTCAGCTTCA 6001 AGTACGGCAA CGGCGTGTGG ATCGGCCGGA CCAAGAGCAC CAACAGCAGA 6051 TCCGGCTTCG AGATGATCTG GGACCCCAAC GGCTGGACCG AGACCGACAG 6101 CAGCTTCTCC GTGAAGCAGG ACATCGTGGC CATCACCGAC TGGTCCGGCT 6151 ACAGCGGCAG CTTCGTGCAG CACCCCGAGC TGACCGGCCT GGACTGCATC 6201 CGGCCCTGCT TTTGGGTGGA GCTGATCAGA GGCAGGCCCA AAGAGAGCAC 6251 CATCTGGACC AGCGGCAGCA GCATCAGCTT TTGCGGCGTG AACAGCGACA 6301 CCGTGGGCTG GTCCTGGCCC GATGGCGCCG AGCTGCCCTT CACCATCGAC 6351 AAGTGAGAGC TAAAGTTCAG ATTGTAGTTA ACGATTCAGA AGAGGCGCCG 6401 TATACTCTAC GCGCGGATCT CTAGAGTCGA CCTGCTCGGG GACGGTGAAG 6451 GTGACAGCAG TCGGTTGGAG CGAGCATCTC TACGGGTGGC ATCCCTGTGA 6501 CCCCTCCCCA GTGCCTCTCC TGGCCCTGGA AGTTGCCACT CCAGTGCCCA 6551 CCAGCCTTGT CCTAATAAAA TTAAGTTGCA TCATTTTGTC TGACTAGGTG 6601 TCCTTCTATA ATATTATGGG GTGGAGGGGG GTGGTATGGA GCAAGGGGCA 6651 AGTTGGGAAG ACAACCTGTA GGGCCTGCGG GGTCTATTGG GAACCAAGCT 6701 GGAGTGCAGT GGCACAATCT TGGCTCACTG CAATCTCCGC CTCCTGGGTT 6751 CAAGCGATTC TCCTGCCTCA GCCTCCCGAG TTGTTGGGAT TCCAGGCATG 6801 CATGACCAGG CTCAGCTAAT TTTTGTTTTT TTGGTAGAGA CGGGGTTTCA 6851 CCATATTGGC CAGGCTGGTC TCCAACTCCT AATCTCAGGT GATCTACCCA 6901 CCTTGGCCTC CCAAATTGCT GGGATTACAG GCGTGAACCA CTGCTCCCTT 6951 CCCTGTCCTT CTGATTTTGT AGGTAACCAC GTGCGGACCG AGCGGCCGTG 7001 CGGACCGAG [SEQ ID NO: !]

(2) CMV-H5 (H5 cassette) Sequence:

1 CGGCCGCACG CGTGGGATCC GAGCTAGTTA TTAATAGTAA TCAATTACGG 51 GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG 101 GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC 151 AATAATGACG TATGTTCCCA TAGTAACGTC AATAGGGACT TTCCATTGAC 201 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA 251 GTGTATCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG 301 GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT 351 GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT 401 TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC 451 AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGC ACCAAAATCA 501 ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG ACGCAAATGG 551 GCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC TCGTTTAGTG 601 AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG 651 AAGACACCGG GACCGATCCA GCCTCCGCGG ATACCGCCGA GACCGCGTCC 701 GCCCCGCGAG CACAGAGCCT CGCCTTTGCC GATCCGCCGC CCGTCCACAC

751 CCGCCGCCAG CTCACCTCGA ATCCCGGCCG GGAACGGTGC ATTGGAACGC

801 GGATTCCCCG TGCCAAGAGT GACGTAAGTA CCGCCTATAG AGTCTATAGG 851 CCCACAAAAA ATGCTTTCTT CTTTTAATAT ACTTTTTTGT TTATCTTATT 901 TCTAATACTT TCCCTAATCT CTTTCTTTCA GGGCAATAAT GATACAATGT

