# Patent

## Title
Optimized influenza vaccines

## Abstract

The invention concerns nucleotides vaccines encoding influenza proteins with few or no glycosylation sites. Since these first introductions of pandemic influenzas the viruses have drifted, accumulating mutations at antigenic sites, but also the N-glycosylation pattern has changed during the drifted years, accumulating N-linked glycosylation sequons that help mask the antigenic sites for recognition by the host immune system. These “naked” initial haemagglutinins induce a broad cross reactivity against widely drifted influenza subtypes. The origin of the DNA or RNA can be both pandemic influenza strains, which codes for proteins which have a naturally low content of glycosylation sites and/or DNA or RNA from non-pandemic influenza strains where the nucleotides have been mutated or changed so it encodes for proteins with less or no glycosylation sites. The invention also discloses DNA or RNA encoding the haemagglutinin (HA) from pandemic influenza A, e.g. the 1918 H1N1 and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus, optionally with the Neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from these pandemic influenza virus included, mixed together with DNA or RNA from non-pandemic influenza A as a vaccine against present day and future influenza A viruses.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national stage of International Patent Application No. PCT/DK2009/000247, filed Nov. 27, 2009.
FIELD OF INVENTION
The invention concerns protein and nucleotides vaccines encoding influenza A proteins with few or no glycosylation sites. The DNA or RNA encodes for haemagglutinin and/or neuraminidase and/or Matrix and/or Nucleoprotein where the origin of the DNA or RNA can be both pandemic influenza strains, which codes for proteins which have a naturally low content of glycosylation sites and DNA or RNA from non-pandemic influenza strains where the nucleotides have been mutated or changed so it encodes for proteins with less glycosylation sites than the seasonal strain. The invention also discloses immunogenic compositions and the use of the haemagglutinin (HA) encoding DNA and/or neuraminidase (NA) encoding DNA expressing the novel 2009 pandemic H1N1v influenza A as a vaccine component optionally combined with DNA encoding the matrix protein (M) and DNA encoding the nucleoprotein (NP) from the 1918 pandemic H1N1 strain and DNA encoding the HA and NA from a recent seasonal H3N2 strain for a combined vaccine against present and future H1N1 and H3N2 infections in humans and swine.
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 H1N1 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 21st 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 (M1) protein layer. The eight segments are associated with nucleoprotein (NP) and three large proteins; polymerase basic 1 (PB1) 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 infectivity for about 100 days at 17° C. and can be stored indefinitely at −50° C. 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 56° C. for 3 hours or 60° C. 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 H1, H2, H3, N1 and N2 have circulated in humans and swine in the last century. The pandemic introduction in humans of these types were 1918 H1N1, 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 acquired the HA, NA and PB1 and HA and PB1 genes from an aquatic source, respectively. In 1977 a strain identical to the H1N1 strains that circulated before 1957 re-emerged. Pigs 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 H1N1 viruses. It has been shown that the rate of accumulating mutations is approximately 4-5×10−3 substitutions per nucleotide per year for HA1 others predict a rate of 5.7×10−3 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 H1N1 influenza A virus strains and an influenza B virus. The normal human vaccine dose is standardised to 15 μg HA protein of each virus component administrated once in normal healthy adults and twice in children and other persons with no pre-existing 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 by 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. In 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 AIV 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 or 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 DNA or RNA. 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 inserted into live viral or bacterial vectors such as Adenovirus, adenoassociated virus, alphavirus, poxviruses, herpes virus etc. 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, NA, 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 Th1 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 have also previously proved effective (18,32).
It has previously been shown that 1918 H1N1 whole inactivated virus vaccine induced partly protection against infection with 1918 H1N1 in mice (28), also recently DNA vaccines encoding the HA from 1918 showed complete protection of mice against a 1918 H1N1 challenge (16) but no protection against present day influenza was demonstrated.
We have demonstrated that gene gun administrated codon optimised plasmid DNA vaccine encoding HA and NA with or without M and NP based on the H1N1 pandemic virus from 1918 induce protection in ferrets against infection with a present day H1N1 virus (Bragstad et al 2009 and PCT/DK2008/000201). This demonstrates a vaccine induced protection not mediated by the usual anti-HA and anti-NA antibodies but by a different immunological mechanism most likely involving cellular immunity. Since the internal proteins M and NP encoded by the DNA and/or the influenza A virus are more conserved among different H1N1 strains than HA and NA it can be expected that the induced immunity to NP and M DNA vaccines are more broadly protective which could extend to also new H1N1 strains. The viruses are separated by a time interval of 89 years and differ by 21.2% in the HA1 protein. 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 H1N1 influenza A gives a high level of cross protection against present day influenza infection.
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 HA1 has been demonstrated by others (13-15) but not as high as with the 21.2% divergence we observe.
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.
Since the novel influenza A (H1N1v) is notably different from other human H1N1 viruses it is assumed that present H1N1 virus immunity and seasonal H1N1 vaccines will not induce efficient protection against the novel strain. The novel H1N1v virus is approximately 5% different in the nucleotide sequence from other known swine H1N2 viruses, while it is nearly 24% different from last seasons circulating human H1N1 viruses.
Thus, in the current situation with a new pandemic H1N1v virus and future variants here off we believe the best vaccine for the current circulating viruses and near future viruses will be a DNA vaccine comprising the HA and NA genes of the new pandemic H1N1v virus, the internal genes of the previous pandemic H1N1 virus from 1918 and the HA and NA genes of the circulating seasonal H3N2 virus. The genes of the H1N1v strain will be included to induce perfect protection against the circulating H1N1v and for future variants of this strain. We expect this strain with variants hereof to be the next seasonal H1N1 viruses in humans. The NP and M internal genes of the 1918 H1N1 pandemic is included as these are the ancestors of all other NP and M genes in human influenza viruses. These highly conserved genes are expected to induce better cross-protection. The HA and NA genes of the seasonal H3N2 virus are included as these will give the best protection against currently circulating H3N2 viruses and future H3N2 viruses. We believe that these gene combinations in a DNA vaccine will be the optimal more universal influenza vaccine at present time and for the nearest future.
We have surprisingly shown that a mix of DNA plasmids of both “initial” pandemic genes with few or no glycosylation sites and optionally present time genes induces the ultimate protection in the sense that the more antigenic sites will be exposed to the host immune system for antibody recognition. Alternatively influenza genes where all or some of the glycosylation codons have been changed/removed so the protein expressed has less or no glycosylation sites can be used in a mixture either themselves or with pandemic influenza DNA to make up a universal influenza vaccine.
SUMMARY OF THE INVENTION
The pandemic influenza A strains were the first introductions of H3N2 and H1N1 subtypes in humans. Since these first introductions the viruses have drifted, accumulating mutations at antigenic sites, but also the N-glycosylation pattern has changed during the drifted years. Accumulating N-linked glycosylation sequons help mask the antigenic sites for recognition by the host immune system. The “naked” initial haemagglutinins, e.g. haemagglutinins with few or no glycosylation, induce a broad cross reactivity against widely drifted influenza subtypes. Our preliminary results show that the 1918 H1N1 DNA vaccines are as good as or better candidates for influenza prophylaxis than annual conventional protein based vaccines in ferrets. The DNA vaccine immune response raised against these “initial” immunogens can cross react with present day viruses, while the opposite may not be true. Also present day influenza DNA used as vaccine show better influenza prophylaxis than annual conventional protein based vaccines against present day viruses. The influenza virus escapes the host immune system by accumulation of mutations especially at the surface glycoproteins HA and NA. Some of these mutations generate additional glycosylation sites which may camouflage the antigenic sites in the gycoproteins and the virus can thereby escape the host immunesystem. The initial pandemic strains possessed a low number of glycosylation sites while the present day viruses have accumulated sites during virus drift. Since the pandemic in 1918 with the H1N1 virus, three additional glycosylation sites have accumulated in the 1999 H1N1 virus included in our experiments, adding the total number of predicted N-glycosylation sites to seven. The current novel pandemic H1N1v virus also only possesses the basic four glycosylation sites as the pandemic 1918 virus.
The present invention discloses that an induced immune response with a DNA vaccine encoding HA and/or NA of the H1N1v influenza A alone or in combination with DNA vaccine encoding NP and M of H1N1 1918 virus gives a high level of cross protection against the novel H1N1v as well as future variants of this H1N1v as well as protection against present day and future H1N1 influenza A virus infections in humans and swine. In combination with DNA vaccine encoding HA and/or NA from an H3N2 strain preferentially from a recent (2005-2008) strain, the DNA vaccine will act as an universal influenza DNA vaccine inducing protective immunity against future circulating H1N1 and H3N2 viruses. The DNA vaccine can be used for humans and animals e.g. pigs to prevent/limit infection and/or spread of infection of human and pig influenza strains.
DETAILED DISCLOSURE OF THE INVENTION
The present invention discloses the use of nucleotides encoding influenza proteins with few or no glycosylation sites for manufacturing an immunogenic composition or a vaccine component against present day and coming influenza A infections in humans and pigs, where the nucleotides encodes for haemagglutinin (HA) and/or Neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP). The nucleotides can be a mix of pandemic DNA or RNA with non-pandemic DNA or RNA.
In a preferred embodiment of the invention the DNA or RNA encodes for 2009 H1N1v HA and NA, 1918 H1N1 M and NP together with seasonal H3N2 HA and NA.
In a preferred embodiment of the invention the DNA or RNA codons are optimized (“humanized”) e.g. the DNA sequence for haemagglutinin and neuraminidase and matrix and nucleoprotein is changed so the sequence coding for said proteins is changed to be optimally expressed in mammalian cells.
In another embodiment the non-pandemic DNA or RNA is mutated so codons are changed so the sequence codes for proteins with less glycosylation sites than the seasonal strain. Preferably some or all the codons coding for Asn-X-Ser of Asn-X-Thr, where X could be any amino acid except proline are changed.
The present invention also discloses the use of nucleotides encoding the haemagglutinin (HA) from pandemic influenza strain(s) (e.g. the 2009 H1N1v, the 1918 H1N1 and/or 1957 H2N2 and/or 1968 H3N2 influenza A virus) as a vaccine component against present day and coming/future H1, H2, H3, N1, N2 containing influenza A infections in humans and swine.
Haemagglutinin and/or neuraminidase and/or matrix protein and/or the nucleoprotein from pandemic influenza virus can optionally be included as proteins and/or DNA vaccines.
In another embodiment the vaccine comprises a mix of influenza proteins or codon optimized DNA encoding the proteins HA and NA from the pandemic 2009 H1N1v and/or Matrix protein and Nucleoprotein from 1918 H1N1 and/or HA and NA from a recent H3N2 2005 virus) as a vaccine component against present day and coming/future H1, H3, N1, N2 containing influenza A infections in humans and swine.
The vaccine is administrated by injection of naked DNA and/or RNA with or without electroporation or inoculated by gene gun. Alternatively the vaccine genes is administrated incorporated into apoptotic cell bodies, or in a live virus such as Adenovirus, poxvirus, adenoassociated virus, alphavirus, herpes virus and/or bacteria such as shigella species or salmonella species or lactobacillus species or lactococcus species. Another way of administration is by saline or buffer injection of naked DNA and/or RNA in a lipid-based formulation with or without an adjuvant for mucosal delivery in the respiratory tract such as intra nasal and/or intratracheal.
Above mentioned vaccine can further comprise above mentioned haemagglutinin (HA) and/or Neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) chosen from SEQ ID NO 2, 4, 6, 8, 10, 12, 14 and 16 or said proteins alone.
In another embodiment the vaccine comprises a sequential vaccination with pandemic DNA/RNA or protein followed by vaccination with non-pandemic DNA/RNA or protein (prime boost).
In another embodiment the vaccine comprises HA with/or without NA from the new circulating 2001 H5N7 low pathogenic (LP) avian influenza virus (AIV) strain (A/Mallard/Denmark/64650/03(H5N7)) as DNA vaccines and/or proteins. The vaccine is intended to protect birds and humans and swine against H5 influenza A strains.
In another embodiment the vaccine comprises HA with or without NA and/or M and/or NP from the newly introduced and circulating March 2006 Denmark H5N1 high pathogenic avian influenza A virus (AIV) strain (A/buzzard/Denmark/6370/06(H5N1)) as DNA vaccines and/or proteins. The vaccine is intended to broadly protect birds and humans and swine against any H5 containing influenza A strains.
The present invention discloses the use of codon optimised pandemic genes in transcriptionally active PCR for generation of transcriptionally active genes for direct use as DNA vaccines. By this the plasmid backbone is not used. The PCR products will contain all components for efficient expression of genes in eukaryotic cells.
Above mentioned vaccines can be used both prophylactic and therapeutic.
Definitions
Pandemic Influenza:
A pandemic virus is a virus which is new in the sense that there is no or little pre-existing immunity against the virus in the human population and therefore the virus will spread to all parts of the world.
Glycosylation:
N-linked glycosylation is a eukaryotic co-translational and posttranslational addition of saccharides to the amide nitrogen of asparagine side chains. A modification step in the synthesis of glycoproteins. These glycosylations are essential for correct folding and stability of the glycoprotein. Glycosylations in, or close to, the antigenic binding sites might interfere with the host cell recognition of the protein. Thereby glycosylations might help mask the protein from the host immune system.
Glycosylation Sites:
Glycosylation sites are here defined as glycosylations exposed on the surface part of the protein. For membrane bound proteins like HA and NA a part of the protein is exposed on the outside of the virus and glycosylation on this part of the protein is important to the virulens of the virus and/or to exposure of immunogenic/antigenic sites. For 1918 H1N1 and the 2009 H1N1v the glycosylation sites are 4 whereas the seasonal H1N1 has 6 glycosylation sites now but may accumulate more in time to come. Similarly For the 1968 H3N2 the glycosylation sites are 6 whereas the seasonal H3N2 has 8 glycosylation sites. Few glycosylation sites must be the basis for the correct folding of the protein and must be present and is always found even in unadapted pandemic strains. These glycosylations sites are conserved. In time the virus protein accumulates more glycosylation sites exposed on the surface to “mask” the immunity and thereby making it more adapted and probably less virulent.
Mutated DNA:
Site directed mutagene on the influenza surface glycoproteins will alter the glycosylation pattern. Nucleotides at sequon sites can be mutated to alter the codons in the tripeptide for the glycosylation site. Thereby selected sequeons can be erased.
Alternatively the protein that should be stripped for glycosylation can be produced in an non-eukaryotic host. Also such unclycosylated vaccine protein may be delivered from such an non-eukaryotic host such as a bacteria such as lactococcus bacteria by delivering the transfected bacteria directly on mucosal surfaces e.g. the nose or per orally to the gut where the organism will produce the vaccine protein in situ.
Haemagglutinin:
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, HA0. This initial molecule has to be cleaved by the host cell proteases into disulfidelinked HA1 (47 kDa) and HA2 (29 kDa) subunits in order for the virus to mediate membrane fusion and subsequent infection. The HA1 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-terminal 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 M1. The M1 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 M1 protein contains a hydrophobic lipid binding domain and a RNP binding domain. Assembly of negative stranded RNA viruses requires localisation of M1 proteins to the plasma membrane. The M1 proteins bind to the cytoplasmic tails of HA, NA and M2, especially NA stimulate the membrane binding by the M1 proteins. M1 together with NS2 is required for export of genomic RNPs from the nucleus, M1 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 M1, disrupting the M1-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 PB1 and PB2 and with a variety of other viral and cellular proteins. The interaction with M1 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 abundant in extracellular fluid and lung tissue during severe influenza A infection.
The 1918 Influenza Virus:
The most severe pandemic this century has been the 1918 H1N1 “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 origin of the 1918 pandemic is debated. Taubenberger et at. 2005 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 (Taubenberger et at. 2005, Antonovics et al 2006). The haemagglutinin (HA) and neuraminidase (NA) genes of the 1918 H1N1 strain did not possess known genetic indicators for high virulence that could have explained the severe disease observed in humans (Reid et al 1999 & 2000). However, the HA (and NA) protein on a backbone of recent human viruses conferred enhanced pathogenicity in mice (Kobasa et al 2004, Tumpey et al 2005b). It might have been the combination of genes more than the HA itself that caused the lethal phenotype (Tumpey et al 2005). The uncertainty about the origin and the mechanisms of high virulence of the 1918 H1N1 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 H1N1 strain showed protection to a lethal challenge of the recombinant 1918 H1N1 virus strain in mice (Kong W et al 2006). The NP and M genes of the 1918 pandemic is the ancestor of NP and M genes of both H1N1, H3N2 and H2N2 viruses circulating today. These genes are also highly conserved and change less than the surface glycoproteins through time.
The 2009 Influenza A H1N1v Virus:
During April 2009 a novel influenza H1N1v virus was discovered in California and also recognised to have caused previous outbreaks in Mexico the same year. The virus was unrelated to previous human influenza viruses both genetically and antigenically. The novel H1N1v strain is more closely related to triple reassorted swine viruses. WHO announced in June that the H1N1v virus was a pandemic virus and as of November 2009, 206 countries and overseas territories or communities have reported laboratory confirmed cases of pandemic influenza H1N1v including over 6770 deaths. Protein influenza vaccines are now available against the new pandemic strain, however these vaccines need to be evaluated as the virus changes. We aim to generate a universal DNA vaccine that will protect even against widely drifted H1n1v viruses.
Seasonal H3N2 Viruses
The first introduction of H3N2 viruses in humans occurred by the pandemic in 1968. This virus was a reassortant between avian H3 viruses and the H2N2 viruses from the 1957 pandemic. Since then, H3N2, and later, H1N1, have co-circulated in the human population causing seasonal outbreaks, and epidemics. The H3N2 viruses have changed enough during time for the protein vaccine composition to change,
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 H1N1 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 H1N1 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 H1N1 strain showed protection to a lethal challenge of the recombinant 1918 H1N1 virus strain in mice (Kong W, Hood C, Yang Z, Wei C, Xu L, Garcia-Sastre A, Tumpey T M, Nabel G J. Protective immunity to lethal challenge of the 1918 pandemic influenza virus by vaccination. PNAS 103(43):15987-91, 2006)
DNA or RNA Vaccines:
In the following the term “DNA vaccines” refers to nucleotide vaccines meaning both DNA vaccines and RNA vaccines. The DNA vaccine can be delivered as RNA or DNA either as “naked” DNA plasmid with or without a lipid-based compound or apoptotic cells to facilitate cellular uptake, provide depot-effect and/or adjuvant effect, including or not a protein adjuvant molecule and/or DNA plasmids encoding such adjuvant protein(s), for enhanced and optimal immune induction. The DNA vaccine genes can be injected with or without a facilitators such as electroporation or delivered by jet air or coated onto particles as with a gene gun or it can be delivered to mucosal surfaces e.g. intranasally and/or intratracheally. The DNA vaccine genes can also be incorporated into a viral or bacterial vector for delivery such as Adenoviruses, poxvirus, adenoassociated virus, alphavirus, cytomegalovirus, and/or bacteria such as lactococcus or lactobacillus or shigelle or salmonella species.
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 Th1 IgG2a response while gene gun delivery tends to initiate a more Th2 IgG1 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 DNA, 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 vector consists of 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 naïve leader sequences may be substituted with optimised leaders increasing translation efficiency.
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 delivered by a viral vector such as Adenovirus, Modified vaccinia virus Ankara (MVA), Vaccinia, Adenoassociated virus (AAV), Alphavirus etc. DNA vaccines could be delivered either by gene gun as in the previous examples or as injection with or without electroporation.
When using DNA vaccine encoding surface proteins (HA and/or NA) of a pandemic strain (e.g. 1918 H1N1 or 1968 H3N2 or 1957 H2N2) in combination with HA and/or NA from present day drifted strains of H1N1 or H3N2 or H2N2 we envision a preferentially induction of cross-reacting antibodies (broadly protective antibodies) to shared more conserved antibody and T cell epitopes. Therefore a broader protection should be obtained when vaccinating with surface proteins either as a DNA vaccine and/or a protein vaccine deriving from the mentioned original pandemic strains together with HA and/or NA from present day virus strains as DNA vaccine and/or a protein vaccine, and importantly this can be done either as a mixture or as a sequential (prime-boost) with pandemic or present day antigens first and present day or pandemic strains at a later time point. As a consequence we will obtain a broader anti-influenza A response by vaccinating (DNA and/or protein) from pandemic strains in individuals already vaccinated with present day protein vaccine. Vise versa we may get a broader anti-influenza coverage against coming influenza strains when primed with HA and/or NA DNA and/or protein from the pandemic strains and boosted with an influenza protein vaccine at any time point in the future.
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 content of C and G, there are an increased possibility of generating both immune stimulatory GpG motifs, but also immune inhibitory GC sequences. Genes engineered using codon optimization is called “humanized” genes and are frequently used in DNA vaccines to enhance expression.
The DNA or RNA sequence for haemagglutinin and neuraminidase and matrix and nucleoprotein is changed so the sequence coding for said proteins is changed to be optimally expressed in humans and swine or the host for which the vaccine is intended.
The invention discloses the use of the novel influenza A (H1N1)v HA and/or NA codon-optimized genes in a DNA immunogenic composition or vaccine and the combination of these novel H1N1 pandemic glycoprotein genes with previous pandemic H1N1 genes encoding internal proteins in a DNA immunogenic composition or vaccine.







