WO2008145129A2 - Influenza vaccines - Google Patents

Abstract

The invention concerns vaccines and the use of the naked DNA and/or RNA molecule encoding hemagglutinin (HA) from pandemic influenza, e.g. the 1918 HlNl and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus and/or the high pathogenic bird pandemic ATV strain (A/buzzard/Denmark/6370/06(H5Nl)) and/or 2001 H5N7 low pathogenic Avian influenza virus (ATV) strain (A/Mallard/Denmark/64650/03(H5N7)) or the March 2006 Denmark H5N1 high pathogenic AIV strain (A/buzzard/Denmark/6370/06(H5Nl)) or the 2008 (A/duck/Denmark/53- 147-8/08 (H7N1)) or the 2004 (A/widegeon/Denmark/66174/G18/04 (H2N3)) as a vaccine component against present day and coming Hl, H2, H3, H5, H7, Nl, N2, N3 containing influenza A infections in humans and/or swine optionally with the naked DNA and/or RNA molecule encoding neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza virus included. If the vaccine components are used as DNA or RNA vaccines with or without the corresponding protein, the codons can optionally be 'humanized' using preferred codons from highly expressed mammalian genes and the administration of this DNA vaccine can be by saline or buffered saline injection of naked DNA or RNA, or injection of DNA plasmid or linear gene expressing DNA fragments coupled to particles. Addition of the matrix protein (M) and/or the nucleoprotein (NP) as protein or DNA from the 1918 influenza strain is also disclosed.

Description

Influenza vaccines

Field of invention

The invention concerns vaccines and the use of the naked DNA and/or

RNA molecules encoding hemagglutinin (HA) from pandemic influenza A, e.g. the 1918 HlNl and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus, as a vaccine component against present day and coming influenza A infections in humans and swine, optionally with the naked DNA and/or RNA molecules encoding neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from these pandemic influenza virus included. The invention also concerns vaccines comprising naked DNA and/or RNA coding HA and/or NA from the new circulating 2001 H5N7 low pathogenic (LP) Avian influenza virus (AIV) strain (A/Mallard/Denmark/64650/03(H5N7)), the newly introduced and circulating March 2006 Denmark H5N1 high pathogenic Avian influenza A virus (AIV) strains A/buzzard/Denmark/6370/06 (H5N1), A/duck/Denmark/53-147-8/08 (H7N1) and A/widegeon/Denmark/66174/G 18/04 (H2N3) .

General background

Influenza is one of the oldest and most common diseases known to man causing between three and five million cases of severe illness and between 250,000 and 500,000 deaths every year around the world. Also swine are susceptible to human and avian influenza virus since they posses both receptors in their respiratory tract. Thus, swine get infection and pneumoni from human influenza strains and may serve as a dangerous mixing vessel for generation of new recombinant influenza strains with pandemic potential.

Influenza rapidly spreads in seasonal epidemics affecting 5-15% of the population and the burden on health care costs and lost productivity are extensive (WHO). Influenza like illness was first described by Hippocrates in the year 412 BC. Up to the 19th century influenza was thought to be a bacterial infection. Virus as the causative agent was first determined in 1931 by Richard Shope. The first known influenza A pandemic was in 1580 and since then there has been 31 pandemics of which three appeared in the 20th century namely the 'Spanish flu' in 1918, the 'Asian flu' in 1957 and the 'Hong Kong flu' in 1968, respectively. The pandemic of 1918 influenza A HlNl was the worst pandemic in newer times causing 20 to 50 million deaths worldwide. The most common form of influenza is seasonal outbreaks and epidemics of variable severity.

Zoonosis of avian influenza virus (AIV) able to infect humans and swine and the spread in Asia, parts of Europe and the Middle East has recently evoked the concern about a pandemic occurring also in the 21^st century. The causative strain of the pandemic will probably be unknown until the pandemic emerges and there will be an urgent need for a vaccine.

Therefore fast diagnosis and characterisation of circulating strains as well as emerging strains, new alternative vaccines approaches and production ways will be required in order to minimise the severity of the pandemic. Since seasonal influenza A vaccines are also produced on eggs an epidemic of highly pathogenic AIV among poultry will also influence the production of seasonal vaccines. Moreover the traditional influenza protein vaccines only have a limited protective effect. Also seasonal vaccines has to be changed every season because of the genetic drift of influenza A virus and the narrow type specific antibody induction by traditional influenza A protein vaccines. Therefore there is a need for new alternative influenza A vaccines with different properties

The influenza virus belongs to the Orthomyxoviridae family. The family includes three genera; influenza A, B and C viruses, identified by antigenic differences in their nucleoprotein (NP) and matrix protein (M).

The influenza A genus is further divided into subtype combinations based on the antigenic differences of the surface glycoproteins haemagglutinin (HA) and neuraminidase (NA). The A strain have evolved to be able to infect several other mammalian species (e.g. horses and swine). Influenza A viruses of all recognised 16 HAs and 9 NAs antigenic subtypes have been recovered from aquatic birds but few infect other animal species indicating that aquatic birds are the natural reservoirs of influenza A.

The influenza A viruses have been the causative agents for the major pandemics and most of the annual outbreaks of epidemic influenza. This invention solely focuses on the influenza A genus. The current nomenclature system for human influenza viruses includes the geographical location of first isolation, strain number, and year of isolation. The antigenic description of HA and NA is given in brackets, e.g. A/Moscow/10/99(H3N2). Nonhuman strains also include the host of origin in the nomenclature, e.g. A/mallard/Denmark/64650/03(H5N7).

The influenza A virus genome consist of eight negative sense single stranded (ss) ribonucleic acid (RNA) segments packed in the viral core comprised of host cell membrane and a matrix 1 (Ml) protein layer. The eight segments are associated with nucleoprotein (NP) and three large proteins; polymerase basic 1 (PBl) and 2 (PB2) protein, and polymerase acidic (PA) protein, which are responsible for RNA replication and transcription. NP encapsulates the RNA and forms ribonucleoprotein (RNP) complexes that protect and stabilise the RNA. Each segment include a sequence of 11-13 nucleotides at the 5' ends and 9-12 nucleotides at the 3^' ends which are highly conserved and similar for A, B and C viruses. The major glycoproteins HA and NA, and the ionchannel M2 protein, are embedded in a host derived lipid bilayer. Influenza viruses are somewhat pleomorphic in shape, but mostly spherical (80-120 nm in diameter).

All subtypes of influenza A are perpetuated in the wild aquatic bird population, believed to be the natural reservoir of influenza. Under normal circumstances an influenza infection in wild ducks is asymptomatic. The virus replicates in the intestinal tract and is excreted in high concentrations with the faeces for a period up to 30 days. An avian influenza virus can persist in water and retain inf ectivity for about 100 days at 17°C and can be stored indefinitely at -50⁰C. The continuous circulation of influenza A viruses might be due to bird overwintering sites in the subtropics. The 2004 H5N1 strains have become very stable and can survive for 6 days at 37°C. The virus is killed by heat at 56⁰C for 3 hours or 60⁰C for 30 minutes. Also disinfectants like formalin and iodine compounds efficiently kill the influenza virus. Avian influenza viruses have been believed to be in evolutionary stasis in its natural host, the virus and the host tolerate each other. Generally no severe clinical symptoms are seen when poultry are infected with avian influenza, and the virus is described as a low pathogenic avian influenza virus (LP AIV). The subtypes H5 and H7 have the potential to become highly pathogenic (HP) to chickens through accumulation of mutations after transmission to poultry. Contrary to previous belief, wild migratory birds might play some role in the transmission of HP AIV. Thousands of wild aquatic birds in Hong Kong 2002 and China 2005 became infected with HP AIV H5N1 and this contributed to the spread of HP H5N1 to Europe and Africa in 2005.

Seasonal influenza strains have been isolated from humans and swine all year round, but in temperate climates it is a winter disease probably because people come together and stay in less ventilated rooms due to the cold weather.

Of the 16 recognised subtypes of HA and 9 NAs only Hl, H2, H3, Nl and N2 have circulated in humans and swine in the last century. The pandemic introduction in humans of these types were 1918 HlNl, 1957 H2N2 ( " Asiatic flu " ) , 1968 H3N2 ( " HongKong Flu " ) and non-pandemic introduction of the reassorted new type H1N2 in 2001, respectively. The antigenicity of human influenza viruses are constantly changing by accumulation of mutations in the HA and NA antigenic sites, thereby making the virus capable of evading the host immune system causing epidemics. Viral mutagenesis is enhanced by the lack of "proof reading" in the replication of RNA. The mutation frequency is approximately one in 100,000 nucleotides. At the northern hemisphere seasonal influenza outbreaks usually occur between October and April and from April to October in the southern hemisphere. The antigenic drift of human influenza viruses are closely monitored by the World Health Organization's global influenza surveillance program. The components of the next seasons influenza vaccine for the northern hemisphere is determined in February based on the knowledge about the current circulating strains, and re-evaluated in September for the southern hemisphere.

Antigenic shift can occur in three ways. Either by direct transmission of an avian strain adapted to humans, genetic reassortment or reintroduction of an "old" strain. The possibility of an avian influenza virus crossing the species barrier and infecting humans directly was not recognised before

1997 when 18 people in Hong Kong became ill with HP AIV H5N1.

The origin of the 1918 pandemic is controversial. Taubenberger et at, (Characterization of the 1918 influenza virus polymerase genes. Nature, 2005, 437:889-893.) suggested based on phylogenetics of the polymerase genes that the virus was entirely of avian origin. However, there are large disagreements about the actual origin of the virus and many still believe that also this pandemic strain is a reassortant between a mammalian and avian virus most likely occurring from swine. If the virus was of avian origin it might imply that the HP avian viruses circulating currently could cause a new pandemic by direct transmission to humans. Antigenic reassortment occurs when viral segments from two antigenic different viruses infecting the same cell. The reassorted virus contains segments of both strains and if the newly introduced segment is HA (and NA) the complete antigenicity of the virus might change and the virus escapes the host immunity. These reassortants might be catastrophic if the virus is capable of efficient replication in the new host. In worst case such a reassorted strain might lead to pandemics, world-spanning infections to which we have no pre-existing immunity. The pandemics of 1957 and 1968 were reassortants that aquired the HA, NA and PBl and HA and

PBl genes from an aquatic source, respectively. In 1977 a strain identical to the HlNl strains that circulated before 1957 re-emerged. Swine are possible "mixing vessels" for reassorted viruses due to their receptor tropism for both α-(2,3) and α-(2,6) linkage to galactose. Other species like chicken and man might also serve as mixing vessels in the light of direct crossover to humans from an avian source after the discovery of α-(2,3) avian like receptor on cells also in humans and chickens.

The interpandemic evolution of influenza viruses has been thought to be caused by progressive antigenic drift due to the mutability of the RNA genome. H3N2 has been the predominant subtype circulating in humans since 1968 and has been in rapid drift as a single lineage while there has been slow replacement of antigenic variants of the HlNl viruses. It has been shown that the rate of accumulating mutations is approximately A- 5xlO^'3 substitutions per nucleotide per year for HAl others predict a rate of 5.7xlO^'3 substitutions per nucleotide per year. The HA and NA might evolve independently from each other and reassortments of the internal genes are also known. Positive selection has been inferred on codons involved at antibody antigenic sites, T-cell epitopes and sites important virus egg growth properties. Recent research on viruses has suggested that the evolution of influenza do not always follow a constant rate, but is characterised by stochastic processes, short intervals of rapid evolution, long intervals of neutral sequence evolution and slow extinction of coexisting virus lineages. The evolution seems also more influenced by reassortment events between co-circulating lineage and viral migration than previously believed.

