WO2008124331A1 - Nouvelles séquences et vaccins à adn contre la grippe aviaire - Google Patents

Nouvelles séquences et vaccins à adn contre la grippe aviaire Download PDF

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WO2008124331A1
WO2008124331A1 PCT/US2008/058510 US2008058510W WO2008124331A1 WO 2008124331 A1 WO2008124331 A1 WO 2008124331A1 US 2008058510 W US2008058510 W US 2008058510W WO 2008124331 A1 WO2008124331 A1 WO 2008124331A1
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sequences
dna
nucleic acid
host
vaccines
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PCT/US2008/058510
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Frederic Kendirgi
Yin Chen
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Cytogenix, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE

Definitions

  • 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, synDNATM, 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.
  • novel genetic sequences preferably DNA
  • 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).
  • HA hemagglutinin
  • NA neuraminidase
  • 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.
  • influenza 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.
  • seasonal influenza outbreaks are responsible for a total cost of over $US 10 billion per year.
  • 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.
  • 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.
  • pandemic strains of influenza grow poorly or kill eggs, and inactivated vaccines appear to be poorly immunogenic.
  • 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.
  • 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.
  • H5N1 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.
  • 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.
  • 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).
  • 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.
  • avian influenza 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).
  • 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.
  • LPAI flu viruses in embryonated chicken eggs.
  • this method cannot be applied for HPAI strains.
  • the highly virulent viruses are difficult to grow and amplify in eggs, as they are lethal to the developing embryo.
  • 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.
  • 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.
  • 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.
  • 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.
  • pDNA purified plasmid
  • 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
  • CpG dinucleotides present in the backbone sequences have been shown to contribute to episomal gene silencing.
  • 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.
  • PCR polymerase chain reaction
  • 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.
  • Phi29 DNA polymerase is a single subunit, proofreading DNA polymerase isolated from the B. subtilis phage ⁇ 29. 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.
  • DNA vaccine cocktails do 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 synDNATM 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.
  • 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 synDNATM 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 synDNATM technology as described above.
  • FIGURE 1 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 ⁇ -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)
  • 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 ⁇ g 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.
  • 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).
  • Trial 2 19 mice/group; challenged with a lethal dose of virulent H5N1 virus (5.3 x 10 3 TCID 50 per mouse).
  • FIGURE 3 Percent change in BODY WEIGHT following challenge with influenza
  • (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).
  • 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 synDNATM, H5 synDNATM, Nl synDNATM, control synDNATM and saline groups.
  • FIGURE 5 Serological analysis of H5 ANTIBODIES in vaccinated mice. Sera from H5N1, H5 and control synDNATM immunized mice were collected prior to challenge (on day
  • 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) synDNATM 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.
  • 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.
  • oligos oligodeoxynucleotides
  • Such nucleic acid molecules may be single- stranded, double-stranded or triplex structures, and may exist as hairpin-like or clover-like structures.
  • 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.
  • 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.
  • the term "expression cassette” is a nucleic acid sequence comprising the necessary sequences for synthesis of a RNA from the nucleic acid template.
  • 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.
  • inactivated or attenuated viruses has been the traditional standby in the preparation of vaccines, this method has limited effectiveness.
  • 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.
  • 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.
  • an artificial culture system such as cell cultures, embryos, or artificial media
  • 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).
  • 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 synDNATM 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.
  • 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.
  • 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 synDNATM to effectively protect against challenges with the influenza A/Vietnam/I 203/04 H5N1 virus.
  • HA hemaglutinin
  • NA neuraminidase
  • the current invention addresses the use of novel codon-opt ⁇ mized H5 and Nl sequences designed for convenient downstream cloning, cloned into optimally designed expression cassettes, together with our own proprietary and biologically active synDNATM 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 synDNATM process described above. Naked DNA was injected without any carrier.
  • 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 ⁇ g 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.
  • H5N1 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.
  • 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.
  • DNA made with our synDNATM process is a bona fide alternative to plasmid vaccines produced from bacteria culture (US Patents 11/792,800 and 12/012,615).
  • 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 synDNATM vaccines in vivo. Once the constructs are made, their immunogenic activity can be tested in chickens.
  • the synDNATM vaccines will be injected into chickens using a conservative intramuscular delivery method. Various amounts of synDNATM 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.
  • 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:
  • 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.
  • (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.
  • 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.
  • 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.
  • 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.
  • stabilizing bases such as phosphorothioate, morpholino groups, PNA, LNA or other similar modifications