TABEL 1





H1N1 Nucleotide and amino acid 


sequences of the codon optimized


genes and the proteins they express.







HA 1918 synthetic gene 0607838, Base on acc.


 No. AF11721: A/South Carolina/1/18


Nucleotide


ATGGAGGCCAGGCTGCTGGTGCTGCTGTGCGCCTTCGCCGCCACCAACG


CCGACACCATCTGCATCGGCTACCACGCCAACAACAGCACCGACACCGT


GGATACCGTGCTGGAGAAGAACGTGACCGTGACCCACAGCGTGAACCTG


CTGGAGGACAGCCACAACGGCAAGCTGTGCAAGCTGAAGGGAATCGCTC


CCCTGCAGCTGGGCAAGTGCAACATCGCCGGCTGGCTGCTGGGCAACCC


CGAGTGCGACCTGCTGCTGACCGCCAGCAGCTGGTCCTACATCGTGGAG


ACCAGCAACAGCGAGAACGGCACCTGCTACCCCGGCGACTTCATCGACT


ACGAGGAGCTGCGGGAGCAGCTGTCCAGCGTGAGCAGCTTCGAGAAGTT


CGAGATCTTCCCCAAGACCAGCTCCTGGCCCAACCACGAGACCACCAAG


GGCGTGACCGCCGCCTGTAGCTACGCCGGAGCCAGCAGCTTCTACAGAA


ACCTGCTGTGGCTGACCAAGAAGGGCAGCAGCTACCCCAAGCTGTCCAA


GAGCTACGTGAACAACAAGGGCAAGGAAGTGCTGGTGCTGTGGGGCGTG


CACCACCCCCCTACCGGCACCGACCAGCAGAGCCTGTACCAGAACGCCG


ACGCCTACGTGAGCGTGGGCAGCAGCAAGTACAACAGAAGGTTCACCCC


CGAGATCGCCGCCAGGCCCAAGGTGCGCGACCAGGCCGGCAGGATGAAC


TACTACTGGACCCTGCTGGAGCCCGGCGACACCATCACCTTCGAGGCCA


CCGGCAACCTGATCGCCCCTTGGTACGCCTTCGCCCTGAACAGGGGCAG


CGGCAGCGGCATCATCACCAGCGACGCCCCCGTGCACGACTGCAACACC


AAGTGCCAGACCCCCCACGGAGCCATCAACAGCAGCCTGCCCTTCCAGA


ACATCCACCCCGTGACCATCGGCGAGTGCCCCAAGTACGTGAGAAGCAC


CAAGCTGAGGATGGCCACCGGCCTGAGGAACATCCCCAGCATCCAGAGC


AGGGGCCTGTTCGGAGCCATCGCCGGATTCATCGAGGGCGGCTGGACCG


GCATGATCGACGGCTGGTACGGCTACCACCACCAGAACGAGCAGGGCAG


CGGCTACGCCGCCGACCAGAAGAGCACCCAGAACGCCATCGACGGCATC


ACCAACAAGGTGAACAGCGTGATCGAGAAGATGAACACCCAGTTCACCG


CCGTGGGCAAGGAGTTCAACAACCTGGAGAGGCGGATCGAGAACCTGAA


CAAGAAGGTGGACGACGGCTTCCTGGACATCTGGACCTACAACGCCGAG


CTGCTGGTGCTGCTGGAGAACGAGAGGACCCTGGACTTCCACGACAGCA


ACGTGAGGAACCTGTACGAGAAGGTGAAGAGCCAGCTGAAGAACAACGC


CAAGGAGATCGGCAACGGCTGCTTCGAGTTCTACCACAAGTGCGACGAC


GCCTGCATGGAGAGCGTGAGAAACGGCACCTACGACTACCCCAAGTACA


GCGAGGAGAGCAAGCTGAACCGGGAGGAGATCGACGGCGTGAAGCTGGA


GAGCATGGGCGTGTACCAGATCCTGGCCATCTACAGCACCGTGGCCAGC


AGCCTGGTGCTGCTGGTGTCCCTGGGAGCCATCAGCTTTTGGATGTGCA


GCAACGGCAGCCTGCAGTGCAGGATCTGCATCTGA


 


Amino acid


MEARLLVLLCAFAATNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNL


LEDSHNGKLCKLKGIAPLQLGKCNIAGWLLGNPECDLLLTASSWSYIVE


TSNSENGTCYPGDFIDYEELREQLSSVSSFEKFEIFPKTSSWPNHETTK


GVTAACSYAGASSFYRNLLWLTKKGSSYPKLSKSYVNNKGKEVLVLWGV


HHPPTGTDQQSLYQNADAYVSVGSSKYNRRFTPEIAARPKVRDQAGRMN


YYWTLLEPGDTITFEATGNLIAPWYAFALNRGSGSGIITSDAPVHDCNT


KCQTPHGAINSSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQS


RGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAIDGI


TNKVNSVIEKMNTQFTAVGKEFNNLERRIENLNKKVDDGFLDIWTYNAE


LLVLLENERTLDFHDSNVRNLYEKVKSQLKNNAKEIGNGCFEFYHKCDD


ACMESVRNGTYDYPKYSEESKLNREEIDGVKLESMGVYQILAIYSTVAS


SLVLLVSLGAISFWMCSNGSLQCRICI.