Vaccination is the preferred choice for influenza prophylaxis. Inactivated influenza vaccines are licensed worldwide while cold-adapted live vaccines are licensed only in Russia and the USA. The preferred prophylaxis of annual influenza infections is vaccination with inactivated protein vaccines from virus propagated in hens' eggs. Thus, the common vaccines are the inactivated vaccine viruses which are propagated in hens' eggs and inactivated by formaldehyde or β-propiolactone. There are three classes of inactivated vaccines; whole, split (chemically disrupted with ether or tributyl phosphate) and subunit (purified surface glycoproteins) administrated intramuscularly or subcutaneously. Whole inactivated influenza vaccine is not currently used due to high levels of side effects. The seasonal influenza vaccine (split and subunit) is trivalent, comprising H3N2 and HlNl influenza A virus strains and an influenza B virus. The normal human vaccine dose is standardised to 15μg HA protein of each virus component administrated once in normal healthy adults and twice in children and other persons with no pre-existig influenza A immunity. The conventional vaccines induce merely a humoral immune response. The protective effect of the traditional protein split vaccine is very limited and because of the continuous evolution of influenza A virus strains and the typespecific antibodies induced ny the conventional vaccines a new vaccine has to be produced every year based on the most recent circulating influenza A strain. Several vaccine improvements are necessary in case of a new emerging human strain. Egg production is too slow (6-12 months) in the case of emerging strains. If this strain is also an AIV virus highly pathogenic (HP) for poultry, egg production might be impossible because the virus kills the egg embryo. Also the availability of eggs might be limited slowdown the vaccine production, hi the case of no pre-existing immunity in the population two vaccinations would be necessary, thereby further delaying the vaccine production. Even if there are no new pandemic influenza A among humans but only spread of a HPV ATV among poultry the shortage of eggs will limit production on eggs of traditional seasonal influenza vaccines. In addition, traditional influenza protein vaccines do not have optimal protection as prophylaxis and no therapeutic effect. Thus, there is a need for new alternative influenza vaccines.

Although DNA vaccines were developed more than 16 yeas ago, clinical trials preceding stage I and II in humans are rare. Two veterinary DNA vaccines however, have been licensed; one for West Nile Virus (in horse) and a second for Infectious Hematopoetic Necrosis virus in Salmon. This demonstrates that DNA vaccines can have good protective effects and that new DNA vaccines are not limited by the size of the animal or species. The great success with DNA vaccines observed for the murine model for first generation DNA vaccines did not translate well to humans, nonetheless; researchers have recently demonstrated protective antibodies levels by a single dose of gene gun administrated HA DNA vaccine to humans. "Nucleic acid immunization" or the commonly preferred name "DNA vaccines" are the inoculation of antigen-encoding DNA or RNA as expression cassettes or expression vectors which may instead be incorporated into viral vectors with the purpose of inducing immunity to the gene product. Thus, in our definition of DNA vaccines we include all kinds of delivery systems for the antigen encoding naked DNA or RNA but exclude viral vector-based delivery. The vaccine gene can be in form of circular plasmid or a linear expression cassette with just the key features necessary for expression (promotor, the vaccine gene and polyadenylation signal). Delivery systems may most often be naked DNA in buffer with or without adjuvant, DNA coupled to nanoparticles and/or formulated into adjuvant containing compounds or insered into live viral or bacterial vectors such as Adenovirus, adenoassociated virus, alphavirus, poxviruses, herpes virus etc.

WO2006063101 describes a pandemic avian influenza vaccine based on an adenovirus vehicle with HA DNA isolated from the avian H5N1 influenza virus isolated during the outbreak in 2003-2005. The vaccine was tested in animals challenged with the same H5N1 influenza virus strain.

DNA vaccines hold great promise since they evoke both humoral and cell-mediated immunity, without the same dangers associated with live virus vaccines. In contrast to live attenuated virus vaccines DNA vaccines may be delivered to same or different tissue or cells than the live virus that has to bind to specific receptors. The production of antigens in their native forms improves the presentation of the antigens to the host immune system. Unlike live attenuated vaccines, DNA vaccines are not infectious and can not revert to virulence. DNA vaccines expressing HA, NA₁ M, NP proteins or combinations of these have proven to induce immune responses comparable to that of a natural viral infection.

DNA vaccines offer many advantages over conventional vaccines. It can be produced in high amounts in short time, abolishing the need for propagation in eggs, it is cost-effective, reproducible and the final product does not require cold storage conditions, because DNA is stable and resistant to the extremes of temperature. All currently licensed inactivated vaccines are efficient at inducing humoral antibody responses but only live attenuated virus vaccines efficiently induce a cytotoxic cellular response as well.

DNA vaccines induce an immune response which is comparable to the response acquired by natural virus infection by activating both humoral and cell-mediated immunity (6, 30). The broad response to DNA vaccines is a result of the encoded genes being expressed by the transfected host cell, inducing both a ThI and Th2 immune responses. The production of antigens in their native form improves the presentation of the antigens to the host immune system. In contrast, the conventional inactivated influenza protein based vaccines only induce a humoral response (Th2), directed against the influenza surface glycoproteins. This type of response is ineffective against drifted virus variants and therefore the virus composition of the seasonal influenza vaccine has to be assessed every season. Antigenic cross-reactive responses are mainly induced by the more conserved influenza proteins like the nucleoprotein (NP) and the matrix (M) protein. By including these genes in a DNA vaccine higher cross reactivity between drifted and heterologous strains have been shown (4, 7, 8, 13).

Influenza infection and symptoms in ferrets are highly comparable to what is observed in humans and is therefore one of the best models for influenza vaccination trials (22). Influenza HA DNA vaccines in ferrets has also previously proved effective (18, 32).

It has previously been shown that 1918 HlNl whole inactivated virus vaccine induced partly protection against infection with 1918 HlNl in mice (28), also recently DNA vaccines encoding the HA from 1918 showed complete protection of mice against a 1918 HlNl challenge (16).

Influenza vaccines that have the ability to induce immune responses able to cross-react with drifted virus variants and even heterologous strains would be of great advantage for both annual vaccine development and in case of emerging new strains.

Summary of the invention

We demonstrate that gene gun administrated codon optimised DNA vaccine in plasmid encoding HA and NA with or without M and NP based on the HlNl pandemic virus from 1918 induce protection in ferrets against infection with a HlNl (A/New Caledonia/20/99(H1N1)) present day virus. The circulating HlNl strain in Europe in the 2006-2007 seasons is New Caledonia-like. The viruses are separated by a time interval of 89 years and differ by 21.2% in the HAl protein. By comparison a similar DNA vaccine encoding the HA and NA of (A/New Caledonia/20/99(H1N1) induced less protection. These results suggest not only a unique ability of the DNA vaccines but also a unique and unexpected feature of the 1918 HA and/or NA in inducing especially broad and efficient protective immunity against even extremely drifted strain variants.

The present invention discloses that an induced immune response with a DNA vaccine encoding HA and/or NA of the 1918 HlNl influenza A gives a high level of cross protection against present day influenza infection. Tests were carried out in ferrets vaccinated with this DNA vaccine synthezised using human preferred codons of the 1918 HlNl influenza and challenged with a contemporary HlNl virus.

The results surprisingly show that the 1918 HlNl DNA vaccines are as good as or better candidates for influenza prophylaxis than annual conventional protein based vaccines which frequently need to be updated to match the circulating influenza virus. DNA vaccination induces broader cross -reactivity against drifted strain and longer memory responses. It has been shown that a similar DNA vaccine may protect against the 1918 HlNl recombinant strain (16). However, our results then suggests that our synthetic DNA vaccine based on the 1918 HlNl sequences protects against extreme drifted variants represented by recent contemporary or seasonal circulating HlNl strains. Thus it is likely that the suggested 1918 HlNl DNA vaccine protects against HlNl strains circulating for up to 89 years and therefore likely also future HlNl variants. This is highly unexpected since traditional protein split vaccines only protects against the strain it is designed from and thus has to be produced from the actual circulating HlNl strains sometimes as frequent as every year. Thus, a DNA vaccine encoding the HA and NA of 1918 HlNl was not expected to protect against such a divergent strain as the present day HlNl, but it does.

Detailed disclosure of the invention

The present invention discloses the use of the naked DNA and/or RNA molecule encoding hemagglutinin (HA) from pandemic influenza, e.g. the 1918 HlNl and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus, as a vaccine component against present day and coming Hl, H2, H3 containing influenza A infections in humans and/or swine.

The naked DNA and/or RNA molecule encoding neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza virus can optionally be included as vaccine components against present day and coming Hl, H2, H3, Nl, N2 containing influenza A infections in humans and/or swine.

The components in the vaccine are the hemagglutinin and/or the neuraminidase and /or matrix protein (M) and/or the nucleoprotein (NP) naked DNA and/or RNA coding for said protein(s) from pandemic influenza strains preferably with a mixture from several pandemic strains.

In a preferred embodiment of the invention the DNA and/or RNA codons are "humanized" e.g. the DNA sequence for hemagglutinin and neuraminidase and Matrix and Nucleoprotein is changed so the sequence coding for said proteins is changed to be optimally expressed in mammalian cells.

The invention also discloses the vaccines against present day and coming human and swine influenza A infection comprising the above mentioned naked DNA and/or RNA coding hemagglutinin and/or neuraminidase and/or a matrix protein and/or the hemagglutinin protein from pandemic influenza, e.g. the 1918 HlNl and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus preferably with a mixture from various strains.

In another embodiment the vaccine comprises naked DNA and/or RNA coding HA and/or NA from the new circulating 2001 H5N7 low pathogenic (LP) Avian influenza virus (ATV) strain (A/Mallard/Denmark/64650/03(H5N7)). The vaccine is intended to protect birds and humans and swine against H5 containing influenza A strains.

In another embodiment the vaccine comprises naked DNA and/or RNA coding HA with and/or NA and/or M and/or NP from the newly introduced and circulating March 2006 Denmark H5N1 high pathogenic

Avian influenza A virus (AIV) strains

(A/buzzard/Denmark/6370/06(H5Nl)) and (A/duck/Denmark/53-147- 8/08(H7Nl) and (A/widegeon/Denmark/66174/G18/04(H2N3)). The vaccine is intended to broadly protect birds and humans and swine against any H5, H7 and/or H2 containing influenza A strains.

All above mentioned vaccines are optionally administered by saline or phosphate buffered saline (PBS) injection of naked DNA or RNA or inoculated by gene gun or injection or is delivered coupled to particles.