951 ATCATGCCTC TTTGCACCAT TCTAAAGAAT AACAGTGATA ATTTCTGGGT 1001 TAAGGCAATA GCAATATTTC TGCATATAAA TATTTCTGCA TATAAATTGT 1051 AACTGATGTA AGAGGTTTCA TATTGCTAAT AGCAGCTACA ATCCAGCTAC 1101 CATTCTGCTT TTATTTTAAG GTTGGGATAA GGCTGGATTA TTCTGAGTCC 1151 AAGCTAGGCC CTTTTGCTAA TCATGTTCAT ACCTCTTATC TTCCTCCCAC 1201 AGCTCCTGGG CAACGTGCTG GTCTGTGTGC TGGCCCATCA CTTTGGCAAA 1251 GAATTGGGAT TCGAACATCG ATTGAATTCG GTACCATGGA TTGGACTTGG 1301 ATCTTATTTT TAGTTGCTGC TGCTACTAGA GTTCATTCTA ACTGGATGGA 1351 AAAGATCGTG CTGCTGTTCG CCATCGTGAG CCTGGTGAAG AGCGACCAGA 1401 TCTGCATCGG CTACCACGCC AACAACAGCA CCGAGCAGGT GGACACCATC 1451 ATGGAAAAAA ACGTGACCGT GACCCACGCC CAGGACATCC TGGAAAAGAA 1501 GCACAACGGC AAGCTGTGCG ACCTGGACGG CGTGAAGCCC CTGATCCTGC 1551 GGGACTGCAG CGTGGCCGGC TGGCTGCTGG GCAACCCCAT GTGCGACGAG 1601 TTCATCAACG TGCCCGAGTG GAGCTACATC GTGGAGAAGG CCAACCCCGT 1651 GAACGACCTG TGCTACCCCG GCGACTTCAA CGACTACGAG GAACTGAAGC 1701 ACCTGCTGTC CCGGATCAAC CACTTCGAGA AGATCCAGAT CATCCCCAAG 1751 AGCAGCTGGT CCAGCCACGA GGCCAGCCTG GGCGTGAGCA GCGCCTGCCC 1801 ATACCAGGGC AAGTCCAGCT TCTTCCGGAA CGTGGTGTGG CTGATCAΆGA 1851 AGAACAGCAC CTACCCCACC ATCAAGCGGA GCTACAACAA CACCAACCAG 1901 GAAGATCTGC TGGTCCTGTG GGGCATCCAC CACCCCAACG ACGCCGCCGA 1951 GCAGACCAAG CTGTACCAGA ACCCCACCAC CTACATCAGC GTGGGCACCA 2001 GCACCCTGAA CCAGCGGCTG GTGCCCCGGA TCGCCACCCG GTCCAAGGTG 2051 AACGGCCAGA GCGGCCGGAT GGAATTCTTC TGGACCATCC TGAAGCCCAA 2101 CGATGCCATC AACTTCGAGA GCAACGGCAA CTTCATCGCC CCCGAGTACG 2151 CCTACAAGAT CGTGAAGAAG GGCGACAGCA CCATCATGAA GAGCGAGCTG 2201 GAATACGGCA ACTGCAACAC CAAGTGCCAG ACCCCCATGG GCGCCATCAA 2251 CAGCAGCATG CCCTTCCACA ACATCCACCC CCTGACCATC GGCGAGTGCC 2301 CCAAGTACGT GAAGAGCAAC