 


NA1 1918 synthetic gene 0607869, Based on acc. 


No.: AF250356: A/Breving mission/1/1918


Nucleotide


ATGAACCCCAACCAGAAGATCATCACCATCGGCAGCATCTGCATGGTGG


TGGGCATCATCAGCCTGATCCTGCAGATCGGCAACATCATCAGCATCTG


GGTGTCCCACAGCATCCAGACCGGCAACCAGAACCACCCCGAGACCTGC


AACCAGTCCATCATCACCTACGAGAACAACACCTGGGTGAACCAGACCT


ACGTGAACATCAGCAACACCAACGTGGTGGCCGGCCAGGACGCCACCTC


CGTGATCCTGACAGGCAACAGCAGCCTGTGCCCCATCAGCGGCTGGGCC


ATCTACAGCAAGGACAACGGCATCAGGATCGGCAGCAAGGGCGACGTGT


TCGTGATCAGAGAGCCCTTCATCAGCTGCAGCCACCTGGAATGCAGGAC


CTTCTTCCTGACCCAAGGAGCCCTGCTGAACGACAAGCACAGCAACGGC


ACCGTGAAGGACAGAAGCCCCTACAGGACCCTGATGAGCTGCCCCGTGG


GCGAGGCTCCCAGCCCCTACAACAGCAGATTCGAGAGCGTGGCCTGGTC


CGCCAGCGCCTGCCACGACGGCATGGGCTGGCTGACCATCGGCATCAGC


GGCCCTGACAACGGGGCCGTGGCCGTGCTGAAGTACAACGGAATCATCA


CCGACACCATCAAGAGCTGGCGGAACAACATCCTGAGGACCCAGGAAAG


CGAGTGCGCCTGCGTGAACGGCAGCTGCTTCACCATCATGACCGACGGC


CCCAGCAACGGCCAGGCCAGCTACAAGATCCTGAAGATCGAGAAGGGCA


AGGTGACCAAGAGCATCGAGCTGAACGCCCCCAACTACCACTACGAGGA


ATGCAGCTGCTACCCCGACACCGGCAAGGTCATGTGCGTGTGCAGGGAC


AACTGGCACGGCAGCAACAGGCCCTGGGTGTCCTTCGACCAGAACCTGG


ACTACCAGATCGGATACATCTGCAGCGGCGTGTTCGGCGACAACCCCAG


GCCCAACGACGGCACCGGCAGCTGCGGCCCTGTGAGCAGCAACGGGGCC


AATGGCATCAAGGGCTTCAGCTTCAGATACGACAACGGCGTGTGGATCG


GCCGCACCAAGAGCACCAGCAGCAGATCCGGCTTCGAGATGATCTGGGA


CCCCAACGGCTGGACCGAGACCGACAGCAGCTTCAGCGTGAGGCAGGAC


ATCGTGGCCATCACCGACTGGTCCGGCTACAGCGGCAGCTTCGTGCAGC


ACCCCGAGCTGACCGGCCTGGACTGCATGAGGCCCTGTTTCTGGGTGGA


GCTGATCAGAGGCCAGCCCAAGGAGAACACCATCTGGACCAGCGGCAGC


AGCATCAGCTTTTGCGGCGTGAACAGCGACACCGTGGGCTGGTCCTGGC


CCGACGGGGCCGAGCTGCCCTTCAGCATCGATAAGTGA


 


Amino acid


MNPNQKIITIGSICMVVGIISLILQIGNIISIWVSHSIQTGNQNHPETC


NQSIITYENNTWVNQTYVNISNYNVVAGQDATSVILTGNSSLCPISGWA


IYSKDNGIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKHSNG


TVKDRSPYRTLMSCPVGEAPSPYNSRFESVAWSASACHDGMGWLTIGIS


GPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTIMTDG


PSNGQASYKILKIEKGKVTKSIELNAPNYHYEECSCYPDTGKVMCVCRD


NWHGSNRPWVSFDQNLDYQIGYICSGVFGDNPRPNDGTGSCGPVSSNGA


NGIKGFSFRYDNGVWIGRTKSTSSRSGFEMIWDPNGWTETDSSFSVRQD


IVAITDWSGYSGSFVQHPELTGLDCMRPCFWVELIRGQPKENTIWTSGS


SISFCGVNSDTVGWSWPDGAELPFSIDK.


 


NP 1918 synthetic gene 0607866, Based on acc. 


No.: AY44935: A/Brevig mission/1/1918


Nucleotide


ATGGCCAGCCAGGGCACCAAGAGAAGCTACGAGCAGATGGAAACCGACG


GCGAGAGGCAGAACGCCACCGAGATCAGGGCCAGCGTGGGCAGGATGAT


CGGCGGCATCGGCAGGTTCTACATCCAGATGTGCACCGAGCTGAAGCTG


TCCGACTACGAGGGCAGGCTGATCCAGAACAGCATCACCATCGAGAGGA


TGGTGCTGTCCGCCTTCGACGAGAGAAGAAACAAGTACCTGGAAGAGCA


CCCCAGCGCCGGCAAGGACCCCAAGAAAACCGGCGGACCCATCTACAGA


AGGATCGACGGCAAGTGGATGAGAGAGCTGATCCTGTACGACAAGGAGG


AAATCAGAAGGATCTGGCGGCAGGCCAACAACGGCGAGGACGCCACAGC


CGGCCTGACCCACATGATGATCTGGCACAGCAACCTGAACGACGCCACC


TACCAGAGGACCAGGGCCCTCGTCAGAACCGGCATGGACCCCCGGATGT


GCAGCCTGATGCAGGGCAGCACACTGCCCAGAAGAAGCGGAGCTGCTGG


AGCCGCCGTGAAGGGCGTGGGCACCATGGTGATGGAACTGATCAGGATG


ATCAAGAGGGGCATCAACGACAGGAACTTTTGGAGGGGCGAGAACGGCA


GAAGGACCAGGATCGCCTACGAGAGGATGTGCAACATCCTGAAGGGCAA


GTTCCAGACAGCCGCCCAGAGGGCCATGATGGACCAGGTCCGGGAGAGC


AGGAACCCCGGCAACGCCGAGATCGAGGACCTGATCTTCCTGGCCAGAA


GCGCCCTGATCCTGAGGGGCAGCGTGGCCCACAAGAGCTGCCTGCCCGC


CTGCGTGTACGGACCCGCCGTGGCCAGCGGCTACGACTTCGAGAGAGAG


GGCTACAGCCTGGTCGCCATCGACCCCTTCAGGCTGCTGCAGAACTCCC


AGGTGTACTCTCTGATCAGGCCCAACGAGAACCCCGCCCACAAGTCCCA


GCTGGTCTGGATGGCCTGCCACAGCGCCGCCTTCGAGGATCTGAGAGTG


AGCAGCTTCATCAGGGGCACCAGAGTGGTGCCCAGGGGCAAGCTGTCCA


CCAGGGGCGTGCAGATCGCCAGCAACGAGAACATGGAAACCATGGACAG


CAGCACCCTGGAACTGAGAAGCAGGTACTGGGCCATCAGGACCAGAAGC


GGCGGCAACACCAACCAGCAGAGGGCCAGCGCCGGACAGATCAGCGTGC


AGCCCACCTTCTCCGTGCAGAGGAACCTGCCCTTCGAGAGGGCCACCAT


CATGGCCGCCTTCACCGGCAACACCGAGGGCAGGACCAGCGACATGAGG


ACCGAGATCATCAGAATGATGGAAAGCGCCAGGCCCGAGGACGTGAGCT


TCCAGGGCAGGGGCGTGTTCGAGCTGTCCGATGAGAAGGCCACCTCCCC


CATCGTGCCCAGCTTCGACATGAGCAACGAGGGCAGCTACTTCTTCGGC


GACAACGCCGAGGAATACGACAACTGA


 


Amino acid


MASQGTKRSYEQMETDGERQNATEIRASVGRMIGGIGRFYIQMCTELKL


SDYEGRLIQNSITIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYR


RIDGKWMRELILYDKEEIRRIWRQANNGEDATAGLTHMMIWHSNLNDAT


YQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRM


IKRGINDRNFWRGENGRRTRIAYERMCNILKGKFQTAAQRAMMDQVRES


RNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGPAVASGYDFERE


GYSLVGIDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRV


SSFIRGTRVVPRGKLSTRGVQIASNENMETMDSSTLELRSRYVVAIRTR


SGGNTNQQRASAGQISVQPTFSVQRNLPFERATIMAAFTGNTEGRTSDM


RTEIIRMMESARPEDVSFQGRGVFELSDEKATSPIVPSFDMSNEGSYFF


GDNAEEYDN.


 


M 1918 synthetic gene 0607868, acc. No.: 


AY130766: A/Brevig mission/1918


Nucleotide


ATGAGTCTTTTAACCGAGGTCGAAACGTACGTTCTCTCTATCGTCCCGT


CAGGCCCCCTCAAAGCCGAGATCGCGCAGAGACTTGAAGATGTCTTTGC


AGGGAAGAACACCGATCTTGAGGCTCTCATGGAATGGCTAAAGACAAGA


CCAATCCTGTCACCTCTGACTAAGGGGATTTTAGGATTTGTGTTCACGC


TCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCTTTGTCCAAAA


TGCCCTTAATGGGAACGGGGATCCAAATAACATGGACAGAGCAGTTAAA


CTGTACAGGAAGCTTAAGAGGGAGATAACATTCCATGGGGCCAAAGAAG


TAGCACTCAGTTATTCCGCTGGTGCACTTGCCAGTTGTATGGGCCTCAT


ATACAACAGGATGGGGACTGTGACCACTGAAGTGGCATTTGGCCTGGTA


TGCGCAACCTGTGAACAGATTGCTGATTCCCAGCATCGGTCTCACAGGC


AAATGGTGACAACAACCAATCCACTAATCAGACATGAGAACAGAATGGT


ACTGGCCAGCACTACGGCTAAGGCTATGGAGCAAATGGCTGGATCGAGT


GAGCAAGCAGCAGAGGCCATGGAGGTTGCTAGTCAGGCTAGGCAAATGG


TGCAGGCGATGAGAACCATTGGGACTCATCCTAGCTCCAGTGCTGGTCT


GAAAGACGATCTTATTGAAAATTTGCAGGCCTACCAGAAACGAATGGGG


GTGCAGATGCAACGATTCAAGTGATCCTCTCGTTATTGCCGCAAGTATC


ATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTCTTTTTTTCA


AATGCATTTATCGTCGCCTTAAATACGGTTTGAAAAGAGGGCCTTCTAC


GGAAGGAGTGCCGGAGTCTATGAGGGAAGAATATCGAAAGGAACAGCAG


AGTGCTGTGGATGTTGACGATGGTCATTTTGTCAACATAGAGCTGGAGT


AAGGCGCC


 


Amino acid


M1 protein


MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTR


PILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDRAVK


LYRKLKREITFHGAKEVALSYSAGALASCMGLIYNRMGTVTTEVAFGLV


CATCEQIADSQHRSHRQMVTTTNPLIRHENRMVLASTTAKAMEQMAGSS


EQAAEAMEVASQARQMVQAMRTIGTHPSSSAGLKDDLIENLQAYQKRMG


VQMQRFK.


 


M2 protein


MSLLTEVETPTRNEWGCRCNDSSDPLVIAASIIGILHLILWILDRLFFK


CIYRRLKYGLKRGPSTEGVPESMREEYRKEQQSAVDVDDGHFVNIELE.


 


HA 2009 H1N1v synthetic gene, based on acc. 