Above mentioned vaccines can be used both prophylactic and therapeutic. Definitions

Hemagglutinin:

The name haemagglutinin is derived from the viruses' ability to agglutinate red blood cells. The envelope glycoprotein HA is a rod-like shaped trimer of identical monomers. The HA protein is synthesised in the infected cell as a single polypeptide chain, HAO. This initial molecule has to be cleaved by the host cell proteases into disulfidelinked HAl (47 kDa) and HA2 (29 kDa) subunits in order for the virus to mediate membrane fusion and subsequent infection. The HAl subunit is the globular domain of the HA molecule which comprise the receptor binding site, responsible for virus attachment to sialic acid receptors on the host cell. The five antigenic sites A, B, C, D and E at the globular head direct the host antibody response. The HA is the primary viral antigen and the only antigen inducing a virus neutralising response in the host. The HA main functions are virion to host cell membrane fusion and fusion of the endocytosed virion with the endosomal membrane allowing release of the genome into the cytoplasm. HA is a prototype 1 integral membrane protein that is targeted to the ER membrane through an N-teπninal signal peptide sequence and cleaved by signal peptidase. The HA2 subunit forms the stem of the molecule. The N-terminus of HA2 (fusion peptide) is hydrophobic and is highly conserved in the HAs of different influenza virus strains, and it is essential in HA fusion activity. The HA is posttranslationally modified by addition of N-linked carbohydrates at asparagine residues (N) on each monomer and palmitic acid to cysteine

(C) residues in the cytoplasmic tail region. HA binds to 5-N-acetyl neuramic acid (sialic acid) on the host cell surface and positions and are essential in determining preferred host cell tropism. Human infectious strains preferentially bind to sialic acid with α-(2,6) linkage to galactose, while avian influenza viruses (AIV) preferentially bind to α-(2,3)

Neuraminidase :

The neuraminidase (NA) is a class II membrane envelope glycoprotein with enzymatic activity. It is a tetramer of identical monomers forming a mushroom-like shape. The hydrophobic stalk region is membrane anchored and the globular head contains the enzyme active site and the three antigenic sites A, B and C of the molecule. Main function is to catalyse the cleavage of glycosidic linkages adjacent to sialic acid. The activity is essential for the progeny virion for efficient release from the surface of the infected cell. Like HA, NA is posttranslational modified with N-linked glycosylations. The NA molecule is target for antiviral drugs like zanamivir and oseltamivir. Inhibition of NA prevents virus release from the infected cell and delays virus propagation. Currently nine subtypes of NA have been recognised.

Matrix proteins:

The matrix proteins consist of two proteins, the ion channel protein M2 and the structural protein Ml. The Ml protein is a matrix protein lining the interior side of the membrane derived from the infected host cell giving structure and rigidity to the membrane. The Ml protein contains a hydrophobic lipid binding domain and a RNP binding domain. Assembly of negative stranded RNA viruses requires localisation of Ml proteins to the plasma membrane. The Ml proteins bind to the cytoplasmic tails of

HA, NA and M2, especially NA stimulate the membrane binding by the Ml proteins. Ml together with NS2 is required for export of genomic RNPs from the nucleus, Ml also inhibits RNA synthesis. The M2 protein is a small homotetramer integral membrane protein, and ion channel, translated from a spliced mRNA in +1 reading frame. The ion channel is activated by the low pH of the endosome allowing protons to enter the interior of the virus leading to conformational changes in Ml, disrupting the Ml-RNP interactions. The M2 ion channel is target for antiviral drugs like amantadine and rimantadine.

Nucleoprotein

The Nucleoprotein (NP) is highly basic and binds the sugar-phosphate backbone of viral RNA in a non-sequence specific manner approximately every 25 nucleotides. NP interacts with both PBl and PB2 and with a variety of other viral and cellular proteins. The interaction with Ml controls the transcriptional activity of RNPs and their intracellular trafficking. NP is mainly responsible for maintaining the structure of RNPs and in regulation of genome transcription and replication, the polymerase can not use naked viral RNA as template. NP associated with viral RNA is abounant in extracellular fluid and lung tissue during seveir influenza A infection.

The 1918 influenza virus:

The most severe pandemic this century has been the 1918 HlNl "Spanish flu" . The virus killed between 40 and 50 million people worldwide during 1918 and 1919 10. Based on preserved specimens all genes have been genetically characterised and the entire virus has now been restored 27. This gives a unique opportunity to elucidate the mechanisms of immunopathogenesis of the pandemic strain.

The pandemic strains of 1957 (H2N2) and 1968 (H3N2) were both a result of genetic reassortment with avian viruses 11,17. The origin of the 1918 pandemic is debated. Taubenberger et at, 26 suggested based on phylogenetic analysis of the polymerase genes that the virus was entirely of avian origin. However, there are large disagreements about the actual origin of the virus and many still believe that this pandemic strain also was a reassortant between a mammalian and avian virus 1,26. The haemagglutinin (HA) and neuraminidase (NA) genes of the 1918 HlNl strain did not possess known genetic indicators for high virulence that could have explained the severeness observed in humans 19,20.

However, the HA (and NA) protein on a backbone of recent human viruses conferred enhanced pathogenicity in mice 12,29. It might have been the combination of genes more than the HA itself that caused the lethal phenotype 27. The uncertainty about the origin and the mechanisms of high virulence of the 1918 HlNl virus has raised questions if it is possible to develop protective immunity to this virus. Recently it has been published that a DNA vaccine encoding the HA of the 1918 HlNl strain showed protection to a lethal challenge of the recombinant 1918 HlNl virus strain in mice (Kong W, Hood C, Yang Z, Wei C, Xu L, Garcia-Sastre A, Tumpey TM, Nabel GJ. Protective immunity to lethal challenge of the 1918 pandemic influenza virus by vaccination. PNAS 103(43):15987-91,2006)

DNA vaccines: DNA vaccines are here defined as naked DNA or RNA, DNA or RNA in solution for direct intramuscular or subcutaneous injection with or without electroporation or coupled to particles, e.g. gold beads for gene gun administration. The DNA can be linear containing only a promoter, the influenza genes and polyadenylation signal or this expression cassette in an expression plasmid..

The administration of DNA vaccine can be by saline or buffered saline injection of naked DNA or RNA, or injection of DNA plasmid or linear gene expressing DNA fragments coupled to particles, or inoculated by gene gun.

The two most common types of DNA vaccine administration are saline injection of naked DNA and gene gun DNA inoculations (DNA coated on solid gold beads administrated with helium pressure). A saline intra muscular injection of DNA preferentially generates a ThI IgG 2a response while gene gun delivery tends to initiate a more Th2 IgGl response. Intramuscular injected plasmids are at risk of being degraded by extracellular deoxyribonucleases, however, the responses induced are often more long-lived than those induced by the gene gun method. Vaccination by gene gun delivery of DNA₁ to the epidermis, has proven to be the most effective method of immunization, probably because the skin contains all the necessary cells types, including professional antigen presenting cells (APC), for eliciting both humoral and cytotoxic cellular immune responses (Langerhans and dendritic cells). Complete protection from a lethal dose of influenza virus has been obtained with as little as 1 μg DNA in mice. The standard DNA vaccine consist of a vector with the gene of interest cloned into a bacterial plasmid engineered for optimal expression in eukaryotic cells. Essential features include; an origin of replication allowing for production in bacteria, a bacterial antibiotic resistance gene allowing for plasmid selection in bacterial culture, a strong constitutive promotor for optimal expression in mammalian cells (promoters derived from cytomegalovirus (CMV) or simian virus provide the highest gene expression), a polyadenylation sequence to stabilise the mRNA transcripts, such as bovine growth hormone (BHG) or simian virus polyadenylation, and a multiple cloning site for insertion of an antigen gene. An intron A sequence improves expression of genes remarkably. Many bacterial DNA vaccine vectors contain unmethylated cytidinephosphate- guanosine (CpG) dinucleotide motifs that may elicit strong innate immune responses in the host. In recent years there have been several approaches to enhance and customise the immune response to DNA vaccine constructs (2nd generation DNA vaccines). For instance dicistronic vectors or multiple gene expressing plasmids have been used to express two genes simultaneously. Specific promoters have been engineered that restrict gene expression to certain tissues, and cytokine/antigen fusion genes have been constructed to enhance the immune response. Furthermore, genes may be codon optimised for optimal gene expression in the host and naϊve leader sequences may be substituted with optimised leaders increasing translation efficiency.

Viral DNA vaccines:

DNA can be delivered by a viral vector such as Adenovirus, Modified vaccinia virus Ankara (MVA), Vaccinia, Adenoassociated virus (AAV), Alphavirus etc. Viral DNA vaccines are not a part of the present study and are not encompassed by this invention.

Codon optimization:

Codon optimization is the complete exchange of the virus codons to those of highly expressed human genes and therefore also mammalian genes that include swine. Codon optimization do not change the encoded amino acids of the protein antigens encoded but may increase the eukaryotic protein expression in mammalian cells. Since genes of highly expressed human proteins has a high conteint of C and G there are an increased possibility of generating both immune stimulatory GpG motofs but also immune inhibitory GC sequences. Genes engineered using codon optimization are called "humanized" genes and are frequently used in DNA vaccines to enhance expression.

The DNA or RNA sequence for hemagglutinin and neuraminidase and Matrix and Nucleoprotein is changed so the sequence coding for said proteins is changed to be optimally expressed in humans.

The invention is the use of the 1918 HA and/or NA codon-optimized genes in a DNA vaccine against all seasonal circulating HlNl influenza A strains including the A/New Caledonia/20/99(H1N1) like virus.

Tabel 1: nucleotide and amino acid sequences of the codon optimized genes and the proteins they express.

Nucleotide ΛTOGAGGCCAGGCTGCTGGTGCTGCTGTGCGCCTTCGCCGCCACCAACGCCGACACCATCTGCATCGGCTACC

CTGCTGGAGGACAGCCACAACGGCAAGCTGTGCAAGCTGAAGGGAATCGCTCCCCTGCAGCTGGGCAAGTGC

AACATCGCCGGCTGGCTGCTGGGCAACCCCGAGTGCGACCTGCTGCTGACCGCCAGCAGCTGGTCCTACATC

GTGGAGACCAGCAACAGCGAGAACGGCACCTGCTACCCCGGCGACTTCATCGACTACGAGGAGCTGCGGGA

GCAGCTGTCCAGCGTGAGCAGCTTCGAGAAGTTCGAGATCTTCCCCAAGACCAGCTCCTGGCCCAACCACGA

GACCACCAAGGGCGTGACCGCCGCCTGTAGCTACGCCGGAGCCAGCAGCTTCTACAGAAACCTGCTGTGGCT

GACCAAGAAGGGCAGCAGCTACCCCAAGCTGTCCAAGAGCTACGTGAACAACAAGGGCAAGGAAGTGCTGG

TGCTGTGGGGCGTGCACCACCCCCCTACCGGCACCGACCAGCAGAGCCTGTACCAGAACGCCGACGCCTACG

TGAGCGTGGGCAGCAGCAAGTACAACAGAAGGTTCACCCCCGAGATCGCCGCCAGGCCCAAGGTGCGCGAC

CAGGCCGGCAGGATGAACTACTACTGGACCCTGCTGGAGCCCGGCGACACCATCACCTTCGAGGCCACCGGC

AACCTGATCGCCCCTTGGTACGCCTTCGCCCTGAACAGGGGCAGCGGCAGCGGCATCATCACCAGCGACGCC

CCCGTGCACGACTGCAACACCAAGTGCCAGACCCCCCACGGAGCCATCAACAGCAGCCTGCCCTTCCAGAAC

ATCCACCCCGTGACCATCGGCGAGTGCCCCAAGTACGTGAGAAGCACCAAGCTGAGGATGGCCACCGGCCTG

AGGAACATCCCCAGCATCCAGAGCAGGGGCCTGTTCGGAGCCATCGCCGGATTCATCGAGGGCGGCTGGACC

GGCATGATCGACGGCTGGTACGGCTACCACCACCAGAACGAGCAGGGCAGCGGCTACGCCGCCGACCAGAA

GAGCACCCAGAACGCCATCGACGGCATCACCAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGT

TCACCGCCGTGGGCAAGGAGTTCAACAACCTGGAGAGGCGGATCGAGAACCTGAACAAGAAGGTGGACGAC

GGCTTCCTGGACATCTGGACCTACAACGCCGAGCTGCTGGTGCTGCTGGAGAACGAGAGGACCCTGGACTTC

CACGACAGCAACGTGAGGAACCTGTACGAGAAGGTGAAGAGCCAGCTGAAGAACAACGCCAAGGAGATCGG

CAACGGCTGCTTCGAGTTCTACCACAAGTGCGACGACGCCTGCATGGAGAGCGTGAGAAACGGCACCTACGA

CTACCCCAAGTACAGCGAGGAGAGCAAGCTGAACCGGGAGGAGATCGACGGCGTGAAGCTGGAGAGCATGG

GCGTGTACCAGATCCTGGCCATCTACAGCACCGTGGCCAGCAGCCTGGTGCTGCTGGTGTCCCTGGGAGCCAT

CAGCTTTTGGATGTGCAGCAACGGCAGCCTGCAGTGCAGGATCTGCATCTGA Amino acid

MEARLLVLLCAFAATNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCKLKGIAPLQLGKCNIA GWLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFIDYEELREQLSSVSSFEKFEIFPKTSSWPNHETTKGVTAA CSYAGASSFYRNLLWLTKKGSSYPKLSKS YVNNKGKE VLVLWGVHHPPTGTDQQSLYQNADAYVSVGSSKYNR RFTPEIAARPKVRDQAGRMNYYWTLLEPGDTITFEATGNLIAPWYAFALNRGSGSGIITSDAPVHDCNTKCQTPHG AINSSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGY AADQKSTQNAIDGΓΓNKVNSVIEKMNTQFTAVGKEFNNLERRIENLNKKVDDGFLDIWTYN AELLVLLENERTLD FHDSMΑMLYEKVKSQLKNNAKEIGNGCFEFYHKCDDACMESVRNGTYDYPKYSEESKLNREEIDGVKLESMGV YQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI.