AGGCTGGTGC TGGCCACCGG CCTGCGGAAC 2351 AGCCCCCAGC GGGAGCGGCG GAGGAAGAAG AGGGGCCTGT TCGGCGCCAT 2401 CGCCGGCTTC ATCGAGGGCG GCTGGCAGGG CATGGTGGAC GGGTGGTACG 2451 GCTACCACCA CAGCAATGAG CAGGGCAGCG GCTACGCCGC CGACAAAGAG 2501 AGCACCCAGA AGGCCATCGA CGGCGTCACC AACAAGGTGA ACAGCATCAT 2551 CGACAAGATG AACACCCAGT TCGAGGCCGT GGGCCGGGAG TTCAACAACC 2601 TGGAACGGCG GATCGAGAAC CTGAACAAGA AAATGGAAGA TGGCTTCCTG 2651 GACGTGTGGA CCTACAACGC CGAGCTGCTG GTGCTGATGG AAAACGAGCG 2701 GACCCTGGAC TTCCACGACA GCAACGTGAA GAACCTGTAC GACAAAGTGC 2751 GGCTGCAGCT GCGGGACAAC GCCAAAGAGC TGGGCAACGG CTGCTTCGAG 2801 TTCTACCACA AGTGCGACAA CGAGTGCATG GAAAGCGTGC GGAACGGCAC 2851 CTACGACTAC CCCCAGTACA GCGAGGAAGC CCGGCTGAAG CGGGAGGAAA 2901 TCAGCGGCGT GAAACTGGAA AGCATCGGCA TCTACCAGAT CCTGAGCATC 2951 TACAGCACCG TGGCCAGCAG CCTGGCCCTG GCCATCATGG TGGCCGGCCT 3001 GAGCCTGTGG ATGTGCAGCA ACGGCAGCCT GCAGTGCCGG ATCTGCATCT 3051 GAGAAATCGA GTTCAGATTG TAGTTAACGA TTCAGAAGAG GCGCCGTATA 3101 CTCTACGCGC GGATCTCTAG AGTCGACCTG CTCGGGGACG GTGAAGGTGA 3151 CAGCAGTCGG TTGGAGCGAG CATCTCTACG GGTGGCATCC CTGTGACCCC 3201 TCCCCAGTGC CTCTCCTGGC CCTGGAAGTT GCCACTCCAG TGCCCACCAG 3251 CCTTGTCCTA ATAAAATTAA GTTGCATCAT TTTGTCTGAC TAGGTGTCCT 3301 TCTATAATAT TATGGGGTGG AGGGGGGTGG TATGGAGCAA GGGGCAAGTT 3351 GGGAAGACAA CCTGTAGGGC CTGCGGGGTC TATTGGGAAC CAAGCTGGAG 3401 TGCAGTGGCA CAATCTTGGC TCACTGCAAT CTCCGCCTCC TGGGTTCAAG 3451 CGATTCTCCT GCCTCAGCCT CCCGAGTTGT TGGGATTCCA GGCATGCATG 3501 ACCAGGCTCA GCTAATTTTT GTTTTTTTGG TAGAGACGGG GTTTCACCAT 3551 ATTGGCCAGG CTGGTCTCCA ACTCCTAATC TCAGGTGATC TACCCACCTT 3601 GGCCTCCCAA ATTGCTGGGA TTACAGGCGT GAACCACTGC TCCCTTCCCT 3651 GTCCTTCTGA TTTTGTAGGT AACCACGTGC GGACCGAGCG