No ACP41105: A/California/04/09


atgaaggctatcctggtggtgctgctgtacaccttcgccaccgccaacg


ccgataccctgtgcatcggctaccacgccaacaacagcaccgacaccgt


ggataccgtgctggaaaagaacgtgaccgtgacccacagcgtgaacctg


ctggaagataagcacaacggcaagctgtgcaagctgagaggcgtggccc


ctctgcacctgggcaagtgcaatatcgccggctggatcctgggcaaccc


cgagtgcgagagcctgagcaccgccagctcttggtcctacatcgtggag


acacccagcagcgacaacggcacctgttaccccggcgacttcatcgact


acgaggaactgcgggagcagctgtccagcgtgtccagcttcgagcggtt


cgagatcttccccaagaccagctcctggcccaaccacgacagcaacaag


ggcgtgaccgccgcctgtcctcacgctggggccaagagcttctacaaga


acctgatctggctggtgaagaagggcaacagctaccccaagctgtccaa


gagctacatcaacgacaagggcaaagaggtgctggtgctgtggggcatc


caccaccctagcaccagcgccgaccagcagagcctgtaccagaacgccg


acacctacgtgttcgtgggcagcagccggtacagcaagaagttcaagcc


cgagatcgccatcagacccaaagtgcgggaccaggaaggccggatgaac


tactactggaccctggtggagcccggcgacaagatcaccttcgaggcca


ccggcaatctggtggtgcccagatacgccttcgccatggaaagaaacgc


cggcagcggcatcatcatcagcgacacccccgtgcacgactgcaacacc


acctgtcagacccccaagggggccatcaacaccagcctgcccttccaga


acatccaccccatcaccatcggcaagtgccctaagtacgtgaagtccac


caagctgagactggccaccggcctgcggaacatccccagcatccagagc


agaggcctgttcggggccattgccggctttatcgagggcggctggaccg


gaatggtggacgggtggtacggctaccaccaccagaatgagcagggcag


cggctacgccgccgacctgaagtccacacagaacgccatcgacgagatc


accaacaaagtgaacagcgtgatcgagaagatgaacacccagttcaccg


ccgtgggcaaagagttcaaccacctggaaaagcggatcgagaacctgaa


caagaaggtggacgacggcttcctggacatctggacctacaacgccgag


ctgctggtgctgctggaaaacgagcggaccctggactaccacgactcca


acgtgaagaatctgtacgagaaagtgcggagccagctgaagaacaacgc


caaagagatcggcaacggctgcttcgagttctaccacaagtgcgacaac


acctgtatggaaagcgtgaagaacggcacctacgactaccccaagtaca


gcgaggaagccaagctgaaccgggaagagatcgacggcgtgaagctgga


aagcacccggatctaccagatcctggccatctacagcaccgtggccagc


tcactggtcctggtcgtgtccctgggcgctatcagcttctggatgtgca


gcaacggcagcctgcagtgccggatctgcatctgaggcgccgagaattc


ttaattaa


 


Amino acid sequence


MKAILVVLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLL


EDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVET


PSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKG


VTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIH


HPSTSADQQSLYQNADTYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNY


YWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTT


CQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSR


GLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEIT


NKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAEL


LVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNT


CMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQILAIYSTVASS


LVLVVSLGAISFWMCSNGSLQCRICI


 


NA 2009 H1N1v synthetic gene, based on acc. 


No ACP41107: A/California/04/09


atgaaccccaaccagaagatcatcaccatcggcagcgtgtgcatgacca


tcggcatggccaacctgatcctgcagatcggcaacatcatcagcatctg


gatcagccacagcatccagctgggcaaccagaaccagatcgagacatgc


aaccagagcgtgatcacctacgagaacaacacctgggtgaaccagacct


acgtgaacatcagcaacaccaacttcgccgctggccagagcgtggtgtc


tgtgaagctggccggcaacagcagcctgtgccctgtgtccggctgggcc


atctacagcaaggacaacagcgtgcggatcggcagcaagggcgacgtgt


tcgtgatccgggagcccttcatcagctgcagccccctggaatgccggac


cttcttcctgacccagggggccctgctgaacgacaagcacagcaacggc


accatcaaggacagaagcccctaccggaccctgatgagctgccccatcg


gcgaggtgcccagcccctacaacagcagattcgagtccgtggcttggag


cgcctctgcctgccacgacggcatcaactggctgacaatcggcatcagc


ggccctgataacggcgctgtggccgtgctgaagtacaacggcatcatca


ccgacacaatcaagagctggcggaacaacatcctgcggacccaggaatc


cgagtgcgcctgcgtgaacggcagctgcttcaccgtgatgaccgacggc


cctagcaatggccaggccagctacaagatcttccggatcgagaagggca


agatcgtgaagtccgtggagatgaacgcccccaactaccactacgagga


atgcagctgctaccccgacagcagcgagatcacctgtgtgtgccgggac


aactggcacggcagcaacagaccctgggtgtccttcaaccagaatctgg


aataccagatcggctacatttgcagcggcatcttcggcgacaaccccag


acccaacgacaagaccggaagctgcggccctgtgtctagcaacggggcc


aacggcgtgaagggcttcagcttcaagtacggcaatggcgtgtggatcg


gccggaccaagagcatcagcagccggaacggcttcgagatgatctggga


ccccaacggctggaccggcaccgacaacaacttcagcatcaagcaggac


atcgtgggcatcaacgagtggagcggctacagcggcagcttcgtgcagc


accctgagctgaccggcctggactgcatccggccctgcttttgggtgga


gctgatcagaggcagacccaaagagaacaccatctggaccagcggcagc


agcatcagcttttgcggcgtgaacagcgacaccgtgggctggtcttggc


ccgatggggccgagctgcccttcaccatcgacaagtgaggcgccgagaa


ttcttaattaa


 


Amino acid sequence


MNPNQKIITIGSVCMTIGMANLILQIGNIISIWISHSIQLGNQNQIETC


NQSVITYENNTWVNQTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWA


IYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNG


TIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGIS


GPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDG


PSNGQASYKIFRIEKGKIVKSVEMNAPNYHYEECSCYPDSSEITCVCRD


NWHGSNRPWVSFNQNLEYQIGYICSGIFGDNPRPNDKTGSCGPVSSNGA


NGVKGFSFKYGNGVWIGRTKSISSRNGFEMIWDPNGWTGTDNNFSIKQD


IVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKENTIWTSGS


SISFCGVNSDTVGWSWPDGAELPFTIDK


 


HA H3N2 seasonal synthetic gene, based on acc. 


No EU103823: A/Wisconsin/67/05


atgaaaaccatcatcgccctgagctacatcctgtgcctggtgttcgccc


agaagctgcccggcaacgacaacagcaccgccaccctgtgcctgggcca


ccacgccgtgcccaacggcaccatcgtcaaaaccattaccaatgatcag


atcgaggtgaccaacgccaccgagctggtgcagagcagcagcaccggcg


gcatctgcgacagcccccaccagatcctggacggcgagaactgcaccct


gatcgacgctctgctcggcgaccctcagtgcgacggcttccagaacaag


aagtgggacctgttcgtcgagcgcagcaaggcctacagcaactgctacc


cctacgacgtgcccgactacgccagcctccgctccctggtcgcctccag


cggcaccctggagttcaacgacgagagcttcaactggaccggcgtgacc


cagaacggcaccagcagcagctgcaagcgccgcagcaacaacagcttct


tcagccgcctgaactggctgacccacctgaagttcaagtaccccgccct


gaacgtgaccatgcccaacaatgagaaattcgacaagctgtacatctgg


ggcgtgcaccaccccgtgaccgacaacgaccagatcttcctgtacgccc


aggccagcggccgcatcaccgtgagcaccaagcgcagccagcagaccgt


gatccccaacatcggcagccgccccaggatccgcaacatccccagccgc


atcagcatctactggaccatcgtgaagcccggcgacatcctgctgatca


actccaccggcaacctgatcgcccccaggggctacttcaagatccgcag


cggcaagagcagcatcatgcgcagcgacgcccccatcggcaagtgcaac


agcgagtgcatcacccccaacggcagcatccccaacgacaagcccttcc


agaacgtgaaccgcatcacctacggagcctgtccccgctacgtgaagca


gaacaccctgaaactggctaccggcatgcggaacgtgcccgagaagcag


acccggggcatcttcggggccatcgccggcttcatcgagaacggctggg


agggcatggtggacgggtggtatggcttccgccaccagaactccgaggg


catcggccaggccgccgacctgaagagcacccaggccgccatcaaccag


atcaacggcaagctgaaccgcctgatcggcaagaccaacgagaagttcc


accagatcgagaaggagtttagcgaggtcgagggccgcatccaggacct


ggagaagtacgtggaggacaccaagatcgacctgtggagctacaacgcc


gagctgctggtcgccctggagaaccagcacaccatcgacctgaccgaca


gcgagatgaacaagctgttcgagcgcaccaagaagcagctgcgcgagaa


cgccgaggacatgggcaacggctgcttcaagatctaccacaagtgcgac


aacgcctgcatcggctccatccgcaacggcacctacgaccacgacgtgt


accgcgacgaggccctgaacaaccgcttccagatcaagggcgtggagct


gaagagcggctacaaggactggatcctgtggatcagcttcgctatcagc


tgcttcctgctgtgcgtggccctgctgggcttcatcatgtgggcctgcc


agaagggcaacatccgctgcaacatctgcatc


 


Amino acid


MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQ


IEVTNATELVQSSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNK


KWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNDESFNWTGVT


QNGTSSSCKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIW


GVHHPVTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSR


ISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCN


SECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQ


TRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAINQ


INGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNA


ELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYHKCD


NACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAIS


CFLLCVALLGFIMWACQKGNIRCNICI


 


NA H3N2 seasonal synthetic gene, based on acc. 


No: ISDN 136490: A/Wisconsin/67/05


atgaaccccaaccagaagatcatcaccatcggatccgtcagcctgacca


tctccaccatctgctttttcatgcagatcgccatcctgatcaccaccgt


gaccctgcacttcaagcagtacgagttcaacagcccccccaacaaccag


gtcatgctgtgcgagcccaccatcatcgagcgcaacatcaccgagatcg


tgtacctgaccaacaccaccatcgagaaagagatctgccccaagctggc


cgagtaccgcaactggtccaagccccagtgcaatatcacaggcttcgcc


cccttcagcaaggacaacagcatccgcctgagcgctggaggggacatct


gggtcacccgcgagccctacgtgagctgcgaccccgacaagtgctacca


gttcgccctcggacaggggaccacactgaataacgtccacagcaacgac


accgtgcacgaccgcaccccctaccgcaccctgctgatgaacgagctgg


gcgtgcccttccacctgggcaccaagcaggtctgcatcgcctggtccag


cagcagctgccacgacggcaaggcctggctgcacgtgtgcgtgaccggc


gacgacaagaacgccaccgccagcttcatctacaacggccgcctggtgg


acagcatcgtgagctggtccaaagagatcctgcgcacccaagaaagcga


gtgcgtctgcatcaacggcacctgcaccgtggtgatgaccgacggcagc


gcctccggcaaggccgacaccaagatcctgttcatcgaagagggcaaga


tcgtgcacaccagcacactgtccggcagcgcccagcacgtggaagagtg


cagctgctacccccgctacctgggcgtgcgctgcgtgtgccgcgacaac


tggaagggcagcaaccgccccatcgtggacatcaacatcaaggactact


ccatcgtgagcagctacgtgtgcagcggcctggtcggcgacacaccccg


caagaacgacagcagctccagcagccactgcctggaccccaacaacgaa


gagggcggccacggcgtgaagggctgggccttcgacgacggcaacgacg


tgtggatgggccgcaccatcagcgagaagctgcggagcggctatgagac


attcaaggtgatcgagggctggtccaaccccaacagcaagctgcagatc


aaccgccaggtgatcgtggaccgcggcaaccgctccggctacagcggca


tcttcagcgtggagggcaagtcctgcatcaaccgctgcttctatgtgga


gctgattcgggggaggaaagaagagaccgaggtcctctggaccagcaac


agcatcgtggtgttctgcggcaccagcggcacctacggcaccggcagct


ggcccgacggggccgacatcaacctgatgcccatctga


 


Amino acid


MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQ


VMLCEPTIIERNITEIVYLTNTTIEKEICPKLAEYRNWSKPQCNITGFA


PFSKDNSIRLSAGGDIWVTREPYVSCDPDKCYQFALGQGTTLNNVHSND


TVHDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCVTG


DDKNATASFIYNGRLVDSIVSWSKEILRTQESECVCINGTCTVVMTDGS


ASGKADTKILFIEEGKIVHTSTLSGSAQHVEECSCYPRYLGVRCVCRDN


WKGSNRPIVDINIKDYSIVSSYVCSGLVGDTPRKNDSSSSSHCLDPNNE


EGGHGVKGWAFDDGNDVWMGRTISEKLRSGYETFKVIEGWSNPNSKLQI


NRQVIVDRGNRSGYSGIFSVEGKSCINRCFYVELIRGRKEETEVLWTSN


SIVVFCGTSGTYGTGSWPDGADINLMPI.









All natural genes applied in the current DNA vaccines are public available as nucleotide and amino acid sequences but can be translated into optimal vaccine DNA genes by synthesis using 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 clinical irrelevant kanamycin resistance gene are included for growth and selection in transfected E. coli for plasmid DNA production.
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 H1N1 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 Calcdonia/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 HA1 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 Calcdonia). This suggest that the 1918 based DNA vaccine induces a much broader protective immunity that protects against influenza A H1N1 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 H1N1 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 H1N1 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 is 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.
One of these features is the accumulation of N-linked glycosylations of the glycoproteins. Since the first introductions of the pandemic H1N1 and H3N2 the glycoproteins have gained several gycosylations (FIG. 1). The oligosaccharides are added to asparagines (N) in the tripeptide sequence Asn-X-Ser of Asn-X-Thr, where X could be any amino acid except proline. If these glycosylations occur in or nearby an antigenic binding site they camouflage the binding sites for recognition by the host antibodies. The glycosylation sites at positions 126 and 144 in the H3 haemagglutinin have been variable since 1999 in Denmark, these sites are located in the HA antigenic site A and are therefore expected to influence on host antibody recognition. These sites and other in or near antigenic sites in HA and/or NA can be erased to give a broader cross-protection than fully glycosylated antigens.
A shown the present time viruses A/New Calcdonia/20/99 and A/Wisconsin/67/05 comprise more predicted N-glycosylated sites than the original pandemic H1N1 or H3N2 strains (FIG. 1).
A DNA vaccine stripped for glycosylations in, and in close vicinity of, the antigenic sites of both HA and NA would induce a broader subtype recognition.
The unusual broad protection we have observed with DNA vaccines based on pandemic strains is due to unique features of an unadapted pandemicstrain e.g. ability of the pandemic genes inducing broader protective antibodies and cellular immunity due to less masked cross-reactive antigenic sites or immune adjuvans effect etc., The influenza virus haemagglutinin accumulates glycosylations and thereby camouflages its antigenic sites for recognition by the host immune system. The good cross protection observed by applying the pandemic genes in the influenza DNA vaccine is partly explained by the few glycosylation sites in these genes which we suggest in the analysis in the Table below:










Asparagines predicted to be N-glycosylated a


Amino acid position











HA H1N1
HA H1N1
HA H1N1v
HA H3N2
HA H3N2


1918
1999
2009
1968
2005





 11
 11
 11
 8
 8


 23
 23
 23
 22
 22


 87
 87
 87
 38
 63


287
125
287
 81
133


481 *
154
481 *
165
144


540 *
286 (287)
540 *
285
165



480 * (481)

483 *
246



539 * (540)


285






483 *





a Amino acid numbering follows H1N1 and H3N2 respectively.