Nucleotide

ATGAACCCCAACCAGAAGATCATCACCATCGGCAGCATCTGCATGGTGGTGGGCATCATCAGCCTGATCCTG

CAGATCGGCAACATCATCAGCATCTGGGTGTCCCACAGCATCCAGACCGGCAACCAGAACCACCCCGAGACC

TGCAACCAGTCCATCATCACCTACGAGAACAACACCTGGGTGAACCAGACCTACGTGAACATCAGCAACACC

AACGTGGTGGCCGGCCAGGACGCCACCTCCGTGATCCTGACAGGCAACAGCAGCCTGTGCCCCATCAGCGGC

TGGGCCATCTACAGCAAGGACAACGGCATCAGGATCGGCAGCAAGGGCGACGTGTTCGTGATCAGAGAGCC

CTTCATCAGCTGCAGCCACCTGGAATGCAGGACCTTCTTCCTGACCCAAGGAGCCCTGCTGAACGACAAGCA

CAGCAACGGCACCGTGAAGGACAGAAGCCCCTACAGGACCCTGATGAGCTGCCCCGTGGGCGAGGCTCCCA

GCCCCTACAACAGCAGATTCGAGAGCGTGGCCTGGTCCGCCAGCGCCTGCCACGACGGCATGGGCTGGCTGA

CCATCGGCATCAGCGGCCCTGACAACGGGGCCGTGGCCGTGCTGAAGTACAACGGAATCATCACCGACACCA

TCAAGAGCTGGCGGAACAACATCCTGAGGACCCAGGAAAGCGAGTGCGCCTGCGTGAACGGCAGCTGCTTCA

CCATCATGACCGACGGCCCCAGCAACGGCCAGGCCAGCTACAAGATCCTGAAGATCGAGAAGGGCAAGGTG

ACCAAGAGCATCGAGCTGAACGCCCCCAACTACCACTACGAGGAATGCAGCTGCTACCCCGACACCGGCAAG

GTCATGTGCGTGTGCAGGGACAACTGGCACGGCAGCAACAGGCCCTGGGTGTCCTTCGACCAGAACCTGGAC

TACCAGATCGGATACATCTGCAGCGGCGTGTTCGGCGACAACCCCAGGCCCAACGACGGCACCGGCAGCTGC

GGCCCTGTGAGCAGCAACGGGGCCAATGGCATCAAGGGCTTCAGCTTCAGATACGACAACGGCGTGTGGATC

GGCCGCACCAAGAGCACCAGCAGCAGATCCGGCTTCGAGATGATCTGGGACCCCAACGGCTGGACCGAGAC

CGACAGCAGCTTCAGCGTGAGGCAGGACATCGTGGCCATCACCGACTGGTCCGGCTACAGCGGCAGCTTCGT

GCAGCACCCCGAGCTGACCGGCCTGGACTGCATGAGGCCCTGTTTCTGGGTGGAGCTGATCAGAGGCCAGCC

CAAGGAGAACACCATCTGGACCAGCGGCAGCAGCATCAGCTTTTGCGGCGTGAACAGCGACACCGTGGGCTG

GTCCTGGCCCGACGGGGCCGAGCTGCCCTTCAGCATCGATAAGTGA

Amino acid

MNPNQKIITIGSICMVVGIISLILQIGNIISIWVSHSIQTGNQNHPETC^QSIITYENNTWVNQTYλT^ISNTNVVAGQD

ATSVILTGNSSLCPISGWAIYSKDNGIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKHSNGTVKDRSPYRTL

MSCPVGEAPSPYNSRFESVAWSASACHDGMGWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACV

NGSCFTIMTDGPSNGQASYKILKIEKGKVTKSIELNAPNYHYEECSCYPDTGKVMCVCRDNWHGSNRPWVSFDQN

LDYQIGYICSGVFGDNPRPNDGTGSCGPVSSNGANGIKGFSFRYDNGVWIGRTKSTSSRSGFEMIWDPNGWTETDS

SFSVRQDrVAITDWSGYSGSFVQHPELTGLDCMRPCFWVELIRGQPKENTrWTSGSSISFCGVNSDTVGWSWPDGA

ELPFSIDK. Nucleotide

ATGGCCAGCCAGGGCACCAAGAGAAGCTACGAGCAGATGGAAACCGACGGCGAGAGGCAGAACGCCACCG

AGATCAGGGCCAGCGTGGGCAGGATGATCGGCGGCATCGGCAGGTTCTACATCCAGATGTGCACCGAGCTGA

AGCTGTCCGACTACGAGGGCAGGCTGATCCAGAACAGCATCACCATCGAGAGGATGGTGCTGTCCGCCTTCG

ACGAGAGAAGAAACAAGTACCTGGAAGAGCACCCCAGCGCCGGCAAGGACCCCAAGAAAACCGGCGGACCC

ATCTACAGAAGGATCGACGGCAAGTGGATGAGAGAGCTGATCCTGTACGACAAGGAGGAAATCAGAAGGAT

CTGGCGGCAGGCCAACAACGGCGAGGACGCCACAGCCGGCCTGACCCACATGATGATCTGGCACAGCAACCT

GAACGACGCCACCTACCAGAGGACCAGGGCCCTCGTCAGAACCGGCATGGACCCCCGGATGTGCAGCCTGAT

GCAGGGCAGCACACTGCCCAGAAGAAGCGGAGCTGCTGGAGCCGCCGTGAAGGGCGTGGGCACCATGGTGA

TGGAACTGATCAGGATGATCAAGAGGGGCATCAACGACAGGAACTTTTGGAGGGGCGAGAACGGCAGAAGG

ACCAGGATCGCCTACGAGAGGATGTGCAACATCCTGAAGGGCAAGTTCCAGACAGCCGCCCAGAGGGCCAT

GATGGACCAGGTCCGGGAGAGCAGGAACCCCGGCAACGCCGAGATCGAGGACCTGATCTTCCTGGCCAGAA

GCGCCCTGATCCTGAGGGGCAGCGTGGCCCACAAGAGCTGCCTGCCCGCCTGCGTGTACGGACCCGCCGTGG

CCAGCGGCTACGACTTCGAGAGAGAGGGCTACAGCCTGGTCGGCATCGACCCCTTCAGGCTGCTGCAGAACT

CCCAGGTGTACTCTCTGATCAGGCCCAACGAGAACCCCGCCCACAAGTCCCAGCTGGTCTGGATGGCCTGCC

ACAGCGCCGCCTTCGAGGATCTGAGAGTGAGCAGCTTCATCAGGGGCACCAGAGTGGTGCCCAGGGGCAAGC

TGTCCACCAGGGGCGTGCAGATCGCCAGCAACGAGAACATGGAAACCATGGACAGCAGCACCCTGGAACTG

AGAAGCAGGTACTGGGCCATCAGGACCAGAAGCGGCGGCAACACCAACCAGCAGAGGGCCAGCGCCGGACA

GATCAGCGTGCAGCCCACCTTCTCCGTGCAGAGGAACCTGCCCTTCGAGAGGGCCACCATCATGGCCGCCTTC

ACCGGCAACACCGAGGGCAGGACCAGCGACATGAGGACCGAGATCATCAGAATGATGGAAAGCGCCAGGCC

CGAGGACGTGAGCTTCCAGGGCAGGGGCGTGTTCGAGCTGTCCGATGAGAAGGCCACCTCCCCCATCGTGCC

CAGCTTCGACATGAGCAACGAGGGCAGCTACTTCTTCGGCGACAACGCCGAGGAATACGACAACTGA

Amino acid

MASQGTKRSYEQMETDGERQNATEIRASVGRMIGGIGRFYIQMCTELKLSDYEGRLIQNSITIERMVLS AFDERRN

KYLEEHPSAGKDPKKTGGPIYRRIDGKWMRELILYDKEEIRRIWRQANNGEDATAGLTHMMIWHSNLNDATYQR

TRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAXTCGVGTMVMELIRMIKRGINDRNFWRGENGRRTRIAYERMC

NILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARS ALILRGSVAHKSCLPACVYGPAVASGYDFEREGYSLV

GIDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGTRWPRGKLSTRGVQIASNENMETMD

SSTLELRSRYWAIRTRSGGNTNQQRASAGQISVQPTFSVQRNLPFERATIMAAFTGNTEGRTSDMRTEIIRMMESAR

PEDVSFQGRGVFELSDEKATSPIVPSFDMSNEGSYFFGDNAEEYDN.

Nucleotide

ATGAGTCTTTTAACCGAGGTCGAAACGTACGTTCTCTCTATCGTCCCGTCAGGCCCCCTCAAAGCCGAGATCG

CGCAGAGACTTGAAGATGTCTTTGCAGGGAAGAACACCGATCTTGAGGCTCTCATGGAATGGCTAAAGACAA

GACCAATCCTGTCACCTCTGACTAAGGGGATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGTGAGCGAGG

ACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAACGGGGATCCAAATAACATGGACAGAGCAGT

TAAACTGTACAGGAAGCTTAAGAGGGAGATAACATTCCATGGGGCCAAAGAAGTAGCACTCAGTTATTCCGC

TGGTGCACTTGCCAGTTGTATGGGCCTCATATACAACAGGATGGGGACTGTGACCACTGAAGTGGCATTTGGC

CTGGTATGCGC AACCTGTGAACAGATTGCTGATTCCCAGCATCGGTCTCACAGGC AAATGGTGACAACAACC

AATCCACTAATCAGACATGAGAACAGAATGGTACTGGCCAGCACTACGGCTAAGGCTATGGAGCAAATGGCT

GGATCGAGTGAGCAAGCAGCAGAGGCCATGGAGGTTGCTAGTCAGGCTAGGCAAATGGTGCAGGCGATGAG

AACCATTGGGACTCATCCTAGCTCCAGTGCTGGTCTGAAAGACGATCTTATTGAAAATTTGCAGGCCTACCAG

AAACGAATGGGGGTGCAGATGCAACGATTCAAGTGATCCTCTCGTTATTGCCGCAAGTATCATTGGGATCTTG

CACTTGATATTGTGGATTCTTGATCGTCTTTTTTTCAAATGCATTTATCGTCGCCTTAAATACGGTTTGAAAAG AGGGCCTTCTACGGAAGGAGTGCCGGAGTCTATGAGGGAAGAATATCGAAAGGAACAGCAGAGTGCTGTGG ATGTTGACGATGGTCATTTTGTCAACATAGAGCTGGAGTAAGGCGCC

Amino acid

Ml protein

MSLLTEVETYVLSΓVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRR RFVQNALNGNGDPNNMDRAXTΑ.YRKLKRERTFHGAKEVALSYSAGALASCMGLIYNRMGTVTTEVAFGLVCAT CEQIADSQHRSHRQMVTTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEVASQARQMVQAMRTIGTHP SSSAGLKDDLIENLQAYQKRMGVQMQRFK.