[SEQ ID NO: 2]

(3) Codon-Optimized H5 (H5-ORF):

1351 TCGTG CTGCTGTTCG CCATCGTGAG CCTGGTGAAG AGCGACCAGA 1401 TCTGCATCGG CTACCACGCC AACAACAGCA CCGAGCAGGT GGACACCATC 1451 ATGGAAAAAA ACGTGACCGT GACCCACGCC CAGGACATCC TGGAAAAGAA 1501 GCACAACGGC AAGCTGTGCG ACCTGGACGG CGTGAAGCCC CTGATCCTGC 1551 GGGACTGCAG CGTGGCCGGC TGGCTGCTGG GCAACCCCAT GTGCGACGAG 1601 TTCATCAACG TGCCCGAGTG GAGCTACATC GTGGAGAAGG CCAACCCCGT 1651 GAACGACCTG TGCTACCCCG GCGACTTCAA CGACTACGAG GAACTGAAGC 1701 ACCTGCTGTC CCGGATCAAC CACTTCGAGA AGATCCAGAT CATCCCCAAG 1751 AGCAGCTGGT CCAGCCACGA GGCCAGCCTG GGCGTGAGCA GCGCCTGCCC 1801 ATACCAGGGC AAGTCCAGCT TCTTCCGGAA CGTGGTGTGG CTGATCAAGA 1851 AGAACAGCAC CTACCCCACC ATCAAGCGGA GCTACAACAA CACCAACCAG 1901 GAAGATCTGC TGGTCCTGTG GGGCATCCAC CACCCCAACG ACGCCGCCGA 1951 GCAGACCAΆG CTGTACCAGA ACCCCACCAC CTACATCAGC GTGGGCACCA 2001 GCACCCTGAA CCAGCGGCTG GTGCCCCGGA TCGCCACCCG GTCCAAGGTG 2051 AACGGCCAGA GCGGCCGGAT GGAATTCTTC TGGACCATCC TGAAGCCCAA 2101 CGATGCCATC AACTTCGAGA GCAACGGCAA CTTCATCGCC CCCGAGTACG 2151 CCTACAAGAT CGTGAAGAAG GGCGACAGCA CCATCATGAA GAGCGAGCTG 2201 GAATACGGCA ACTGCAACAC CAAGTGCCAG ACCCCCATGG GCGCCATCAA 2251 CAGCAGCATG CCCTTCCACA ACATCCACCC CCTGACCATC GGCGAGTGCC 2301 CCAAGTACGT GAAGAGCAAC AGGCTGGTGC TGGCCACCGG CCTGCGGAAC 2351 AGCCCCCAGC GGGAGCGGCG GAGGAAGAAG AGGGGCCTGT TCGGCGCCAT 2401 CGCCGGCTTC ATCGAGGGCG GCTGGCAGGG CATGGTGGAC GGGTGGTACG 2451 GCTACCACCA CAGCAATGAG CAGGGCAGCG GCTACGCCGC CGACAAAGAG 2501 AGCACCCAGA AGGCCATCGA CGGCGTCACC AACAAGGTGA ACAGCATCAT 2551 CGACAAGATG AACACCCAGT TCGAGGCCGT GGGCCGGGAG TTCAACAACC 2601 TGGAACGGCG GATCGAGAAC CTGAACAAGA AAATGGAAGA TGGCTTCCTG 2651 GACGTGTGGA CCTACAACGC CGAGCTGCTG GTGCTGATGG AAAACGAGCG 2701 GACCCTGGAC TTCCACGACA GCAACGTGAA GAACCTGTAC GACAAAGTGC 2751 GGCTGCAGCT GCGGGACAAC GCCAAAGAGC TGGGCAACGG CTGCTTCGAG 2801 TTCTACCACA AGTGCGACAA CGAGTGCATG GAAAGCGTGC GGAACGGCAC 2851 CTACGACTAC CCCCAGTACA GCGAGGAAGC CCGGCTGAAG CGGGAGGAAA 2901 TCAGCGGCGT GAAACTGGAA AGCATCGGCA TCTACCAGAT CCTGAGCATC 2951 TACAGCACCG TGGCCAGCAG CCTGGCCCTG GCCATCATGG TGGCCGGCCT 3001 GAGCCTGTGG ATGTGCAGCA ACGGCAGCCT GCAGTGCCGG ATCTGCATCT