* Positions also positively predicted; however, the sequons are positioned in the HA2 region, not likely to be glycosylated.






The pandemic glycoproteins of a current pandemic either alone or in combination with previous pandemic genes or seasonal influenza virus genes in a DNA vaccine will induce optimal protection against the current pandemic virus and the following drifted versions of the same subtype.
The DNA or RNA sequence for haemagglutinin and neuraminidase, e.g. from non-pandemic seasonal strains, can be changed so the sequence coding for said proteins is changed to code for said proteins with less or no glycosylation sites.
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, or a lipid-based formulation for more efficient delivery
The universal seasonal influenza A DNA vaccine should comprise H1N1 HA and/or NA glycoproteins from the most recent H1N1 pandemic, H3N2 HA and/or NA glycoproteins from the last H3N2 pandemic and/or the most recent epidemic together with the genes coding for the internal genes. The internal genes NP and/or M from one of the pandemics e.g. H1N1 1918 that has shown protection in our animal model.
The DNA construct used is a modified WRG7079 plasmid (Backbone MVLF #1528). Our synthetic flu genes codon optimised for humans have been inserted between the Kas1 and Mlu1 restriction sites in the expression vector.







TABEL 2





H3N2 and H2N2 nucleotide and  


amino acid sequences of the


genes (not codon optimized).







HA H3N2 Acc. No.: AB295605: A/Aichi/2/1968(H3N2)


Nucleotide


ATAATTCTATTAATCATGAAGACCATCATTGCTTTGAGCTACATTTTCT


GTCTGGCTCTCGGCCAAGACCTTCCAGGAAATGACAACAGCACAGCAAC


GCTGTGCCTGGGACATCATGCGGTGCCAAACGGAACACTAGTGAAAACA


ATCACAGATGATCAGATTGAAGTGACTAATGCTACTGAGCTAGTTCAGA


GCTCCTCAACGGGGAAAATATGCAACAATCCTCATCGAATCCTTGATGG


AATAGACTGCACACTGATAGATGCTCTATTGGGGGACCCTCATTGTGAT


GTTTTTCAAAATGAGACATGGGACCTTTTCGTTGAACGCAGCAAAGCTT


TCAGCAACTGTTACCCTTATGATGTGCCAGATTATGCCTCCCTTAGGTC


ACTAGTTGCCTCGTCAGGCACTCTGGAGTTTATCACTGAGGGTTTCACT


TGGACTGGGGTCACTCAGAATGGGGGAAGCAATGCTTGCAAAAGGGGAC


CTGGTAGCGGTTTTTTCAGTAGACTGAACTGGTTGACCAAATCAGGAAG


CACATATCCAGTGCTGAACGTGACTATGCCAAACAATGACAATTTTGAC


AAACTATACATTTGGGGGGTTCACCACCCGAGCACGAACCAAGAACAAA


CCAGCCTGTATGTTCAAGCATCAGGGAGAGTCACAGTCTCTACCAGGAG


AAGCCAGCAAACTATAATCCCGAATATCGAGTCCAGACCCTGGGTAAGG


GGTCTGTCTAGTAGAATAAGCATCTATTGGACAATAGTTAAGCCGGGAG


ACGTACTGGTAATTAATAGTAATGGGAACCTAATCGCTCCTCGGGGTTA


TTTCAAAATGCGCACTGGGAAAAGCTCAATAATGAGGTCAGATGCACCT


ATTGATACCTGTATTTCTGAATGCATCACTCCAAATGGAAGCATTCCCA


ATGACAAGCCCTTTCAAAACGTAAACAAGATCACATATGGAGCATGCCC


CAAGTATGTTAAGCAAAACACCCTGAAGTTGGCAACAGGGATGCGGAAT


GTACCAGAGAAACAAACTAGAGGCCTATTCGGCGCAATAGCAGGTTTCA


TAGAAAATGGTTGGGAGGGAATGATAGACGGTTGGTACGGTTTCAGGCA


TCAAAATTCTGAGGGCACAGGACAAGCAGCAGATCTTAAAAGCACTCAA


GCAGCCATCGACCAAATCAATGGGAAATTGAACAGGGTAATCGAGAAGA


CGAACGAGAAATTCCATCAAATCGAAAAGGAATTCTCAGAAGTAGAAGG


GAGAATTCAGGACCTCGAGAAATACGTTGAAGACACTAAAATAGATCTC


TGGTCTTACAATGCGGAGCTTCTTGTCGCTCTGGAGAATCAACATACAA


TTGACCTGACTGACTCGGAAATGAACAAGCTGTTTGAAAAAACAAGGAG


GCAACTGAGGGAAAATGCTGAAGACATGGGCAATGGTTGCTTCAAAATA


TACCACAAATGTGACAACGCTTGCATAGAGTCAATCAGAAATGGGACTT


ATGACCATGATGTATACAGAGACGAAGCATTAAACAACCGGTTTCAGAT


CAAAGGTGTTGAACTGAAGTCTGGATACAAAGACTGGATCCTGTGGATT


TCCTTTGCCATATCATGCTTTTTGCTTTGTGTTGTTTTGCTGGGGTTCA


TCATGTGGGCCTGCCAGAGAGGCAACATTAGGTGCAACATTTGCATTTG


AGTGTATTAGTAATTA


 


Amino acid


MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQ


IEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNE


TWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVT


QNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIW


GVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIESRPWVRGLSSR


ISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCI


SECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQ


TRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQ


INGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNA


ELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCD


NACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAIS


CFLLCVVLLGFIMWACQRGNIRCNICI


 


NA H3N2 Acc. No.: AB29606: A/Aichi/2/1968(H3N2)


Nucleotide


GAAAATGAATCCAAATCAAAAGATAATAACAATTGGCTCTGTCTCTCTC


ACCATTGCAACAGTATGCTTCCTCATGCAGATTGCCATCCTGGTAACTA


CTGTAACATTGCATTTTAAGCAATATGAGTGCGACTCCCCCGCGAGCAA


CCAAGTAATGCCGTGTGAACCAATAATAATAGAAAGGAACATAACAGAG


ATAGTGTATTTGAATAACACCACCATAGAGAAAGAGATATGCCCCAAAG


TAGTGGAATACAGAAATTGGTCAAAGCCGCAATGTCAAATTACAGGATT


TGCACCTTTTTCTAAGGACAATTCAATCCGGCTTTCTGCTGGTGGGGAC


ATTTGGGTGACGAGAGAACCTTATGTGTCATGCGATCATGGCAAGTGTT


ATCAATTTGCACTCGGGCAGGGGACCACACTAGACAACAAACATTCAAA


TGACACAATACATGATAGAATCCCTCATCGAACCCTATTAATGAATGAG


TTGGGTGTTCCATTTCATTTAGGAACCAGGCAAGTGTGTATAGCATGGT


CCAGCTCAAGTTGTCACGATGGAAAAGCATGGCTGCATGTTTGTATCAC


TGGGGATGACAAAAATGCAACTGCTAGCTTCATTTATGACGGGAGGCTT


GTGGACAGTATTGGTTCATGGTCTCAAAATATCCTCAGAACCCAGGAGT


CGGAATGCGTTTGTATCAATGGGACTTGCACAGTAGTAATGACTGATGG


AAGTGCTTCAGGAAGAGCCGATACTAGAATACTATTCATTGAAGAGGGG


AAAATTGTCCATATTAGCCCATTGTCAGGAAGTGCTCAGCATGTAGAAG


AGTGTTCCTGTTATCCTAGATATCCTGGCGTCAGATGTATCTGCAGAGA


CAACTGGAAAGGCTCTAATAGGCCCGTCGTAGACATAAATATGGAAGAT


TATAGCATTGATTCCAGTTATGTGTGCTCAGGGCTTGTTGGCGACACAC


CTAGAAACGACGACAGATCTAGCAATAGCAATTGCAGGAATCCTAATAA


TGAGAGAGGGAATCAAGGAGTGAAAGGCTGGGCCTTTGACAATGGAGAT


GACGTGTGGATGGGAAGAACGATCAGCAAGGATTTACGCTCAGGTTATG


AAACTTTCAAAGTCATTGGTGGTTGGTCCACACCTAATTCCAAATCGCA


GATCAATAGACAAGTCATAGTTGACAGCGATAATCGGTCAGGTTACTCT


GGTATTTTCTCTGTTGAGGGCAAAAGCTGCATCAATAGGTGCTTTTATG


TGGAGTTGATAAGGGGAAGGAAACAGGAGACTAGAGTGTGGTGGACCTC


AAACAGTATTGTTGTGTTTTGTGGCACTTCAGGTACCTATGGAACAGGC


TCATGGCCTGATGGGGCGAACATCAATTTCATGCCTATATAAGCTTTCG


CAATTTTAGA


 


Amino acid


MNPNQKIITIGSVSLTIATVCFLMQIAILVTTVTLHFKQYECDSPASNQ


VMPCEPIIIERNITEIVYLNNTTIEKEICPKVVEYRNWSKPQCQITGFA


PFSKDNSIRLSAGGDIWVTREPYVSCDHGKCYQFALGQGTTLDNKHSND


TIHDRIPHRTLLMNELGVPFHLGTRQVCIAWSSSSCHDGKAWLHVCITG


DDKNATASFIYDGRLVDSIGSWSQNILRTQESECVCINGTCTVVMTDGS


ASGRADTRILFIEEGKIVHISPLSGSAQHVEECSCYPRYPGVRCICRDN


WKGSNRPVVDINMEDYSIDSSYVCSGLVGDTPRNDDRSSNSNCRNPNNE


RGNQGVKGWAFDNGDDVWMGRTISKDLRSGYETFKVIGGWSTPNSKSQI


NRQVIVDSDNRSGYSGIFSVEGKSCINRCFYVELIRGRKQETRVWWTSN


SIVVFCGTSGTYGTGSWPDGANINFMPI


 


HA H2N2 Acc. No.: CY022013:


 A/Albany/20/1957(H2N2)


Nucleotide


ATAGACAACCAAAAGCAAAACAATGGCCATCATTTATCTCATTCTCCTG


TTCACAGCAGTGAGAGGGGACCAGATATGCATTGGATACCATGCCAATA


ATTCCACAGAGAAGGTCGACACAATTCTAGAGCGGAACGTCACTGTGAC


TCATGCCAAGGACATTCTTGAGAAGACCCATAACGGAAAGTTATGCAAA


CTAAACGGAATCCCTCCACTTGAACTAGGGGACTGTAGCATTGCCGGAT


GGCTCCTTGGAAATCCAGAATGTGATAGGCTTCTAAGTGTGCCAGAATG


GTCCTATATAATGGAGAAAGAAAACCCGAGAGACGGTTTGTGTTATCCA


GGCAGCTTCAATGATTATGAAGAATTGAAACATCTCCTCAGCAGCGTGA


AACATTTCGAGAAAGTAAAGATTCTGCCCAAAGATAGATGGACACAGCA


TACAACAACTGGAGGTTCACGGGCCTGCGCGGTGTCTGGTAATCCATCA


TTCTTCAGGAACATGATCTGGCTGACAAAGAAAGGATCAAATTATCCGG


TTGCCAAAGGATCGTACAACAATACAAGCGGAGAACAAATGCTAATAAT


TTGGGGGGTGCACCATCCCAATGATGAGACAGAACAAAGAACATTGTAC


CAGAATGTGGGAACCTATGTTTCCGTAGGCACATCAACATTGAACAAAA


GGTCAACCCCAGACATAGCAACAAGGCCTAAAGTGAATGGACTAGGAAG


TAGAATGGAATTCTCTTGGACCCTATTGGATATGTGGGACACCATAAAT


TTTGAGAGTACTGGTAATCTAATTGCACCAGAGTATGGATTCAAAATAT


CGAAAAGAGGTAGTTCAGGGATCATGAAAACAGAAGGAACACTTGGGAA


CTGTGAGACCAAATGCCAAACTCCTTTGGGAGCAATAAATACAACATTG


CCTTTTCACAATGTCCACCCACTGACAATAGGTGAGTGCCCCAAATATG


TAAAATCGGAGAAGTTGGTCTTAGCAACAGGACTAAGGAATGTTCCCCA


GATTGAATCAAGAGGATTGTTTGGGGCAATAGCTGGTTTTATAGAAGGA


GGATGGCAAGGAATGGTTGATGGTTGGTATGGATACCATCACAGCAATG


ACCAGGGATCAGGGTATGCAGCGGACAAAGAATCCACTCAAAAGGCATT


TGATGGAATCACCAACAAGGTAAATTCTGTGATTGAAAAGATGAACACC


CAATTTGAAGCTGTTGGGAAAGAATTCAGTAACTTAGAGAGAAGACTGG


AGAACTTGAACAAAAAGATGGAAGACGGGTTTCTAGATGTGTGGACATA


CAATGCTGAGCTTCTAGTTCTGATGGAAAATGAGAGGACACTTGACTTT


CATGATTCTAATGTCAAGAATCTGTATGATAAAGTCAGAATGCAGCTGA


GAGACAACGTCAAAGAACTAGGAAATGGATGTTTTGAATTTTATCACAA


ATGTGATGATGAATGCATGAATAGTGTGAAAAACGGGACGTATGATTAT


CCCAAGTATGAAGAAGAGTCTAAACTAAATAGAAATGAAATCAAAGGGG


TAAAATTGAGCAGCATGGGGGTTTATCAAATCCTTGCCATTTATGCTAC


AGTAGCAGGTTCTCTGTCACTGGCAATCATGATGGCTGGGATCTCTTTC


TGGATGTGCTCCAACGGGTCTCTGCAGTGCAGGATCTGCATATGATTAT


AAGTCATTTTATAATTAA


 