M2 protein

MSLLTEVETPTRNEWGCRCNDSSDPLVIAASIIGILHLILWILDRLFFKCIYRRLKYGLKRGPSTEGVPESMREEYRK EQQSAVDVDDGHFVNIELE.

The 1918 HA and NA amino sequences are public available (GenBank A/south Carolina/1/18 AF117241, A/Brevig Mission/1/18 AF250356) and can be translated into DNA using standard optimal codons for eukaryotic mammalian expression using standard expression vectors (key features: CMV promoter, intron A, Kozac sequence, vaccine gene inclusive its secretion sequence, stop codon, PolyAdenylation) A kanamycin resistance gene are included for growing and selection of transfected E.coli for plasmid DNA production.

DNA vaccination with the 1918 HlNl HA and NA synthetic codon optimized genes using gene gun standard conditions induces protective immunity to present day circulating influenza A virus as exemplified using A/New Caledonia/20/99(H1N1) virus challenge in DNA vaccinated ferrets (Mustela Putorius Furo). This is highly surprising since the two virus are separated by more than 80 years of antigenic drift and show about 21% difference in the HAl protein. Normally a protective protein vaccine must be based upon the amino acid sequence of the circulating seasonal influenza A strain to induce protection. Moreover the protection by the 1918 DNA vaccine against 2007 circulating strain is more consistent than the traditional protein vaccine based on the homologous circulating strain (New Caledonia). This suggest that the 1918 based DNA vaccine induces a much broader protective immunity that protects against influenza A HlNl strains from 1918 to present time and perhaps beyond. The unusual broad protection may be due to a unique amino acid sequence in the 1918 HA and/or NA proteins inducing broader protective antibodies to special epitopes or cellular immunity or immune adjuvans effect, or a particular gene expression or particular immune induction by the optimized nucleotide sequence of the particular 1918 HlNl genes, or some or all of these factors in combination.

The advantages are that a limited number of vaccine components delivered as a DNA vaccine either as naked DNA or RNA as plasmid or linear encoding sequences or incorporated into recombinant virus for more efficient delivery

The discovery of a broad protection induced by the pandemic influenza A strain 1918 HlNl may suggest that a similar good protection may be obtained against circulating H2 strains using DNA vaccines based on HA and/or NA from the 1958 H2N2 pandemic strain and against circulating H3 strains using DNA vaccines based in HA and/or NA from the 1968 pandemic strain.

The unusual broad and/or efficient protection obtained using a pandemic influenza A strain instead of the present day circulating strains may me due to special features in the sequence of the first new pathogenic and spreading virus. These features may gradually vain by accumulation of sequence changes during years of adaptation to the human and swine population.

If the protective feature is contained in the encoded amino acid sequence of the HA and/or NA 1918 and not the nucleotide sequence then the HA and/or NA protein(s) from 1918 may be used alone as an alternative to DNA or in combination with the DNA vaccine for immunization or vaccinations.

The use of the DNA vaccine components may serve as an adjuvans for the protein components and thus the protein and the DNA can be preferentially administered together as a mixed vaccine. As a more universal DNA and/or protein vaccine against contemporary influenza in humans and/or swine a mixture may be used of HA and NA from the 1918 HlNl pandemic strain plus HA and/or NA from the 1957 H2N2 pandemic strain plus HA from the 1968 H3N2 pandemic strain, where the N2 component is similar to the NA of the preferred earlier 1957 H2N2 strain..

Tabel 2: nucleotide and amino acid sequences of the codon optimized genes and the proteins they express (not codon optimized).

Nucleotide

ATAATTCTATTAATCATGAAGACCATCATTGCTTTGAGCTACATTTTCTGTCTGGCTCTCGGCCAAGACCTTCCA

GGAAATGACAACAGCACAGCAACGCTGTGCCTGGGACATCATGCGGTGCCAAACGGAACACTAGTGAAAACAATC

ACAGATGATCAGATTGAAGTGACTAATGCTACTGAGCTAGTTCAGAGCTCCTCAACGGGGAAAATATGCAACAAT

CCTCATCGAATCCTTGATGG AATAGACTGCACACrGATAGATGCTCTATTGGGGGACCCTCATTGTGATGTTTTT

CAAAATGAGACATGGGACCTTTTCGTTGAACGCAGCAAAGCTTTCAGCAACTGTTACCCTTATGATGTGCCAGAT

TATGCCTCCCTTAGGTCACTAGTTGCCTCGTCAGGCACTCTGGAGTTTATCACTGAGGGTTTCACTTGGACTGGG

GTCACTCAGAATGGGGGAAGCAATGCTTGCAAAAGGGGACCTC^TAGCGGTTTTTTCAGTAGACTGAACTGGTTG

ACCAAATCAGGAAGCACATATCCAGTGCTGAACGTGACTATGCCAAACAATGACAATTTTGACAAACTATACATT

TCTACCAGGAGAAGCCAGCAAACTATAATCCCGAATATCGAGTCCAGACCCTGGGTAAGGGGTCTGTCTAGTAGA

ATAAGCATCTATTGGACAATAGTTAAGCCGGGAGACGTACTGGTAATTAATAGTAATGGGAACCTAATCGCTCCT

CGGGGTTATTTCAAAATGCGCACTGGG AAAAGCTCAATAATGAGGTCAGATGCACCTATTGATACCTGTATTTCT

GAATGCATCACTCCAAATGGAAGCATTCCCAATGACAAGCCCTTTCAAAACGTAAACAAGATCACATATGGAGCA

TGCCCCAAGTATGTTAAGCAAAACACCCTGAAGTTGGCAACAGGGATGCGGAATGTACCAGAGAAACAAACTAGA

GGCCTATTCGGCGCAATAGCAGGTTTCATAGAAAATGGTTGGGAGGGAATGATAGACGGTTGGTACGGTTTCAGG

AGAATTCAGGACCTCGAGAAATACGTTGAAGACACTAAAATAGATCTCTGGTCTTACAATGCGGAGCTTCTTGTC

CTGAGGGAAAATGCTGAAGACATGGGCAATGGTTGCTTCAAAATATACCACAAATGTGACAACGCTTGCATAGAG

TCAATCAGAAATGGGACTTATGACCATGATGTATACAGAGACGAAGCATTAAACAACCGGTTTCAGATCAAAGGT

GTTGAACTGAAGTCTGGATACAAAGACTGGATCCTGTGGATTTCCTITCCCATATCATGCTTTTTGCTTTGTGTT

GTTTTGCTGGGGTTCATCATGTGGGCCTGCCAGAGAGGCAACATTAGGTGCAACATTTGCATTTGAGTGTATTAG

TAATTA

Amino acid

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKT

ΓΓDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDL

FVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPG

SGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRV

TVSTRRSQQTIIPNIESRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKM RTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLAT

GMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQ

INGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQ

HTIDLTDSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYR

DEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCWLLGFIMWACQRGNIRCNICI

Nucleotide

GAAAATGAATCCAAATCAAAAGATAATAACAATTGGCTCTGTCTCTCTCACCATTGCAACAGTATGCTTCCTCAT

GCAGATTGCCATCCTGGTAACTACTGTAACATTGCATTTTAAGCAATATGAGTGCGACTCCCCCGCGAGCAACCA

AGTAATGCCGTGTGAACCAATAATAATAGAAAGGAACATAACAGAGATAGTGTATTTGAATAACACCACCATAGA

GAAAGAGATATGCCCCAAAGTAGTGGAATACAGAAATTGGTCAAAGCCGCAATGTCAAATTACAGGATTTGCACC

TTTTTCTAAGGACAATTCAATCCGGCTTTCTGCTGGTGGGGACATTTGGGTGACGAGAGAACCTTATGTGTCATG

CGATCATGGCAAGTGTTATCAATTTGCACTCGGGCAGGGGACCACACTAGACAACAAACATTCAAATGACACAAT

ACATGATAGAATCCCTCATCGAACCCTATTAATGAATGAGTTGGGTGTTCCATTTCATTTAGGAACCAGGCAAGT

GTGTATAGCATGGTCCAGCTCAAGTTGTCACGATGGAAAAGCATGGCTGCATGTTTGTATCACTGGGGATGACAA

AAATGCAACTGCTAGCTTCATTTATGACGGGAGGCTTGTGGACAGTATTGGTTCATGGTCTCAAAATATCCTCAG

AGCCGATACTAGAATACTATTCATTGAAGAGGGGAAAATTGTCCATATTAGCCCATTGTCAGGAAGTGCTCAGCA TGTAGAAGAGTGTTCCTGTTATCCTAGATATCCTGGCGTCAGATGTATCTGCAGAGACAACTGGAAAGGCTCTAA TAGGCCCGTCGTAGACATAAATATGGAAGATTATAGCATTGATTCCAGTTATGTGTGCTCAGGGCTTGTTGGCGA

GAAAGGCTGGGCCTTTGACAATGGAGATGACGTGTGGATGGGAAGAACGATCAGCAAGGATTTACGCTCAGGTTA

TGAAACTTTCAAAGTCATTGGTGGTTGGTCCACACCTAATTCCAAATCGCAGATCAATAGACAAGTCATAGTTGA

CAGCGATAATCGGTCAGGTTACTCTGGTATTΓTCTCTGTTGAGGGCAAAAGCTGCATCAATAGGTGCTTTTATGT

GGAGTTGATAAGGGGAAGGAAACAGGAGACTAGAGTGTGGTGGACCTCAAACAGTATTGTTGTGTTTTGTGGCAC

TTCAGGTACCTATGGAACAGGCTCATGGCCTGATGGGGCGAACATCAATTTCATGCCTATATAAGCTTTCGCAAT

TTTAGA

Amino acid

MNPNQKIITIGSVSLTIATVCFLMQIAILVTTVTLHFKQYECDS

PASNQVMPCEPIΠERNITEIVYLNNTTIEKEICPKWEYRNWSKPQCQITGFAPFSK

DNSIRLSAGGDIWVTREPYVSCDHGKCYQFALGQGTTLDNKHSNDTIHDRIPHRTLLM

NELGVPFHLGTRQVCIAWSSSSCHDGKAWLHVCITGDDKNATASFIYDGRLVDSIGSW

SQNILRTQESECVCINGTCTWMTDGSASGRADTRILFIEEGKIVHISPLSGSAQHVE

ECSCYPRYPG VRCICRDNWKGSNRPWDINMEDYSIDSSYVCSGLVGDTPRNDDRSSN

SNCRNPNNERGNQG VKGWAFDNGDDVWMGRTISKDLRSGYETFKVIGGWSTPNSKSQI

NRQVΓVDSDNRSGYSGIFSVEGKSCINRCFYVELIRGRKQETRVWWTSNSΓWFCGTS GTYGTGSWPDGANINFMPI

Nucleotide

ATAGACAACCAAAAGCAAAACAATGGCCATCATTTATCTCATTCTCCTGTTCACAGCAGTGAGAGGGGACCAGAT ATGCATTGGATACCATGCCAATAATTCCACAGAGAAGGTCGACACAATTCTAGAGCGGAACGTCACTGTGACTCA TGCCAAGGACATTCTTGAGAAGACCCATAACGG AAAGTTATGC AAACTAAACGGAATCCCTCCACTTGAACTAGG GGACTGTAGCATTGCCGGATGGCTCCTTGGAAATCCAGAATGTGATAGGCTTCTAAGTGTGCCAGAATGGTCCTA TATAATGGAGAAAGAAAACCCGAGAGACGGTTTGTGTTATCCAGGCAGCTTCAATGATTATGAAGAATTGAAACA