[SEQ ID NO: 3]

(4) CMV-Nl (Nl cassette):

1 CGGCCGCACG CGTGGGATCC GAGCTAGTTA TTAATAGTAA TCAATTACGG 51 GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG 101 GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC 151 AATAATGACG TATGTTCCCA TAGTAACGTC AATAGGGACT TTCCATTGAC 201 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA 251 GTGTATCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG 301 GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT 351 GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT 401 TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC 451 AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGC ACCAAAATCA 501 ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG ACGCAAATGG 551 GCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC TCGTTTAGTG 601 AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG 651 AAGACACCGG GACCGATCCA GCCTCCGCGG ATACCGCCGA GACCGCGTCC

701 GCCCCGCGAG CACAGAGCCT CGCCTTTGCC GATCCGCCGC CCGTCCACAC

751 CCGCCGCCAG CTCACCTCGA ATCCCGGCCG GGAACGGTGC ATTGGAACGC

801 GGATTCCCCG TGCCAAGAGT GACGTAAGTA CCGCCTATAG AGTCTATAGG

851 CCCACAAAAA ATGCTTTCTT CTTTTAATAT ACTTTTTTGT TTATCTTATT

901 TCTAATACTT TCCCTAATCT CTTTCTTTCA GGGCAATAAT GATACAATGT

951 ATCATGCCTC TTTGCACCAT TCTAAAGAAT AACAGTGATA ATTTCTGGGT

1001 TAAGGCAATA GCAATATTTC TGCATATAAA TATTTCTGCA TATAAATTGT

1051 AACTGATGTA AGAGGTTTCA TATTGCTAAT AGCAGCTACA ATCCAGCTAC

1101 CATTCTGCTT TTATTTTAAG GTTGGGATAA GGCTGGATTA TTCTGAGTCC

1151 AAGCTAGGCC CTTTTGCTAA TCATGTTCAT ACCTCTTATC TTCCTCCCAC

1201 AGCTCCTGGG CAACGTGCTG GTCTGTGTGC TGGCCCATCA CTTTGGCAAA

1251 GAATTGGGAT TCGAACATCG ATTGAATTCG GTACCATGGA TTGGACTTGG

1301 ATCTTATTTT TAGTTGCTGC TGCTACTAGA GTTCATTCTA ACTGGATGAA

1351 CCCCAACCAG AAGATCATCA CCATCGGCAG CATCTGCATG GTGACCGGCA

1401 TCGTGAGCCT GATGCTGCAG ATCGGCAACA TGATCAGCAT CTGGGTGTCC

1451 CACAGCATCC ACACCGGCAA CCAGCACCAG AGCGAGCCCA TCAGCAACAC

1501 CAACTTTCTG ACCGAGAAGG CCGTGGCCAG CGTGAAGCTG GCCGGCAACA

1552 GCAGCCTGTG CCCCATCAAC GGCTGGGCCG TGTACAGCAA GGACAACAGC

1601 ATCCGGATCG GCAGCAAGGG CGATGTGTTC GTGATCCGGG AGCCCTTCAT

1651 CAGCTGCAGC CACCTGGAAT GCCGGACCTT CTTCCTGACC CAGGGGGCCC

1701 TGCTGAACGA CAAGCACAGC AACGGCACCG TGAAGGACAG AAGCCCCCAC

1751 CGGACCCTGA TGAGCTGCCC CGTGGGCGAG GCCCCCAGCC CCTACAACAG

1801 CCGGTTCGAG AGCGTGGCCT GGTCCGCCAG CGCCTGCCAC GACGGCACCA

1851 GCTGGCTGAC CATCGGCATC AGCGGCCCTG ACAACGGCGC CGTGGCCGTG

1901 CTGAAGTACA ACGGCATCAT CACCGACACC ATCAAGAGCT GGCGGAACAA

1951 CATCCTGCGG ACCCAGGAAA GCGAGTGCGC CTGCGTGAAC GGCAGCTGCT

2001 TCACCGTGAT GACCGACGGC CCCAGCAACG GCCAGGCCAG CCACAAGATC

2051 TTCAAGATGG AAAAGGGCAA GGTGGTGAAG AGCGTGGAGC TGGACGCCCC

2101 CAACTACCAC TACGAGGAAT GCAGCTGCTA CCCCAACGCC GGCGAGATCA

2151 CCTGCGTGTG CCGGGACAAC TGGCACGGCA GCAACCGGCC CTGGGTGTCC

2201 TTCAACCAGA ACCTGGAATA CCAGATCGGC TACATCTGCA GCGGCGTGTT

2251 CGGCGACAAC CCCAGGCCCA ACGATGGCAC CGGCAGCTGC GGCCCTGTGA

2301 GCAGCAACGG CGCCTACGGC GTGAAGGGCT TCAGCTTCAA GTACGGCAAC