Amino acid


MAIIYLILLFTAVRGDQICIGYHANNSTEKVDTILERNVTVTHAKDILE


KTHNGKLCKLNGIPPLELGDCSIAGWLLGNPECDRLLSVPEWSYIMEKE


NPRDGLCYPGSFNDYEELKHLLSSVKHFEKVKILPKDRWTQHTTTGGSR


ACAVSGNPSFFRNMIWLTKKGSNYPVAKGSYNNTSGEQMLIIWGVHHPN


DETEQRTLYQNVGTYVSVGTSTLNKRSTPDIATRPKVNGLGSRMEFSWT


LLDMWDTINFESTGNLIAPEYGFKISKRGSSGIMKTEGTLGNCETKCQT


PLGAINTTLPFHNVHPLTIGECPKYVKSEKLVLATGLRNVPQIESRGLF


GAIAGFIEGGWQGMVDGWYGYHHSNDQGSGYAADKESTQKAFDGITNKV


NSVIEKMNTQFEAVGKEFSNLERRLENLNKKMEDGFLDVWTYNAELLVL


MENERTLDFHDSNVKNLYDKVRMQLRDNVKELGNGCFEFYHKCDDECMN


SVKNGTYDYPKYEEESKLNRNEIKGVKLSSMGVYQILAIYATVAGSLSL


AIMMAGISFWMCSNGSLQCRICI


 


NA H2N2 Acc. No.: CY022015:


 A/Albany/20/1957(H2N2)


Nucleotide


TGAAAATGAATCCAAATCAAAAGATAATAACAATTGGCTCTGTCTCTCT


CACCATTGCAACAGTATGCTTCCTCATGCAGATTGCCATCCTGGCAACT


ACTGTGACATTGCATTTTAAACAACATGAGTGCGACTCCCCCGCGAGCA


ACCAAGTAATGCCATGTGAACCAATAATAATAGAAAGGAACATAACAGA


GATAGTGTATTTGAATAACACCACCATAGAGAAAGAGATTTGCCCCGAA


GTAGTGGAATACAGAAATTGGTCAAAGCCGCAATGTCAAATTACAGGAT


TTGCACCTTTTTCTAAGGACAATTCAATCCGGCTTTCTGCTGGTGGGGA


CATTTGGGTGACGAGAGAACCTTATGTGTCATGCGATCCTGGCAAGTGT


TATCAATTTGCACTCGGGCAAGGGACCACACTAGACAACAAACATTCAA


ATGGCACAATACATGATAGAATCCCTCACCGAACCCTATTAATGAATGA


GTTGGGTGTTCCATTTCATTTAGGAACCAAACAAGTGTGTGTAGCATGG


TCCAGCTCAAGTTGTCACGATGGAAAAGCATGGTTGCATGTTTGTGTCA


CTGGGGATGATAGAAATGCGACTGCCAGCTTCATTTATGACGGGAGGCT


TGTGGACAGTATTGGTTCATGGTCTCAAAATATCCTCAGGACCCAGGAG


TCGGAATGCGTTTGTATCAATGGGACTTGCACAGTAGTAATGACTGATG


GAAGTGCATCAGGAAGAGCCGATACTAGAATACTATTCATTAAAGAGGG


GAAAATTGTCCATATCAGCCCATTGTCAGGAAGTGCTCAGCATATAGAG


GAGTGTTCCTGTTACCCTCGATATCCTGACGTCAGATGTATCTGCAGAG


ACAACTGGAAAGGCTCTAATAGGCCCGTTATAGACATAAATATGGAAGA


TTATAGCATTGATTCCAGTTATGTGTGCTCAGGGCTTGTTGGCGACACA


CCCAGGAACGACGACAGCTCTAGCAATAGCAATTGCAGGGATCCTAACA


ATGAGAGAGGGAATCCAGGAGTGAAAGGCTGGGCCTTTGACAATGGAGA


TGATGTATGGATGGGAAGAACAATCAACAAAGATTCACGCTCAGGTTAT


GAAACTTTCAAAGTCATTGGTGGTTGGTCCACACCTAATTCCAAATCGC


AGGTCAATAGACAGGTCATAGTTGACAACAATAATTGGTCTGGTTACTC


TGGTATTTTCTCTGTTGAGGGCAAAAGCTGCATCAATAGGTGCTTTTAT


GTGGAGTTGATAAGGGGAAGGCCACAGGAGACTAGAGTATGGTGGACCT


CAAACAGTATTGTTGTGTTTTGTGGCACTTCAGGTACTTATGGAACAGG


CTCATGGCCTGATGGGGCGAACATCAATTTCATGCCTATATAAGCTTTC


GCAATTTTAGAAAA


 


Amino acid


MNPNQKIITIGSVSLTIATVCFLMQTAILATTVTLHFKQHECDSPASNQ


VMPCEPIIIERNITEIVYLNNTTIEKEICPEVVEYRNWSKPQCQITGFA


PFSKDNSIRLSAGGDIWVTREPYVSCDPGKCYQFALGQGTTLDNKHSNG


TIHDRIPHRTLLMNELGVPFHLGTKQVCVAWSSSSCHDGKAWLHVCVTG


DDRNATASFIYDGRLVDSIGSWSQNILRTQESECVCINGTCTVVMTDGS


ASGRADTRILFIKEGKIVHISPLSGSAQHIEECSCYPRYPDVRCICRDN


WKGSNRPVIDINMEDYSIDSSYVCSGLVGDTPRNDDSSSNSNCRDPNNE


RGNPGVKGWAFDNGDDVWMGRTINKDSRSGYETFKVIGGWSTPNSKSQV


NRQVIVDNNNWSGYSGIFSVEGKSCINRCFYVELIRGRPQETRVWWTSN


SIVVFCGTSGTYGTGSWPDGANINFMPI









To further investigate the principle of a pandemic DNA vaccine, we designed a DNA vaccine based on the pandemic HA and NA genes of the Hong Flu 1968 H3N2 virus. Again, the pandemic DNA vaccine induced protection against a contemporary H3N2 virus infection. More rapid virus clearance and high antibody levels against the challenge strain was experienced.
Nucleotides and Proteins Covered by the Present Invention:













Nucleotides and proteins
SEQ ID NO


















Synthetic 1918 H1N1 HA DNA
1



1918 H1N1 HA protein
2



Synthetic 1918 H1N1 NA DNA
3



1918 H1N1 NA protein
4



Synthetic 1918 H1N1 NP DNA
5



1918 H1N1 NP protein
6



Synthetic 1918 H1N1 M DNA
7



1918 H1N1 M1 protein
8



1918 H1N1 M2 protein
9



Synthetic 2009 H1N1v HA DNA
10



2009 H1N1v HA protein
11



Synthetic 2009 H1N1v NA DNA
12



2009 H1N1v NA protein
13



Synthetic seasonal H3N2 HA DNA
14



Seasonal H3N2 HA protein
15



Synthetic seasonal H3N2 NA DNA
16



Seasonal H3N2 NA protein
17












FIGURE LEGENDS
FIG. 1: Predicted N-glycosylation sites in the haemagglutinin protein. N-glycosylated sequons predicted in HA of the pandemic a) H1N1 1918 (A/South Carolina/1/18(H1N1) and b) H3N2 1968 (A/Aichi/2/1968(H3N2)) compared to present time c) H1N1 (A/New Calcdonia/20/99(H1N1)) and d) H3N2 (A/Wisconsin/67/05) viruses above 0.5 threshold value.
FIG. 2: Initial H1N1 DNA vaccine IgG antibody and virus titer results. Ferrets were vaccinated as in example 2 with codon optimised HA+NA 1918 H21N1 DNA and HA+NA+NP+M 1918 H1N1 or 1999 H1N1 HA+NA non-codon optimized present time virus DNA and challenged with a virus from 1947. (A) Mean serum specific IgG antibody response (ELISA) in ferrets to influenza A of A/New Calcdonia/20/99(H1N1) days after viral challenge and (B) number of viral RNA copies (real time RT/PCR) in nasal wash in days after challenge. Six ferrets in each group.
FIG. 3: HA+NA 1918 H1N1 pandemic H1N1 DNA vaccinated ferrets challenged with 1999 H1N1 virus circulating season 2007. (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.
FIG. 4: Hemadsorption as a measure of functional protein expression in mammalian cells of codon optimized HA from 1918 H1N1 (HA 1918), avian H5N7 (HA H5N7) and 1968 H3N2 (HA H3N2) compared to non-codon optimized 1918 H1N1 (HA NC)
FIG. 5: H3N2 2007/08 virus titre in nasal washings from ferrets vaccinated with either 1968 H3N2 DNA or 2005 H3N2 DNA (circulating sesong 2007/8)
FIG. 6: H3N2 2007/08 influenza A virus specific IgG antibodies in ferret sera post challenge.
FIG. 7: Haemagglutination inhibition assay against A: 1968 (A/Aichi/2/68) and B: 2007/08 (A/Wisconsin/67/05) H3N2 virus.
FIG. 8: 2007 H1N1 Virus titer in ferret nasal washings after A/New Calcdonia/20/99 infection.
FIG. 9: Haemagglutination inhibition assay against A/new Calcdonia/20/99(H1N1) virus.
FIG. 10: H1N1 2007 (A/New Calcdonia/20/99 influenza specific IgG antibodies in ferret sera post challenge.
FIG. 11: Pandemic H1N1v DNA vaccine in swine clear seasonal swine h1n1 virus infection
Pigs were vaccinated as described in example 15, and TCID50/ml virus was calculated based on a standard curve of known concentrations
FIG. 12: Pandemic H1N1v DNA vaccine in swine induce haemagglutination inhibitory antibodies that is triggered by seasonal swine H1N1 virus from 1993.
HI antibodies against the pandemic human H1N1v 2009 was measured as described for the ferret studies in example 4b
FIG. 13: Pandemic H1N1v DNA vaccine in swine prevent fever after challenge with a seasonal swine H1N1 virus from 1993.