TCTCCTCAGCAGCGTGAAACATTTCGAGAAAGTAAAGATTCTGCCCAAAGATAGATGGACACAGCATACAACAAC

TGGAGGTTCACGGGCCTGCGCGGTGTCTGGTAATCCATCATTCTTCAGGAACATGATCTGGCTGACAAAGAAAGG

ATCAAATTATCCGGTTGCCAAAGGATCGTACAACAATACAAGCGGAGAACAAATGCTAATAATTTGGGGGGTGCA

CCATCCCAATGATGAGACAGAACAAAGAACATTGTACCAGAATGTGGGAACCTATGTTTCCGTAGGCACATCAAC

TTGGACCCTATTGGATATGTGGGACACCATAAATTTTGAGAGTACTGGTAATCTAATTGCACCAGAGTATGGATT CAAAATATCGAAAAGAGGTAGTTCAGGGATCATGAAAACAGAAGGAACACTTGGGAACTGTGAGACCAAATGCCA AACTCCTTTGGGAGCAATAAATACAACATTGCCTTTTCACAATGTCCACCCACTGACAATAGGTGAGTGCCCCAA ATATGTAAAATCGGAGAAGTTGGTCTTAGCAACAGGACTAAGGAATGTTCCCCAGATTG AATCAAGAGGATTGTT TGGGGCAATAGCTC^JTTTTATAGAAGGAGGATGGCAAGGAATGGTTGATGGTTGGTATGGATACCATCACAGCAA

TTCTGTGATTGAAAAGATGAACACCCAATTTGAAGCTGTTGGGAAAGAATTCAGTAACTTAGAGAGAAGACTGGA GAACTTGAACAAAAAGATGGAAGACGGGTTTCTAGATGTGTGGACATACAATGCTGAGCTTCTAGTTCTGATGGA AAATGAGAGGACACTTGACTTTCATGATTCTAATGTCAAGAATCTGTATGATAAAGTCAGAATGCAGCTGAGAGA CAACGTCAAAGAACTAGGAAATGGATGTTTTGAATTTTATCACAAATGTGATGATGAATGCATGAATAGTGTGAA

GAGCAC^ATGCKXKJTTTATCAAATCCTTGCCATTTATGCTACAGTAGCAGGTTCTCTGTCACTGGC AATCATGAT GGCTGGGATCTCTTTCTGGATGTGCTCCAACGGGTCTCTGCAGTGCAGGATCTGCATATGATTATAAGTCATTTT ATAATTAA

Amino acid

MAIIYLILLFTAVRGDQICIGYHANNSTEKVDTILERNVTVTHA KDILEKTHNGKLCKLNGIPPLELGDCSIAGWLLGNPECDRLLSVPEWSYIMEKENPRD GLCYPGSFND YEELKHLLSSVKHFEKVKILPKDRWTQHTTTGGSRACAVSGNPSFFRN

MIWLTKKGSNYPVAKGSYNNTSGEQMLIIWGVHHPNDETEQRTLYQNVGTYVSVGTST

LNKRSTPDIATRPKVNGLGSRMEFSWTLLDMWDTINFESTGNLIAPEYGFKISKRGSS

GIMKTEGTLGNCETKCQTPLGAINTTLPFHNVHPLTIGECPKYVKSEKLVLATGLRNV

PQIESRGLFGAIAGFIEGGWQGMVDGWYGYHHSNDQGSGYAADKESTQKAFDGITNKV

NSWEKMNTQFEAVGKEFSNLERRLENLNKKMEDGFLDVWTYNAELLVLMENERTLDF

HDSNVKNLYDKVRMQLRDNVKELGNGCFEFYHKCDDECMNSVKNGTYDYPKYEEESKL

NRNEIKGVKLSSMGVYQILAIYATVAGSLSLAIMMAGISFWMCSNGSLQCRICI

Nucleotide

TGAAAATGAATCCAAATCAAAAGATAATAACAATTGGCTCTGTCTCTCTCACCATTGCAACAGTATGCTTCCTCA TGCAGATTGCCATCCTGGCAACTACTGTGACATTGCATTTTAAACAACATGAGTGCGACTCCCCCGCGAGCAACC

AGAAAGAGATTTGCCCCGAAGTAGTGGAATACAGAAATTGGTCAAAGCCGCAATGTCAAATTACAGGATTTGCAC CTΓTTTCTAAGGACAATTCAATCCGGCTTTCTGCTGGTGGGGACATTTGGGTGACGAGAGAACCTTATGTGTCAT

TACATGATAGAATCCCTCACCGAACCCTATTAATGAATGAGTTGGGTGTTCCATTTCATTTAGGAACCAAACAAG TGTGTGTAGCATGGTCCAGCTCAAGTTGTCACGATGGAAAAGCATGGTTGCATGTTTGTGTCACTGGGGATGATA GAAATGCGACTGCCAGCTTCATTTATGACGGGAGGCTTGTGGACAGTATTGGTTCATGGTCTCAAAATATCCTCA GGACCCAGGAGTCGGAATGCGTTTGTATCAATGGGACTTGCACAGTAGTAATGACTGATGGAAGTGCATCAGGAA GAGCCGATACTAGAATACTATTCATTAAAGAGGGGAAAATTGTCCATATCAGCCCATTGTCAGGAAGTGCTCAGC ATATAGAGGAGTGTTCCTGTTACCCTCGATATCCTGACGTCAGATGTATCTGCAGAGACAACTGGAAAGGCTCTA ATAGGCCCGTTATAGACATAAATATGGAAGATTATAGCATTGATTCCAGTTATGTGTGCTCAGGGCTTGTTGGCG

TGAAAGGCTGGGCCTTTGACAATGGAGATGATGTATGGATGGGAAGAACAATCAACAAAGATTCACGCTCAGGTT

ATGAAACTTTCAAAGTCATTGGTGGTTGGTCCACACCTAATTCCAAATCGCAGGTCAATAGACAGGTCATAGTTG

ACAACAATAATTGGTCTGGTTACTCTGGTATTITCTCTGTTGAGGGCAAAAGCTGCATCAATAGGTGCTTTTATG

TGGAGTTGATAAGGGGAAGGCCACAGGAGACTAGAGTATGGTGGACCTCAAACAGTATTGTTGTGTTTTGTGGCA

CTTCAGGTACTTATGGAACAGGCTCATGGCCTGATGGGGCGAACATCAATTTCATGCCTATATAAGCTTTCGCAA

TTTTAGAAAA

Amino acid

MNPNQKIITIGSVSLTIATVCFLMQIAILATTVTLHFKQHECDS

PASNQVMPCEPIIIERNITEIVYLNNTTIEKEICPEWEYRNWSKPQCQITGFAPFSK

DNSIRLSAGGDIWVTREPYVSCDPGKCYQFALGQGTTLDNKHSNGTIHDRIPHRTLLM

NELGVPFHLGTKQVCVAWSSSSCHDGKAWLHVCVTGDDRNATASFIYDGRLVDSIGSW

SQNILRTQESECVCINGTCTWMTDGSASGRADTRILFIKEGKIVHISPLSGSAQHIE

ECSCYPRYPDVRCICRDNWKGSNRPVIDINMEDYSIDSSYVCSGLVGDTPRNDDSSSN

SNCRDPNNERGNPGVKGWAFDNGDDVWMGRTINKDSRSGYETFKVIGGWSTPNSKSQV

NRQVαVDNNNWSGYSGIFSVEGKSCINRCFYVELIRGRPQETRVWWTSNSrVVFCGTSGTYGTGSWPDGANINFMPI

Figure legends

Figure 1. (A) Mean serum specific IgG antibody response (ELISA) to influenza A of A/New Caledonia/20/99(H1N1) days after viral challenge and (B) number of viral RNA copies (real time RTVPCR) in nasal wash in days after challenge. Six ferrets in each group.

Figure 2 : 1918 pandemic HlNl DNA vaccinated ferrets challenged with 2007 HlNl

(A) Fever at day 2 post challenge; (B) Body weight loss by day 4 post challenge; (C) Virus titre in nasal washings at day 7 post challenge; (D) Clinical score for illness based on a scoring table for sneezing, nasal discharge and activity level.

Figure 3: Hemadsorption as a measure of functional protein expression in mamalian cells of codon optimized HA from 1918 HlNl(HA 1918), avian H5N7 (HA H5N7) and 1968 H3N2 (HA H3N2) compared to non- codon optimized 1918 HlNl (HA NC) Examples

Example 1: Construction of expression vectors The 1918 pandemic HlNl genes were designed from nucleotide sequences published in GenBank (HA: A/South Carolina/1/18 AFl 17241, and NA, NP and M: A/Brevig Mission/1/18 AF250356, AY744035 and AY130766, respectively). The genes were made synthetically and designed to include the appropriate restriction enzymes and Kozak sequence (GCCACC), -1 base upstream from the start codon, for efficient cloning and transcription in the WRG7079 expression vector (PowderJect,

Madison, WI). The genes were synthesised using only codons from highly expressed human genes 5 (codon optimised). By this the nucleotide codons are altered (humanised), but the encoded amino acids are identical to those encoded by the viral RNA. The genes were further cloned individually into the WRG7079 expression vector. Key elements in the expression vector are a kanamycin resistance gene, cytomegalovirus immediate-early promotor, intron A, and polyadenylation signal. The tissue plasminogen activator (tPA) signal sequence in the original WRG7079 expression vector, used to target proteins to a secretory pathway, was excised in favour of the influenza signal sequence located in the 1918 HA and NA genes. We wanted to apply the same vector for expression of also the internal genes NP and Ml that do not have secretory signals and which are naturally located inside the virus and inside the infected cells, therefore the tPA secretory signal of the WRG7079 was removed.

Viral RNA from the A/New Caledonia/20/99(H1N1) MDCK ceU cultivated virus was isolated by QIAamp® Viral RNA Mini Kit (QIAGEN, Hilden, Germany) and RT-PCR was performed as previously described 2 by OneStep® RT-PCR Kit (QIAGEN). The primers were designed to amplify the coding gene of HA and NA. The same restriction sites and Kozak sequence were included in the primers as for the 1918 HlNl constructs (HA NC F: 5^'-caacgcgtgccaccatgaaagcaaaactactgg-3^', HA NC R: 5^'- tcggcgcctcagatgcatattctacactgc -3^', NA NC F: 5'- caacgcgtgccaccatgaatccaaatc-3^', NA NC R: 5'-tcg gcgccctacttgtcaatggtgaa cggc-3^'). The RT-PCR products were purified from an agarose gel by the GFX™ PCR DNA and Gel Band Purification Kit (Amersham Biosciences, Piscataway, USA) prior to sequencing. Purified PCR products were sequenced directly. The sequencing reaction was performed by ABI PRISM® BigDye™ Terminators v3.1 Cycle

Sequencing Kit (Applied Biosystems, Foster City, California, USA) as described previously (2). The development of the sequences was performed on an automatic ABI PRISM® 3130 genetic analyzer (Applied Biosystems) with 80 cm capillaries. Consensus sequences were generated in SeqScape® Software v2.5 (Applied Biosystems). Sequence assembly, multiple alignment and alignment trimming were performed with the BioEdit software v.7.0.5 9. The PCR products were further restriction enzyme digested and cloned into the WRG7079 expression vector in DH5α bacteria. Endotoxin free DNA purification of the vaccine clones were prepared by EndoFree Plasmid Giga Kit (QIAGEN). All inserts and vaccine clones were control sequenced.