2351 GGCGTGTGGA TCGGCCGGAC CAAGAGCACC AACAGCAGAT CCGGCTTCGA

2401 GATGATCTGG GACCCCAACG GCTGGACCGA GACCGACAGC AGCTTCTCCG

2451 TGAAGCAGGA CATCGTGGCC ATCACCGACT GGTCCGGCTA CAGCGGCAGC

2501 TTCGTGCAGC ACCCCGAGCT GACCGGCCTG GACTGCATCC GGCCCTGCTT

2551 TTGGGTGGAG CTGATCAGAG GCAGGCCCAA AGAGAGCACC ATCTGGACCA

2601 GCGGCAGCAG CATCAGCTTT TGCGGCGTGA ACAGCGACAC CGTGGGCTGG

2651 TCCTGGCCCG ATGGCGCCGA GCTGCCCTTC ACCATCGACA AGTGAGAGCT

2701 AAAGTTCAGA TTGTAGTTAA CGATTCAGAA GAGGCGCCGT ATACTCTACG

2751 CGCGGATCTC TAGAGTCGAC CTGCTCGGGG ACGGTGAAGG TGACAGCAGT

2801 CGGTTGGAGC GAGCATCTCT ACGGGTGGCA TCCCTGTGAC CCCTCCCCAG

2851 TGCCTCTCCT GGCCCTGGAA GTTGCCACTC CAGTGCCCAC CAGCCTTGTC

2901 CTAATAAAAT TAAGTTGCAT CATTTTGTCT GACTAGGTGT CCTTCTATAA

2951 TATTATGGGG TGGAGGGGGG TGGTATGGAG CAAGGGGCAA GTTGGGAAGA

3001 CAACCTGTAG GGCCTGCGGG GTCTATTGGG AACCAAGCTG GAGTGCAGTG

3051 GCACAATCTT GGCTCACTGC AATCTCCGCC TCCTGGGTTC AAGCGATTCT

3101 CCTGCCTCAG CCTCCCGAGT TGTTGGGATT CCAGGCATGC ATGACCAGGC

3151 TCAGCTAATT TTTGTTTTTT TGGTAGAGAC GGGGTTTCAC CATATTGGCC

3201 AGGCTGGTCT CCAACTCCTA ATCTCAGGTG ATCTACCCAC CTTGGCCTCC

3251 CAAATTGCTG GGATTACAGG CGTGAACCAC TGCTCCCTTC CCTGTCCTTC

3301 TGATTTTGTA GGTAACCACG TGCGGACCGA [SEQ ID NO: 4]

(5) Codon-Optimized Nl (Nl-ORF)

1356 ATGGA TTGGACTTGG 1301 ATCTTATTTT TAGTTGCTGC TGCTACTAGA GTTCATTCTA ACTGGATGAA 1351 CCCCAACCAG AAGATCATCA CCATCGGCAG CATCTGCATG GTGACCGGCA 1401 TCGTGAGCCT GATGCTGCAG ATCGGCAACA TGATCAGCAT CTGGGTGTCC 1451 CACAGCATCC ACACCGGCAA CCAGCACCAG AGCGAGCCCA TCAGCAACAC 1501 CAACTTTCTG ACCGAGAAGG CCGTGGCCAG CGTGAAGCTG GCCGGCAACA 1551 GCAGCCTGTG CCCCATCAAC GGCTGGGCCG TGTACAGCAA GGACAACAGC 1601 ATCCGGATCG GCAGCAAGGG CGATGTGTTC GTGATCCGGG AGCCCTTCAT 1651 CAGCTGCAGC CACCTGGAAT GCCGGACCTT CTTCCTGACC CAGGGGGCCC 1701 TGCTGAACGA CAAGCACAGC AACGGCACCG TGAAGGACAG AAGCCCCCAC 1751 CGGACCCTGA TGAGCTGCCC CGTGGGCGAG GCCCCCAGCC CCTACAACAG 1801 CCGGTTCGAG AGCGTGGCCT GGTCCGCCAG CGCCTGCCAC GACGGCACCA 1851 GCTGGCTGAC CATCGGCATC AGCGGCCCTG ACAACGGCGC CGTGGCCGTG 1901 CTGAAGTACA ACGGCATCAT CACCGACACC ATCAAGAGCT GGCGGAACAA 1951 CATCCTGCGG ACCCAGGAAA GCGAGTGCGC CTGCGTGAAC GGCAGCTGCT 2001 TCACCGTGAT GACCGACGGC CCCAGCAACG GCCAGGCCAG CCACAAGATC 2051 TTCAAGATGG AAAAGGGCAA GGTGGTGAAG AGCGTGGAGC TGGACGCCCC 2101 CAACTACCAC TACGAGGAAT GCAGCTGCTA CCCCAACGCC GGCGAGATCA 2151 CCTGCGTGTG CCGGGACAAC TGGCACGGCA GCAACCGGCC CTGGGTGTCC 2201 TTCAACCAGA ACCTGGAATA CCAGATCGGC TACATCTGCA GCGGCGTGTT 2251 CGGCGACAAC CCCAGGCCCA ACGATGGCAC CGGCAGCTGC GGCCCTGTGA 2301 GCAGCAACGG CGCCTACGGC GTGAAGGGCT TCAGCTTCAA GTACGGCAAC 2351 GGCGTGTGGA TCGGCCGGAC CAAGAGCACC AACAGCAGAT CCGGCTTCGA 2401 GATGATCTGG GACCCCAACG GCTGGACCGA GACCGACAGC AGCTTCTCCG 2451 TGAAGCAGGA CATCGTGGCC ATCACCGACT GGTCCGGCTA CAGCGGCAGC 2501 TTCGTGCAGC ACCCCGAGCT GACCGGCCTG GACTGCATCC GGCCCTGCTT 2551 TTGGGTGGAG CTGATCAGAG GCAGGCCCAA AGAGAGCACC ATCTGGACCA 2601 GCGGCAGCAG CATCAGCTTT TGCGGCGTGA ACAGCGACAC CGTGGGCTGG 2651 TCCTGGCCCG ATGGCGCCGA GCTGCCCTTC ACCATCGACA AGT

[SEQ ID NO: 5]