Fever after infection with A/Swine/Denmark/19126/93(H1N1) virus was measured everyday post challenge.
EXAMPLES
Example 1: Construction of Expression Vectors
Influenza DNA vaccine genes were designed from nucleotide sequences published in GenBank (Table 1) 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, Wis.). The genes were synthesised using only codons from highly expressed human/ferret 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 or the pKCMV vector. Key elements in the expression vectors 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 M1 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.
Genes from the A/New Caledonia/20/99(H1N1) virus applied in the first line of experiments was not designed synthetically or codon optimized. Viral RNA from the A/New Caledonia/20/99(H1N1) MDCK cell 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 H1N1 constructs (HA NC F: 5′-caacgcgtgccaccatgaaagcaaaactactgg-3′ (SEQ ID NO: 26), HA NC R: 5′-tcggcgcctcagatgcatattctacactgc-3′ (SEQ ID NO: 27), NA NC F: 5′-caacgcgtgccaccatgaatccaaatc-3′ (SEQ ID NO: 28), NA NC R: 5′-tcg gcgccctacttgtcaatggtgaacggc-3′ (SEQ ID NO: 29)). The RT-PCR products were purified from an agarose gel by the GFXTM 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® BigDyeTM Terminators v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, Calif., 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: Immunizations and Challenge
In the first line of H1N1 DNA vaccine experiments the ferrets (Mustela Putorius Furo), approximately seven months old, were divided in groups by using a chip-tag identification for dogs (E-vet, pet-id, Haderslev, Denmark), five to 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 H1N1 plasmid DNA vaccinated, (2) HA, NA, NP and M (all codon optimised) 1918 H1N1 plasmid DNA vaccinated, (3) empty plasmid vaccinated (negative vaccine control) and (4) HA and NA (not codon optimised) A/New Calcdonia/20/99(H1N1) plasmid DNA vaccinated (positive vaccine control). Ferrets were challenged with 1×107 TCID50 A/Fourth Mounth/1/47(H1N1) virus.
To investigate the protection against challenge with a contemporary H1N1 virus in comparison with the conventional vaccine in a second experiment, five groups of five animals were vaccinated as follows: (1) HA and NA 1918 H1N1 DNA vaccinated, (2) HA and NA A/New Calcdonia/20/99(H1N1) DNA vaccinated, (3) NP and M 1918 H1N1 DNA vaccinated, (4) conventional trivalent protein vaccine (Influvac, Solvay Pharmaceuticals), (5) empty plasmid vaccinated (negative vaccine control). These animals received two shots of DNA vaccine for each vaccination and were challenged with 1×107 TCID50 A/New Calcdonia/20/99
To compare the protection of 1918 DNA vaccinated ferrets with naïve untreated ferrets in the third experiment, two groups of five animals were vaccinated as follows: (1) HA and NA 1918 H1N1 DNA vaccinated, (2) Unvaccinated naïve animals. The DNA vaccinated animals in this experiment received four shots of DNA vaccine for each vaccination. The ferrets were challenged with 1×107 TCID50 A/New Calcdonia/20/99
To evaluate the H3N2 pandemic DNA vaccine against present time H3N2 viruses ferrets were divided into four groups, six animals in each group. The groups were vaccinated as follows: (1) HA and NA 1968 H3N2 DNA vaccine (2) HA and NA 2007/08 H3N2 DNA vaccine (A/Wisconsin/67/05(H3N2), (3) conventional trivalent protein vaccine (Influvac), (4) Unvaccinated naïve animals. The DNA vaccinated animals in this experiment received four shots of DNA vaccine for each vaccination and were challenged with 1×107 TCID50 A/Wisconsin/67/05(H3N2).
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 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, Calif.) 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 10-14 days after third immunisation by  ̃1×107 TCID50/ml of either A/Fouth Mounth/1/47 (H1N1), A/New Calcdonia/20/99(H1N1), or A/Wisconsin/65/2005(H3N2) virus in 100 μl PBS administrated into the nostrils with a syringe. Blood serum was collected sequentially post-challenge from vena cava 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 Virus
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 liters 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 μl Milli-Q H2O. The RT-PCR reactions were performed with oligonucleotide sequences as described by Spackman et al., (23). Extracted material (5 μl) was added to 20 μl 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 50° C., 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 H1N1 or H3N2 virus with known concentration.
Example 4: Serum Antibody Determined by ELISA
ELISA plates (96 wells) were coated with 100 μl, split influenza vaccine (Vaxigrip 2007-08, or Influvac 2006-07) diluted 1:100 in 35 mM NaHCO3 pH 9.6 and 15 mM Na2CO3 over night at 4° C. 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 4C: Haemagglutination Inhibition Assay
Ferret sera were treated with receptor destroying enzyme (RDE(II), Seiken, Japan) as described in by the manufacturer. One part of ferret serum was blood-absorbed by 20 parts of packed guinea pig red blood cells and incubated for one hour at 4° C. followed by centrifugation. Viruses were titrated by a haemagglutination assay according to the protocols of the WHO [9] with 0.75% guinea pig red blood cells in U-bottom plates (U96 MicroWell Plates, Nunc) and incubated for one hour. Virus was standardised to 100% haemagglutination endpoint titre of 8 haemagglutination units (HAU). The haemagglutination inhibition (HI) assay was performed according to the protocols of WHO [9] with 0.75% guinea pig red blood cells in U-bottom 96 well plates (U96 MicroWell Plates, Nunc) and the HI titres read as the reciprocal of the last dilution of sera that completely inhibits haemagglutination.
Example 5: Results from Influenza Challenged Ferrets Receiving Our Initial H1N1 Influenza DNA Vaccines in Ferrets
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 Calcdonia/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. 2) but challenged with a virus from 1947. Ferrets vaccinated with HA+NA DNA A/New Calcdonia/20/99(H1N1) induced lower specific serum antibody titre on day seven. It is possible that a higher 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 a A/Forth Mounth/1/47(H1N1) virus (FIG. 2). Partial protection was observed in ferrets vaccinated with HA+NA A/New Calcdonia/20/99(H1N1) DNA plasmids.
The data clearly show that DNA immunisations based on genes from the 1918 H1N1 pandemic strain induce strong specific antibody response and protect ferrets completely against infection with a H1N1 strain from 1947. Thus challenge with a drifted influenza H1N1 virus trigger recognition of antibodies generated by a heterologous virus based DNA vaccine. The 1918 genes indused full protection against the 1947 strain, while the 1999 genes were slightly less efficient. No negative or positive effects on the humoral immune response or protection was observed by including the NP and M 1918 genes in the HA+NA DNA vaccination since the protection from infection already was nearly 100%.
Example 6: Second H1N1 Influenza DNA Vaccine Experiment in Ferrets; Challenge with A/New Calcdonia/20/99 (H1N1) Virus
We compared conventional protein H1N1 split vaccine (two immunizations) versus 1918 H1N1 HA and NA codon optimized DNA vaccine versus codon optimized New Calcdonia H1N1 HA and NA versus codon optimized M and NP from 1918 H1N1 virus, versus empty DNA vaccine vector using three immunizations. Ferrets were challenged with A/New Calcdonia/20/99(H1N1) virus intra nasally and virus quantitated in nasal washings by real-time RT/PCR assay as in example 3.
Ferret antibodies are examined for ELISA antibodies and HI antibody as described in examples 4 and 4c.
Example 7: Mouse Antibody Experiments
Codon optimized versus non-codon optimized HA and NA DNA vaccines from New Calidonia H1N1 (shows the difference between codon optimization and non-optimization) versus codon optimized HA and NA from 1918 H1N1 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 8: Protein Expression Experiments
Codon optimized versus non-codon optimized HA and NA DNA vaccines from New Calidonia H1N1 (shows the difference between codon optimization and non-optimization) versus codon optimized HA and NA from 1918 H1N1 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.
Example 9: Cytokine Induction Experiments
Codon optimized versus non-codon optimized HA and NA DNA vaccines from New Calidonia H1N1 (shows the difference between codon optimization and non-optimization) versus codon optimized HA and NA from 1918 H1N1 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 H1N1 1918 HA and NA as compared to the codon optimised H1N1 New Calcdonia HA and NA to examine special cytokine induction by the 1918 genes.
Example 10: 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 11: Mouse DNA Vaccine Delivery Experiments
Codon optimized HA and/or NA DNA vaccines from 1918 H1N1 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 12: Pig DNA Vaccine Delivery Experiments
Codon optimized HA and/or NA DNA vaccines from 1918 H1N1 strain is inoculated in pigs as expression plasmids or PCR products (from example 14) and challenged with a present day New Calcdonia-like H1N1 strain and protection against disease and immune induction are measured. Thus the H1N1, H2N2 and/or H3N2 DNA vaccines may be applied in pigs that are susceptible to human influenza A pathogenic viruses.
Example 13: Results of Second H1N1 DNA Vaccine Experiment in Ferrets Compared to Conventional Vaccine
In the second experiment (Example 6) the efficiency of the 1918 DNA vaccine induced protection against a nearly 90 year drifted virus, A/New Calcdonia/20/99(H1N1), was evaluated. The protection was compared against the protection induced by the conventional trivalent protein vaccine from 2006-2007 for which A/New Calcdonia/20/99 was one of the three vaccine components. The ability of the different vaccines to prevent infection or clearance of virus infection was measured by influenza A virus titres in nasal washings of to ten days after challenge.
Low virus titre levels were measured for all vaccinated groups; however, all DNA vaccinated groups had a reduction in virus titre throughout the infection, and the HA+NA 1918 DNA vaccinated group had a significant (P<0.05) reduction in virus titre from day four to day five post challenge with A/New Calcdonia/20/99 (FIG. 8). The HA+NA A/New Calcdonia/20/99 (H/N NC) DNA vaccine was the most effective vaccine in preventing A/New Calcdonia/20/99(H1N1) virus infection and virus clearance. 1918 DNA vaccine with the internal proteins M and NP alone seemed more effective than the HA+NA 1918 DNA vaccine in providing cross-protection against the extremely drifted virus challenge. Interestingly, no significant change in virus titre from day three to day six was observed for the group vaccinated with the conventional protein vaccine or the empty plasmid (FIG. 8). The negative control group vaccinated with empty plasmid had virus infection still at day seven (below cut-off value 10 TCID50/mL) and one animal in the conventional vaccine group still possessed high virus titer at day 7. By day ten no virus could be detected in any groups (not shown).
The third experiment comparing HA+NA 1918 DNA vaccinated ferrets with naïve, unvaccinated, ferrets showed similar high levels of virus titre for both groups until day 6. However, at day 7 post infection the HA+NA 1918 DNA vaccinated ferrets had more efficient reduction in virus titre (P=0.13) compared to the naïve ferrets (P=0.18) and a lower virus load at day seven compared to the naïve group (P=0.61). Only three of five ferrets in the 1918 DNA vaccinated group had detectable virus load at day seven compared to five of five animals in the naïve group (FIG. 3c). DNA vaccinated ferrets also showed lower rive in body temperature than unvaccinated ferrets (FIG. 3a) and also scored less on clinical symptoms compared to the naïve ferrets (FIG. 3c). There were no difference in weight loss (FIG. 3b)
Influenza Specific Antibody Response after DNA Vaccination
A significant higher influenza A IgG titer (example 4) compared to the negative control group was observed at the day of challenge for the H/N 1918 DNA vaccinated ferrets, indicating induction of influenza specific antibodies after DNA vaccination (FIG. 10). At day five post infection, both H/N 1918 and H/N 1999 DNA vaccinated animals and conventional vaccine vaccinated animals showed vaccine induced influenza specific antibodies (FIG. 10). The H/N 1918 DNA vaccinated animals had comparable recall antibody titre to the conventional vaccine vaccinated animals at day seven post infection (FIG. 10). Influenza specific antibodies for the NP/M 1918 DNA vaccinated group was not expected measured in this ELISA assay pre-challenge due to the use of conventional influenza vaccine as antigen.
Induction of Naturalising Antibodies after DNA Vaccination
Haemagglutination inhibition assay (example 4b) correlates with virus neutralisation assay and measures how well sera from vaccinated animals inactivate influenza virus binding of red blood cells. Only ferrets vaccinated with the H/N 1999 H1N1 DNA vaccine had significant HI titre against the A/New Calcdonia/20/99(H1N1) virus after DNA vaccination at the day of challenge (FIG. 9). Neither H/N 1918 nor NP/M 1918 were expected to give titres before infection due to the long drift and accumulation of mutations at the HA receptor-binding site between the 1918 and 1999 H1N1 viruses. Antibodies against NP or M are not neutralising. The H/N 1999 DNA vaccine gave a better recall response of neutralising antibodies than the conventional trivalent protein vaccine (FIG. 9). At day five after infection 60% of the H/N 1999 H1N1 DNA vaccinated ferrets had seroconverted (HI>40), compared to 40% of the ferrets in the conventional vaccine group. Also a >2.5 fold increase in HI MGT was accomplished after vaccination measured the day of challenge (FIG. 9).
Example 14: Results Pandemic H3N2 DNA Vaccine in Ferrets
To prove the principle of broad cross reactivity obtained by using particularly the pandemic surface proteins as DNA vaccines we evaluated the protection against contemporary H3N2 virus challenge after vaccination with codon optimised HA and NA from the 1968 H3N2 pandemic Hong Flu flu as DNA vaccine in ferrets.
Ferrets were vaccinated (Example 2) with gene gun (PMED) three times, two weeks apart, with HA+NA DNA vaccine based on either 1968 virus or 2007/08 virus (A/Wisconsin/67/05(H3N2)). Control groups were vaccinated twice, three weeks apart, with the conventional trivalent protein vaccine. The negative control group did not receive any vaccination prior to challenge. All ferrets were challenged two weeks after the last vaccination.
Ferrets nostrils were flushed post challenge with 1 ml PBS and washings were stored immediately at −80° C. Virus titre were measured by real-time RT-PCR (Example 3) on the matrix gene of influenza A and correlated against a standard curve of known H3N2 2007/08 virus TCID50/ml in MDCK cells. DNA vaccinated groups had reduction in virus titre during virus incubation not observed for the control groups. Only the conventional vaccinated ferrets and the non-vaccinated ferrets had virus titre present at day 12 post infection (FIG. 5).
Ferret blood was collected at different time points post challenge and sera was analysed for H3N2 2007/08 influenza virus specific IgG antibodies by ELISA (Example 4). The DNA vaccines induced 2007/08 influenza virus specific IgG in sera after vaccination as did the conventional vaccine (FIG. 6). The 1968 DNA vaccinated ferrets generated high cross reactive antibodies against the contemporary challenge virus A/Wisconsin/67/05(H3N2). The response directed against the challenge virus can not be seen before day 7 in the naïve group. The level of influenza specific antibodies in the 2007/08 HA+NA DNA vaccinated group is comparable to the level observed for the conventional vaccine group.
Blood sera collected at different time points post challenge were measured for A/Aichi/2/68 and A/Wisconsin/67/05 H3N2 virus haemagglutination inhibitory (HI) antibodies by a HI assay (Example 4b). Titres were measured as the last sera dilution giving 100% inhibition of 4 haemagglutination units (HAU) virus in 25 μl (FIG. 7).
A) High sera HI titres against 1968 virus after HA+NA 1968 DNA vaccination were observed. The antibodies generated after HA+NA 2007/08 DNA or conventional vaccine could not cross react with the 1968 virus. B) High cross reactive HI titres against 2007/08 virus in pools after 1968 DNA vaccination was observed. The 2007/08 DNA vaccinated ferrets had higher HI antibodies against 2007/08 virus after vaccination than ferrets immunised with the 2007/08 conventional trivalent protein vaccine.
Vaccination with 1968 H3N2 HA induced protection against both 1968 and drifted H3N2 virus strain from more present day (2007).
Example 15: Pandemic H1N1v1 Influenza DNA Vaccine in Swine
Pigs and ferrets was vaccinated with the DNA vaccine mix containing HA and NA encoding plasmids from H1N1v with or without DNA plasmids encoding M and NP from H1N1 1918 with or without DNA plasmids encoding HA and NA from an seasonal H3N2 strain (A/Wisconsin/67/05) using the synthesised genes with human preferred codons for high expression in humans, ferrets and swine. Haemagglutinin Inhibition (HI) titre levels and/or neutralising antibody titres and/or total IgG antibodies and/or protection against heterologous or homologous virus challenge as measured by faster virus clearance in respiratory tract after one or two DNA immunisations was evaluated (example 3, 4 and 4b).
To assess the cross-protective immunity in swine after vaccination with influenza DNA vaccine based on the HA and NA genes of the new 2009 pandemic H1N1v virus with or without the NP and M genes of the pandemic 1918 H1N1 virus either delivered with gene gun or by electroporation we vaccinated four 6 weeks old pigs, twice, three weeks apart and challenged 10 weeks after last immunisation with a seasonal swine H1N1 virus (A/swine/Denmark/19126/93) 1×107 EID50. Four pigs was included as naïve animals, not receiving vaccination.
One pig vaccinated with gene gun received both HA and NA H1N1v beads and NP and M 1918 H1N1 beads. The other animal in the gene gun group received a double dose of HA and NA H1N1v beads.
One pig vaccinated with electroporation received both HA and NA 2009 H1N1v DNA injections and NP and M 1918 H1N1 DNA injections, while the other pig in the electroporation group received double injections of HA and NA 2009 H1N1v DNA.
The pigs were vaccinated as follows:
Three Groups:















Gr1
Gene Gun



Gr2
Electroporation



Gr3
Naïve unvaccinated









Details for Vaccination of Group 1 and 2:















Gene Gun Group 1


Pig 1
HA + NA H1N1v beads



Dorsal side of each ear: two shots



Inner side of each thigh: two shots



NP + M 1918



Dorsal side of each ear: two shots



Inner side of each thigh: two shots


Pig 2
HA + NA H1N1v beads



Dorsal side of each ear: four shots



Inner side of each thigh: four shots



Electroporation Group 2


Pig 3
HA H1N1v



Dorsal side of each ear: One injections 50 μl, one pulse



Inner side of each thigh: One injections 50 μl, one pulse



NA H1N1v



Dorsal side of each ear: One injections 50 μ1, one pulse



Inner side of each thigh: One injections 50 μ1, one pulse



NP 1918



Dorsal side of each ear: One injections 50 μl, one pulse



Inner side of each thigh: One injections 50 μl, one pulse



M 1918



Dorsal side of each ear: One injections 50 μl, one pulse



Inner side of each thigh: One injections 50 μ1, one pulse


Pig 4
HA H1N1v



Dorsal side of each ear: two injections 50 μl, one pulse



Inner side of each thigh: two injections 50 μ1, one pulse



NA H1N1v



Dorsal side of each ear: two injections 50 μl, one pulse



Inner side of each thigh: two injections 50 μl, one pulse









    
    
        Immunisations:
        
            Gene Gun Vaccination:
            Helium pressure:  ̃420 psi
            One shot  ̃2 ug DNA
            Pig1 receives a total of 16 shots
            Pig2 receives a total of 16 shots
            1st vaccination day 0
            2nd vaccination day 21
            Electroporation
            Settings:
            0.2 A, one pulse (several pulses in one pulsing), 52 ms/pulse, One injection contains 50 μl of 2 ug/μl DNA (100 ug DNA in one injection).
            Pig 3 receives 16 injections and 16 pulses
            Pig 4 receives 16 injections and 16 pulses
            1st vaccination day 0
            2nd vaccination day 21
        
        
    
    


All groups were challenged 10 weeks after last immunisation with 1×107 TCID50 seasonal A/swine/Denmark/19126/93(H1N1) virus.

    
    
        Samplings: Blood and nasopharyngeal swabs were collected sequentially after challenge and the animals were euthanized 14 days after challenge

Results

Virus Titre in Nasopharyngeal Swabs:

    
    


All DNA vaccinated pigs were able to clear the heterologous virus challenge more efficiently than the naïve pigs not receiving vaccine (FIG. 11). The only vaccine able to prevent infection was HA and NA H1N1v DNA administrated by electroporation.
Cross-Reactive Haemagglutination Inhibition of Pandemic H1N1v Virus
HA and NA H1N1v DNA vaccine administrated by electroporation were able to induce an HI tire against the H1N1v above 40, 10 days after second vaccination (FIG. 12). The pig was able to maintain the titre 10 weeks after second vaccination. All DNA vaccinated pigs demonstrated significant cross-reactive HI antibodies against the H1N1v virus from five days after challenge with the heterologous swine seasonal H1N1 virus from 1993. These results indicate that challenge with a different strain than the one comprising the DNA vaccine is able to trigger some common HI antibodies after DNA vaccination, inducing efficient cross reactive HA neutralising antibodies.
Fever
The four pigs immunized with the pandemic H1N1v DNA vaccine did not show rise in body temperature during the infection with the seasonal swine virus from 1993; however, the un-vaccinated pigs did develop fever at day 1 post challenge (FIG. 13).
Example 16: Use of Transcriptionally Active PCR Products from Influenza A as DNA Vaccines Instead of Plasmid DNA
PCR products can be obtained using the codon optimised vaccine genes from pandemic influenza A strain transcriptionally active PCR products as template and the PCR products them selves can be used as a DNA vaccine. To be transcritionally functional the PCR products will contains all necessary signal components (promoter, Kozak and terminator) for efficient transcription of a desired influenza gene or partial gene in the host. In this way no plasmid backbone e.g. with antibiotic resistance marker and unnecessary DNA would be needed.
Example 17: DNA Vaccine Encoding Glycosylation-Free Influenza Proteins
Influenza genes are site directed mutated in a plasmid backbone to remove desired N-linked glycosylations at antigenic sites in the HA and NA gene. The desired glycosylations that should be removed are all or some of those that has been added to the pandemic strains during the evolution and drift leading to present day circulating Influenza A strains of H1N1, H2N2, H3N2 types, respectively. Completely glycosylation-stripped proteins can also be produced in this way.
Example 18: Composition of a Universal Influenza DNA Vaccine
A DNA vaccine is composed containing codon optimized genes of influenza H1H1, H3N2 and influenza B: For the H1N1 and H3N2 there should be HA and NA from an original pandemic strain and one contemporary strain e.g. New Calcdonia/20/99(H1N1) and/or Wisconsin/67/05 (H3N2) possibly mutated to express less glycosylated protein. In addition, to obtain even broader cross reactivity, the M and NP genes from pandemic H1N1 should be included. This will result in a trivalent vaccine that will induce long time protection against all H3 and H1 subtypes including most influenza B strains. The universal DNA vaccine could either be a pool of all eleven plasmids or a mix of bicistronic vectors. Alternatively the pandemic vaccine components could be given as DNA vaccine as a prime and the conventional trivalent protein vaccine on the market could serve as a boost. The goal is a vaccine that would work a lifetime, at least seasonal vaccinations would be unnecessary.
Example 19: Universal H1N1 and H3N2 Influenza DNA Vaccine
We have previously demonstrated that different influenza DNA vaccines based on a subtype combination of influenza, e.g. H1N1 or H1N1v or H3N2 are effective in inducing protection against the given subtype of viruses.
However, the optimal influenza vaccine would comprise components of both H1N1 and H3N2. We therefore designed a “universal influenza DNA vaccine” which is based on the HA and NA from the recent pandemic H1N1v, the internal genes NP and M of the previous pandemic H1N1 from 1918, and the HA and NA genes from a seasonal H3N2 virus. This example focus on the “universal influenza DNA vaccine” administrated together with a helper plasmid serving as an adjuvant. If delivered intranasally the flu DNA together with the helper plasmid will be embedded in a lipid-formulation for optimal delivery to the mucus. The immune response induced by the universal influenza DNA vaccine administrated either intranasally or by gene gun or by electroporation will be evaluated in an animal model. The protection and cross protection against homologous and heterologous virus challenge will be investigated.
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## Claims

1. An immunogenic composition against influenza A infection in a human or a pig, comprising isolated nucleic acid sequences encoding influenza proteins, wherein the nucleic acid sequences encode for haemagglutinin (HA) and/or neuramidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP), and wherein the nucleic acid sequences comprise a mix of (a) DNA or RNA from a pandemic influenza virus or DNA or RNA from a non-pandemic influenza virus wherein the DNA or RNA from the non-pandemic virus is mutated, the mutated sequence encoding a protein with fewer glycosylation sites than the non-mutated sequence, wherein said mutated sequence encodes Asn-Y-Ser in place of Asn-X-Ser in at least one occurrence of Asn-X-Ser in said non-mutated sequence,
wherein X is, individually, in each occurrence of Asn-X-Ser, any amino acid except proline, and
Y is, individually, in each occurrence of Asn-Y-Ser, any amino acid other than X, and
(b) DNA or RNA from a different subtype influenza virus is from a H3N2 influenza virus.

2. The immunogenic composition according to claim 1, wherein the immunogenic composition is formulated for administration by injection of isolated naked nucleic acid sequences.

3. The immunogenic composition according to claim 1, wherein the DNA or RNA encoding at least one influenza protein from a H3N2 influenza virus selected from SEQ ID NOs: 15 and 17.

4. An immunogenic composition against influenza A infection in a human or a pig, comprising isolated nucleic acid sequences encoding influenza proteins, wherein the nucleic acid sequences encode for haemagglutinin (HA) and/or neuramidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP), and wherein the nucleic acid sequences comprise a mix of (a) DNA or RNA from a pandemic influenza virus or DNA or RNA from a non-pandemic influenza virus wherein the DNA or RNA from the non-pandemic virus is mutated, the mutated sequence encoding a protein with fewer glycosylation sites than the non-mutated sequence, wherein said mutated sequence encodes Asn-Y-Thr in place of Asn-X-Thr in at least one occurrence of Asn-X-Thr in said non-mutated sequence,
wherein X is, individually, in each occurrence of Asn-X-Thr, any amino acid except proline, and
Y is, individually, in each occurrence of Asn-Y-Thr, any amino acid other than X, and
(b) DNA or RNA from a different subtype influenza virus is from a H3N2 influenza virus.

5. The immunogenic composition according to claim 3, wherein the DNA or RNA encodes SEQ ID NOs: 15 and 17.

6. The immunogenic composition according to claim 1, wherein the DNA or RNA codons of at least one sequence are optimized.

7. The immunogenic composition according to claim 1, comprising nucleic acid sequences selected from SEQ ID NOs: 1, 3, 5, 7, 10, 12, 14, and 16.

8. The immunogenic composition according to claim 1, wherein the isolated nucleic acid sequences encode at least one sequence selected from SEQ ID NOs: 2, 4, 6, 8, 9, 11, 13, 15, and 17.

9. The immunogenic composition according to claim 1, wherein the immunogenic composition further comprises an adjuvant.

10. The immunogenic composition according to claim 4, wherein the DNA or RNA codons of at least one sequence are optimized.

11. The immunogenic composition according to claim 4, comprising nucleic acid sequences selected from SEQ ID NOs: 1, 3, 5, 7, 10, 12, 14, and 16.

12. The immunogenic composition according to claim 4, wherein the isolated nucleic acid sequences encode at least one sequence selected from SEQ ID NOs: 2, 4, 6, 8, 9, 11, 13, 15, and 17.

13. The immunogenic composition according to claim 4, wherein the immunogenic composition further comprises an adjuvant.

14. The immunogenic composition according to claim 4, wherein the immunogenic composition is formulated for administration by injection of isolated naked nucleic acid sequences.

15. The immunogenic composition according to claim 4, wherein the DNA or RNA encoding at least one influenza protein from a H3N2 influenza virus selected from SEQ ID NOs: 15 and 17.

16. The immunogenic composition according to claim 15, wherein the DNA or RNA encodes SEQ ID NOs: 15 and 17.