Example 2: Immunisations

A total of 24 ferrets (Mustela Putorius Furo), approximately seven months old, were divided in four groups by using a chip-tag identification for dogs (E -vet, pet-id, Haderslev, Denmark), six animals in each group. All animals were kept together and fed a standard diet with food and water ad libitum. The animals were housed according to the Danish Animal Experimentation Act and kept at level II biosecurity facilities at the Faculty of Life Sciences, Copenhagen. The acclimatisation period was nine days.

Four groups of six ferrets were vaccinated as follows; (1) HA (codon optimised gene) and NA (codon optimised gene) 1918 HlNl plasmid DNA vaccinated, (2) HA, NA, NP and M (all codon optimised) 1918 HlNl plasmid DNA vaccinated, (3) empty plasmid vaccinated (negative vaccine control) and (4) HA and NA (not codon optimised) A/New Caledonia/20/99(H1N1) plasmid DNA vaccinated (positive vaccine control). All ferrets received four standard gene gun shots onto shaved abdomen. HA and NA DNA mixed vaccines were given in two shots and NP and M DNA mixed vaccines were given in two shots. Therefore groups 1 and 4 receiving only HA and NA DNA vaccine were additionally shot twice with empty plasmid DNA, ensuring that all animals had received the same amount of DNA and the same number of shots. The ferrets were gene gun (Helios, Bio-Rad, Hercules, CA) inoculated (400 psi compressed helium) on shaved abdominal skin, using 2 μg plasmid DNA- coated gold particles (1.6 μm-sized particles), 80-95% coating efficiency each shot. Each ferret received four shots, three times biweekly. Ferrets were challenged ten days after third immunisation by IxIO⁷ 50% egg infectious dose (EID50) of A/New Caledonia/20/99(H1N1) virus in 100 ul PBS administrated into the nostrils with a syringe. Blood serum was collected at day -2, 3, 5 and 7 post-challenge from vena jugularis of anesthetised animals (tiletamine/zolazepam (zoletil-mix for cats)). Animals were terminated with pentobarbital.

Example 3: Quantitative real time RT-PCR assay for influenza A.

At the day of blood serum collection the nostrils of each ferret were flushed with 1 ml PBS and the flushing were frozen down immediately for real-time RT-PCR analysis. Two hundred micro litres of nasal wash were extracted on an automated MagNA Pure LC Instrument applying the MagNa Pure LC Total Nucleic Acid Isolation Kit (Roche diagnostics,

Basel, Switzerland). The extracted material was eluated in 200 ul Milli-Q H2O. The RT-PCR reactions were performed with oligonucleotide sequences as described by Spackman et ah, (23). Extracted material (5 ul) was added to 20 ul of master mix consisting of 10 nM of each primer and 2 nM of the Taqman probe labelled with FAM in the 5' end and black hole quencher 1 in the 3' end together with reagents from the OneStep® RT-PCR Kit (QIAGEN, Hilden, Germany) according to the manufacturer. Target sequences were amplified on the MX3005 system from Stratagene with the following program: 20 min 50⁰C, 15 min 95°C and 40 cycles of 15 sec 95°C and 60 sec at 55°C. The content of viral genomes in the samples was determined using a standard curve developed by amplifying dilution of HlNl with known concentration. Example 4: Serum antibody determined by ELISA ELISA plates (96 wells) were coated with 100 μl, split influenza vaccine (Vaxigrip, Sanofi Pasteur, Belgium) diluted 1:100 in 35 mM NaHCO3 pH 9.6 and 15 mM Na2CO3 over night at 4⁰C. Wells were blocked with 1 % PBS/BSA for 30 minutes at room temperature. Plates were washed with

0.05% PBS/tween (PBST). Sera 1:100 were diluted in 0.1 % BSA/PBST two-folds in the plate and incubated for one hour at room temperature. The plates were washed and incubated with 100 μl biotinylated rabbit anti-ferret IgG diluted 1:250 for one hour in room temperature, washed, and incubated with 100 μl 1:1,000 horseradish peroxidase (HRP) streptavidin (DakoCytomation, Glostrup, Denmark). After 30 minutes the plates were washed and 100 μl of hydrogen peroxide with OPD was added. The reaction was stopped by adding 50 μl 0.5 M H₂SO₄ and read at OD492 nm.

Example 5: Results

Ferrets were negative for influenza specific antibodies seven days before start of immunisations as measured by ELISA.

High IgG specific serum antibodies (to A/New Caledonia/20/99(H1N1) in ELISA) were observed at day seven post-challenge in ferrets vaccinated with both HA+NA 1918 (two plasmids) and HA+NA+NP+M 1918 DNA vaccines (four plasmids) (Fig.l). Ferrets vaccinated with HA+NA DNA A/New Caledonia/20/99(H1N1) induced lower specific serum antibody titre on day seven. It is possible that ahigher antibody response could have been observed at later time points if the experiment had not been terminated at day seven after challenge for practical reasons. At day five post-challenge the ferrets vaccinated with empty plasmid (negative vaccine control) showed high viral load in nasal washing measured as viral RNA copies in the nasal washings, indicating no protection against the viral challenge. However, ferrets vaccinated with HA+NA 1918 and HA+NA, NP+M 1918 DNA vaccines were completely protected from infection with an A/New Caledonia/20/99(H1N1) like virus (Fig. 1). Partial protection was observed in ferrets vaccinated with HA+NA A/New Caledonia/20/99(H1N1) DNA plasmids.

The data clearly show that DNA gene gun immunisations based on genes from the 1918 HlNl pandemic strain induce strong specific antibody response and protect ferrets completely against infection with a HlNl strain that has drifted by 89 years. No negative or positive effects on the humoral immune response or protection was observed by including the NP and M genes in the HA+NA DNA vaccination since the protection from infection already was already 100%.

The A/South Carolina/1/18 and A/New Caledonia/20/99 are 21.2% different in the HAl protein and possess eight substitutions at residues involved in antigenic sites 3 (1918 to New Caledonia); Cb S83P, Sa T128V and K160N, Sb S156G, Q193H and D196N, CaI N207S and A224E.

DNA vaccines do have the ability of immune stimulatory mechanisms. This might be one reason why we observe such a good induced cross reactivity and protection against challenge infection. Cross-protection and cross -reactivity induced by DNA vaccines of strains differing by 11-13% in HAl has been demonstrated by others (13-15) but not as high as the 21.2% we observe.

Example 6: 1918 pandemic HlNl DNA vaccinated ferrets were challenged with 2007 HlNl:

Vaccine production and vaccinations and assays were carried out as described above.

A total of 10 ferrets (Mustela Putorius Furo), approximately seven months old, were divided in two groups by using a chip-tag identification for dogs (E-vet, pet-id, Haderslev, Denmark), five animals in each group. All animals were kept together and fed a standard diet with food and water ad libitum. The animals were housed according to the Danish Animal Experimentation Act and kept at level II biosecurity facilities at the Faculty of Life Sciences, Copenhagen. The acclimatisation period was one day. Two groups of five ferrets were vaccinated as follows; (1) HA (codon optimised gene) and NA (codon optimised gene) 1918 HlNl plasmid DNA vaccinated, (2) non-vaccinated, naϊve animals. HA and NA DNA mixed vaccines were given in four shots. The ferrets were gene gun (Helios, Bio-Rad, Hercules, CA) inoculated (400 psi compressed helium) on shaved abdominal skin, using 2 μg plasmid DNA-coated gold particles

(1.6 um-sized particles), 80-95% coating efficiency each shot. Vaccinated ferrets received four shots, three times biweekly. Ferrets were challenged ten days after third immunisation by IxIO⁷ 50% egg infectious dose (EID50) of A/New Caledonia/20/99(H1N1) virus in 1000 ul PBS administrated into the nostrils with a syringe. Blood serum was collected at day -48, 0, 5, 7 and 12 post-challenge from vena jugularis of anesthetised animals (tiletamine/zolazepam (zoletil-mix for cats)). Animals were terminated with pentobarbital. The 1918 DNA vaccinated ferrets had a lower temperature rise than the unvaccinated group (p=0.2) at the day of maximal temperature rise, day 2 post challenge (Figure 2A).

No difference in weight loss between the vaccinated and the unvaccinated animals was observed at the day of maximal weight loss, day 4 post challenge (Figure 2B). Vaccinated animals displayed fewer influenza symptoms than unvaccinated animals measured by sneezing, nasal discharge and activity level (p=0.065) (Figure 2C). Ferrets in both groups had high virus load post infection measured by quantitative realtime RT-PCR, however by day 7 post infection the 1918 DNA vaccinated ferrets better cleared their virus infection than the unvaccinated ferrets (p=0.63 ) (Figure 2D).

Example 7: Challenge with New Calidonia HlNl in ferrets: Traditional protein HlNl New Caledonia vaccine plus/minus DDA/TDB adjuvans versus 1918 HlNl HA plus NA DNA vaccine (versus empty DNA vaccine vector) using two DNA immunizations (instead or the usual 3 DNA immunizations)

Traditional protein HlNl split vaccine (two immunizations) versus 1918 HlNl HA plus NA codon optimized DNA vaccine versus codon optimized and non-codon-otimized New Calidonia HlNl HA and NA versus codon optimized M and NP from 1918 HlNl virus (versus empty DNA vaccine vector) using three immunizations. Ferrets are challenged with HlNl New Caledonia-like virus intra nasally and virus quantitated in basal washings by real-time RT/PCR assay. Ferret antibodies will be examined for ELISA antibodies and HI antibody reactions to HlNl, H2N2, H3N2, H5N7, and/or H5N1.

Example 8: Mouse antibody experiments:

Codon optimized versus non-codon optimized HA and NA DNA vaccines from New Calidonia HlNl (shows the difference between codon optimization and non-optimization) versus codon optimized HA and NA from 1918 HlNl strain is inoculated in mice. Antibody titers and epitope mapping of induced antibodies is done by overlapping peptides in ELISA and cross-reactions measured to other influenza A virus.

Example 9: Protein expression experiments:

Codon optimized versus non-codon optimized HA and NA DNA vaccines from New Calidonia HlNl (shows the difference between codon optimization and non-optimization) versus codon optimized HA and NA from 1918 HlNl strain is expressed in mammalian cell lines in vitro and standard radio immuno precipitation (RIPA) are done with polyclonal influenza A antibodies to examine the improved protein expression obtained by codon optimization. Codon optimized from 1918 HlNl, H5N7 and H3N2 strain versus non-codon optimized HA DNA vaccines from 1918 HlNl strain is expressed in mammalian cell lines in vitro and hemadsorption is measured. This shows that the Hl is functionally expressed better when codons are optimized (Fig.3).

Example 10: Cytokine induction experiments: Codon optimized versus non-codon optimized HA and NA DNA vaccines from New Calidonia HlNl (shows the difference between codon optimization and non-optimization) versus codon optimized HA and NA from 1918 HlNl strain is added onto mammalian peripheral blood monocytes (PBMCs) in vitro and measurements of resulting cytokine production is measured in the cell supernatant to examine the innate immune induction (adjuvant effect) obtained by codon optimization and by the codon optimised HlNl 1918 HA and NA as compared to the codon optimised HlNl New Caledonia HA and NA to examine special cytokine induction by the 1918 genes.

Example 11: 1918 HA and NA protein vaccine experiments: Proteins are produced by the DNA vaccine plasmids and used as a protein vaccine in mice or ferrets as compared to DNA vaccination and to traditional protein split vaccine to measure the immune induction of 1918 proteins versus DNA vaccine.