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Abstract

La présente invention concerne de nouvelles séquences conçues pour protéger un hôte contre une souche virulente du virus H5N1 influenza A (grippe aviaire) et comprend des séquences à codon optimisé codant pour les protéines virales antigéniques hémagglutinine (par exemple, H5) et neuraminadase (par exemple, N1). Les séquences peuvent être utilisées individuellement, conjointement ou associées à d'autres séquences pour entraîner une réponse immunitaire protectrice lorsqu'elles sont confrontées au virus (en tant que vaccin à ADN). La présente invention concerne également des cassettes d'expression comprenant les séquences à codon optimisé H5 et/ou N1 en plus d'autres séquences optionnelles telles que des séquences promoteurs, des séquences de fin de transcription et autres séquences utilisées pour améliorer l'expression et la présentation de l'antigène exprimé à l'intérieur de l'hôte. Les séquences et cassettes d'expression peuvent être utilisées sous diverses formes à condition que l'acide nucléique puisse être exprimé, traité et présenté de façon appropriée dans l'hôte pour induire une réponse immunitaire. Certaines formes utiles comprennent des plasmides, des particules virales, des brins linéaires de formes à brins doubles et multimères, des répétitions en tandem d'une ou de deux séquences et des formes circulaires tronquées qui peuvent contenir simplement une ou plusieurs cassettes d'expression utiles avec une ou plusieurs des séquences à codon optimisé.
PCT/US2008/058510 2007-04-03 2008-03-27 Nouvelles séquences et vaccins à adn contre la grippe aviaire WO2008124331A1 (fr)

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Cited By (4)

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WO2010136476A1 (fr) * 2009-05-28 2010-12-02 Abbott Biologicals B.V. Analyse d'agents étrangers
US8133723B2 (en) 2007-11-12 2012-03-13 The Trustees Of The University Of Pennsylvania Vaccines against multiple subtypes of influenza virus
WO2016130031A1 (fr) 2015-02-10 2016-08-18 Instytut Biochemii I Biofizyki Pan Vaccin à adn contre le virus de la grippe h5n1, séquence de nucléotides modifiée et utilisation de la séquence de nucléotides modifiée dans la fabrication d'un vaccin
US10363302B2 (en) 2010-01-26 2019-07-30 The Trustees Of The University Of Pennsylvania Influenza nucleic acid molecules and vaccines therefrom

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DATABASE GENBANK [online] 22 July 2001 (2001-07-22), "Cloning vector pCMV-MCS", Database accession no. (AF369966) *
HATCHETTE T.F. ET AL.: "Influenza A viruses in feral Canadian ducks: extensive reassortment in nature", JOURNAL OF GENERAL VIROLOGY, vol. 85, 2004, pages 2327 - 2337 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8133723B2 (en) 2007-11-12 2012-03-13 The Trustees Of The University Of Pennsylvania Vaccines against multiple subtypes of influenza virus
US9592285B2 (en) * 2007-11-12 2017-03-14 The Trustees Of The University Of Pennsylvania Vaccines against multiple subtypes of influenza virus
US10076565B2 (en) 2007-11-12 2018-09-18 The Trustees Of The University Of Pennsylvania Vaccines against multiple subtypes of influenza virus
WO2010136476A1 (fr) * 2009-05-28 2010-12-02 Abbott Biologicals B.V. Analyse d'agents étrangers
AU2010251950B2 (en) * 2009-05-28 2014-12-18 Bgp Products B.V. Extraneous agents testing
TWI597499B (zh) * 2009-05-28 2017-09-01 亞培生物股份有限公司 體外病原體檢驗技術
US10363302B2 (en) 2010-01-26 2019-07-30 The Trustees Of The University Of Pennsylvania Influenza nucleic acid molecules and vaccines therefrom
WO2016130031A1 (fr) 2015-02-10 2016-08-18 Instytut Biochemii I Biofizyki Pan Vaccin à adn contre le virus de la grippe h5n1, séquence de nucléotides modifiée et utilisation de la séquence de nucléotides modifiée dans la fabrication d'un vaccin

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