Example 12: Mouse DNA vaccine delivery experiments: Codon optimized HA and/or NA DNA vaccines from 1918 HlNl strain is inoculated in mice as expression plasmids or as a linear piece of DNA containing the necessary components for vaccine gene expression but without the rest of the plasmid to rule out any effect of the rest of the plasmid.

Example 13: Swine DNA vaccine delivery experiments: Codon optimized HA and/or NA DNA vaccines from 1918 HlNl strain is inoculated in swine as expression plasmids and challenge with a present day New Caledonia-like HlNl strain and protection against disease and immune induction are measured.

References:

1. Antonovics, J., M. E. Hood, and C. H. Baker. 2006. Molecular virology: Was the 1918 flu avian in origin? Nature 440:E9.

2. Bragstad, K., P. H. Jorgensen, K. J. Handberg, S. Mellergaard, S. Corbet, and A. Fomsgaard. 2005. New avian influenza A virus subtype combination H5N7 identified in Danish mallard ducks. Virus. Res. 109:181-190. 3. Caton, A. J., G. G. Brownlee, J. W. Yewdell, and W. Gerhard. 1982.

The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (Hl subtype). CeU 31:417-427.

4. Chen, Z., S. e. Kadowaki, Y. Hagiwara, T. Yoshikawa, K. Matsuo, T. Kurata, and S. i. Tamura. 2000. Cross-protection against a lethal influenza virus infection by DNA vaccine to neuraminidase. Vaccine

18:3214-3222.

5. Corbet, S., L. Vinner, D. M. Hougaard, K. Bryder, H. V. Nielsen, C. Nielsen, and A. Fomsgaard. 2000. Construction, biological activity, and immunogenicity of synthetic envelope DNA vaccines based on a primary, CCR5-tropic, early HIV type 1 isolate (BX08) with human codons. AIDS

Res. Hum. Retroviruses 16:1997-2008.

6. Davis, H. L., B. A. Demeneix, B. Quantin, J. Coulombe, and R. G. Whalen. 1993. Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle. Hum. Gene Ther. 4:733-740. 7. Donnelly, J. J., A. Friedman, D. Martinez, D. L. Montgomery, J. W.

Shiver, S. L. Motzel, J. B. Ulmer, and M. A. Liu. 1995. Preclinical efficacy of a prototype DNA vaccine: enhanced protection against antigenic drift in influenza virus. Nat. Med 1:583-587.

8. Epstein, S. L., W. p. Kong, J. A. Misplon, C. Y. Lo, T. M. Tumpeγ, L. Xu, and G. J. Nabel. 2005. Protection against multiple influenza A subtypes by vaccination with highly conserved nucleoprotein. Vaccine

23:5404-5410.

9. Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser 41:95-98. 10. Johnson, N. P. and J. Mueller. 2002. Updating the accounts: global mortality of the 1918-1920 "Spanish" influenza pandemic. Bull. Hist Med 76:105-115.

11. Kawaoka, Y., S. Krauss, and R. G. Webster. 1989. Avian-to-human transmission of the PBl gene of influenza A viruses in the 1957 and 1968 pandemics. J Virol 63:4603-4608.

12. Kobasa, D., A. Takada, K. Shinya, M. Hatta, P. Halfmann, S. Theriault, H. Suzuki, H. Nishlmura, K. Mitamura, N. Sugaya, T. Usui, T. Murata, Y. Maeda, S. Watanabe, M. Suresh, T. Suzuki, Y. Suzuki, H. Feldmann, and Y. Kawaoka. 2004. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature 431:703-707.

13. Kodihalli, S., H. Goto, D. L. Kobasa, S. Krauss, Y. Kawaoka, and R. G. Webster. 1999. DNA vaccine encoding hemagglutinin provides protective immunity against H5N1 influenza virus infection in mice. J.

Virol. 73:2094-2098.

14. Kodihalli, S., J. R. Haynes, H. L. Robinson, and R. G. Webster. 1997. Cross-protection among lethal H5N2 influenza viruses induced by DNA vaccine to the hemagglutinin. J. Virol. 71:3391-3396. 15. Kodihalli, S., D. L. Kobasa, and R. G. Webster. 2000. Strategies for inducing protection against avian influenza A virus subtypes with DNA vaccines. Vaccine 18:2592-2599.

16. Kong, W. p., C. Hood, Z. y. Yang, C. J. Wei, L. Xu, A. Garcia-Sastre, T. M. Tumpey, and G. J. Nabel. 2006. Protective immunity to lethal challenge of the 1918 pandemic influenza virus by vaccination. PNAS

103:15987-15991.

17. Lindstrom, S. E., N. J. Cox, and A. Klimov. 2004. Genetic analysis of human H2N2 and early H3N2 influenza viruses, 1957-1972: evidence for genetic divergence and multiple reassortment events. Virology 328:101- 119.

18. Ljungberg, K., C. Kolmskog, B. Wahren, G. van Amerongen, M. Baars, A. Osterhaus, A. Iinde, and G. Rimmelzwaan. 2002. DNA vaccination of ferrets with chimeric influenza A virus hemagglutinin (H3) genes. Vaccine 20:2045-2052. 19. Reid, A. H., T. G. Fanning, J. V. Hultin, and J. K. Taubenberger.

1999. Origin and evolution of the 1918 "Spanish" influenza virus hemagglutinin gene. Proc. Natl. Acad. Sci. U. S. A 96:1651-1656.

20. Reid, A. H., T. G. Fanning, T. A. Janczewski, and J. K. Taubenberger. 2000. Characterization of the 1918 "Spanish" influenza virus neuraminidase gene. Proc. Natl. Acad. Sci U. S. A 97:6785-6790.

21. Seo, S. H., E. Hoffmann, and R. G. Webster. 2002. Lethal H5N1 influenza viruses escape host anti-viral cytokine responses. Nat. Med. 8:950-954. 22. Smith, H. and C. Sweet. 1988. Lessons for human influenza from pathogenicity studies with ferrets. Rev. Infect Dis 10:56-75.

23. Spackman, E., D. A. Senne, T. J. Myers, L. L. Bulaga, L. P. Garber, M. L. Perdue, K. Lohman, L. T. Damn, and D. L. Suarez. 2002. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin.

Microbiol. 40:3256-3260.

24. Talon, J., C. M. Horvath, R. Polley, C. F. Basler, T. Muster, P. Palese, and A. Garcia-Sastre. 2000. Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NSl protein. J. Virol. 74:7989-7996.

25. Tamura, S., T. Tanimoto, and T. Kurata. 2005. Mechanisms of broad cross -protection provided by influenza virus infection and their application to vaccines. Jpn. J Infect Dis 58:195-207.

26. Taubenberger, J. K., A. H. Reid, R. M. Lourens, R. Wang, G. Jin, and T. G. Fanning. 2005. Characterization of the 1918 influenza virus polymerase genes. Nature 437:889-893.

27. Tumpey, T. M., C. F. Basler, P. V. Aguilar, H. Zeng, A. Solorzano, D. E. Swayne, N. J. Cox, J. M. Katz, J. K. Taubenberger, P. Palese, and A. Garcia-Sastre. 2005. Characterization of the reconstructed 1918 Spanish influenza pandemic virus. Science 310:77-80.

28. Tumpey, T. M., A. Garcia-Sastre, J. K. Taubenberger, P. Palese, D. E. Swayne, and C. F. Basler. 2004. Pathogenicity and immunogenicity of influenza viruses with genes from the 1918 pandemic virus. Proc. NaU. Acad. Sci U. S. A 101:3166-3171. 29. Tumpey, T. M., A. Garcia-Sastre, J. K. Taubenberger, P. Palese, D. E. Swayne, M. J. Pantin-Jackwood, S. Schultz-Cherry, A. Solorzano, N. Van Rooijen, J. M. Katz, and C. F. Basler. 2005. Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice. J Virol 79:14933-14944.

30. Ulmer, J. B., T. M. Fu, R. R. Deck, A. Friedman, L. Guan, C. DeWitt, X. Uu, S. Wang, M. A. Liu, J. J. Donnelly, and M. J. Caulfield. 1998. Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA. J Virol 72:5648-5653.

31. Wang, X., M. Li, H. Zheng, T. Muster, P. Palese, A. A. Beg, and A. Garcia-Sastre. 2000. Influenza A Virus NSl Protein Prevents Activation of NF-kappa B and Induction of Alpha/Beta Interferon. J. Virol. 74:11566- 11573. 32. Webster, R. G., E. F. Fynan, J. C. Santoro, and H. Robinson. 1994.

Protection of ferrets against influenza challenge with a DNA vaccine to the haemagglutinin. Vaccine 12:1495-1498.

Claims

Claims

1. The use of the naked DNA and/or RNA molecule encoding hemagglutinin (HA) from pandemic influenza, e.g. the 1918 HlNl and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus, as a vaccine component against present day and coming Hl, H2, H3 containing influenza A infections in humans and/or swine.

2. The use according to claim 1, where the naked DNA and/or RNA molecule plasmid DNA encoding neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza virus is included, as a vaccine component against present day and coming Nl, N2 containing influenza A infections in humans and/or swine.

3. The use according to claim 3 where the DNA or RNA codons are humanized, using codons of highly expressed human proteins.

4. The use according to claim 1-3 where an adjuvant is included.

5. The use according to claim 1-4 where the vaccine is administrated by saline injection of naked DNA and/or RNA or inoculated by gene gun or delivered coupled to particles.

6. A vaccine for use in humans and/or swine comprising the naked DNA and/or RNA molecule encoding hemagglutinin (HA) from pandemic influenza e.g. the 1918 HlNl and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus.

7. A vaccine according to claim 6, further comprising the naked DNA and/or RNA molecule encoding Neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza virus.

8. A vaccine according to claim 6-7, where the DNA or RNA codons are humanized using codons of highly expressed human proteins.

9. A vaccine according to claim 6-8, where the vaccine further comprises an adjuvant.

10. A vaccine according to claim 6-9, where the vaccines are used as therapeutic vaccine in already infected humans

11. The use of naked DNA and/or RNA molecule encoding HA and/or NA from 2001 H5N7 low pathogenic Avian influenza virus (AIV) strain (A/MaUard/Denmark/64650/03(H5N7)) or the March 2006 Denmark H5N1 high pathogenic AIV strain (A/buzzard/Denmark/6370/06(H5Nl)) or the 2008 (A/duck/Denmark/53- 147-8/08 (H7N1)) or the 2004 (A/widegeon/Denmark/66174/G18/04 (H2N3)) as vaccine component(s) against present day and coming H5, H7 or H2 containing influenza A infections in humans and/or swine and/or chickens.

12. The use according to claim 11, where the DNA or RNA codons are "humanized" .

13. A vaccine for use in humans and/or swine and/or chickens comprising the naked DNA and/or RNA molecule encoding HA and/or NA from 2001 H5N7 low pathogenic Avian influenza virus (AIV) strain (A/Mallard/Denmark/64650/03(H5N7)) or the March 2006 Denmark

H5N1 high pathogenic AIV strain (A/buzzard/Denmark/6370/06(H5Nl)) or the 2008 (A/duck/Denmark/53-147-8/08 (H7N1)) or the 2004 (A/widegeon/Denmark/66174/G 18/04 (H2N3)) against present day and coming H5, H7 or H2 containing influenza A infections in humans and/or swine and/or chickens.

14. A vaccine according to claim 13, where the vaccine is administered by saline injection of naked DNA and/or RNA or inoculated by gene gun where the DNA is coupled to particles.

15. A vaccine according to claim 13, where the DNA or RNA codons are humanized.

16. A vaccine according to claims 13-15, where the vaccine further comprises an adjuvant.

17. A vaccine according to claim 13-16, where the vaccines are used as a therapeutic vaccine in already infected humans