# Patent

## Title
Influenza vaccines

## Abstract

Described herein are vaccines and the use of naked DNA and/or RNA encoding hemagglutinin (HA) from pandemic influenza, e.g., the 1918 H1N1 and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus, as a vaccine component against present day and coming H1, H2, H3, H5, N1, N2 containing influenza A infections in humans and swine optionally with the naked DNA and/or RNA encoding Neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza virus included. If the vaccine components are used as DNA or RNA vaccines with or without the corresponding protein, the codons can optionally be “humanized” using preferred codons from highly expressed mammalian genes and the administration of this DNA vaccine can be by saline or buffered saline injection of naked DNA or RNA, or injection of DNA plasmid or linear gene expressing DNA fragments coupled to particles. Addition of the matrix protein (M) and/or the nucleoprotein (NP) from the 1918 influenza strain is also disclosed.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a division of U.S. patent application Ser. No. 12/156,456, filed May 30, 2008, which claims the benefit under 35 USC 119(e) of U.S. Patent Application No. 60/934,117, filed Jun. 11, 2007.


BACKGROUND OF THE INVENTION
The invention concerns therapeutic and prophylactic vaccines for humans and swine, for influenza A infections in humans and swine.
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 possess both receptors in their respiratory tract. Because swine get infection and pneumonia from human influenza strains, they may serve as a dangerous mixing vessel for the 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 (World Health Organization (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. Since then there have 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 recent times causing 20 to 50 million deaths worldwide. Influenza most commonly presents as seasonal outbreaks and epidemics of variable severity.
Zoonosis of avian influenza virus (MV) able to infect humans and swine and its 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 such a pandemic will probably be unknown until the pandemic emerges, at which time 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 methods will be required in order to minimise the severity of the pandemic.
Since seasonal influenza A vaccines are 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 have 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 hemagglutinin (HA) and neuraminidase (NA). The A strain has 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 which indicates 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. 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 ion channel 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 feces 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 2004H5N1 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. Disinfectants like formalin and iodine compounds can also 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. However, 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, 1957H2N2 (“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. In the northern hemisphere seasonal influenza outbreaks usually occur between October and April. In the southern hemisphere, these outbreaks usually occur between April and October. The antigenic drift of human influenza viruses is closely monitored by WHO'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 al., (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. 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. However, there is consideration disagreement about the actual origin of the virus and many still believe that also this 1918 pandemic strain is a reassortant between a mammalian and avian virus most likely occurring from swine. Antigenic reassortment occurs when viral segments from two antigenic different viruses infect 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. The reassortant might be catastrophic if the virus is capable of efficient replication in the new host. In the worst case, such a reassorted strain might lead to a pandemic, world-spanning infection to which there is no pre-existing immunity in the human population. 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 a-(2,3) and a-(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 a-(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 for virus egg growth properties. Recent research on viruses has suggested that the evolution of influenza does not always follow a constant rate, but is characterised by stochastic processes, short intervals of rapid evolution, and 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 administered 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 type specific 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 and slow down 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, a shortage of eggs will limit production of traditional seasonal influenza vaccines on eggs. 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 years ago, clinical trials proceeding to 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 Hematopoietic 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 which may be incorporated into viral vectors with the purpose of inducing immunity to the gene product. Thus, as used herein, DNA vaccines refer to all kinds of delivery systems for the antigen encoding naked DNA or RNA but exclude viral vector-based delivery. The vaccine gene can be in form of circular plasmid or a linear expression cassette with just the key features necessary for expression (promoter, 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, adeno-associated 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.
WO2006063101 describes a pandemic avian influenza vaccine based on an adenovirus vehicle with HA DNA isolated from the avian H5N1 influenza virus isolated during the outbreak in 2003-2005. The vaccine was tested in animals challenged with the same H5N1 influenza virus strain.
DNA vaccines induce an immune response which may be 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). However, cellular immunity alone cannot protect against infection since this requires antibodies.
Influenza infection and symptoms in ferrets are highly comparable to what is observed in humans. Therefore ferrets are one of the best models for influenza vaccination trials (22). Influenza HA DNA vaccines in ferrets has also previously proved effective (18,32).
It has previously been shown that H1N1 whole inactivated virus vaccine induced partly protection against infection with 1918 in mice (28). Also, recently, a DNA vaccine encoding the HA from 1918 showed complete protection of mice against a 1918 H1N1 challenge (16).
Influenza vaccines that have the ability to induce immune responses able to cross-react with drifted virus variants and even heterologous strains would be of great advantage for both annual vaccine development and in cases of emerging new strains.
SUMMARY OF THE INVENTION
The present invention provides regimens and compositions containing the hemagglutinin (HA) from pandemic influenza A, e.g. the 1918 H1N1 and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus, useful as a therapeutic or prophylactic vaccine component against present day and future influenza A strains. The invention may optionally provide naked DNA and/or RNA molecules encoding the Neuraminidase (NA), matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza virus. The invention also concerns vaccines comprising naked DNA and/or RNA coding HA and/or NA from the new circulating 2001 H5N7 low pathogenic (LP) Avian influenza virus (AIV) strain (A/Mallard/Denmark/64650/03(H5N7)), the newly introduced and circulating March 2006 Denmark H5N1 high pathogenic Avian influenza A virus (AIV) strains A/buzzard/Denmark/6370/06 (H5N1), A/duck/Denmark/53-147-8/08 (H7N1) and A/widegeon/Denmark/66174/G18/04 (H2N3).
The data herein demonstrates that gene gun administrated codon optimised DNA vaccine in plasmid encoding HA and NA with or without M and NP based on the H1N1 pandemic virus from 1918 induced protection in ferrets against infection with a H1N1 (A/New Caledonia/20/99(H1N1)) present day virus. The circulating H1N1 strain in Europe in the 2006-2007 seasons is New Caledonia-like. The viruses are separated by a time interval of 89 years and differ by 21.2% in the HA1 protein. By comparison, a similar DNA vaccine encoding the HA and NA of (A/New Caledonia/20/99 (H1N1)) induced less protection. These results suggest not only a unique ability of the DNA vaccines but also a unique and unexpected feature of the 1918 HA and/or NA in inducing especially broad and efficient protective immunity against even extremely drifted strain variants.
The present invention discloses that an induced immune response with a DNA vaccine encoding HA and/or NA of the 1918 H1N1 influenza A gives a high level of cross protection against present day influenza infection. Tests were carried out in ferrets vaccinated with this DNA vaccine synthesised using human preferred codons of the 1918 H1N1 influenza and challenged with a contemporary H1N1 virus.
The results surprisingly show that the 1918 H1N1 DNA vaccines are as good as or better candidates for influenza prophylaxis than annual conventional protein based vaccines which frequently need to be updated to match the circulating influenza virus. DNA vaccination induces broader cross-reactivity against drifted strain and longer memory responses. It has been shown that a similar DNA vaccine may protect against the 1918 H1N1 recombinant strain (16). However, the results herein then suggest that the synthetic DNA vaccine based on the 1918 H1N1 sequences protects against extreme drifted variants represented by recent contemporary or seasonal circulating H1N1 strains. Thus it is likely that the suggested 1918 H1N1 DNA vaccine protects against H1N1 strains circulating for up to 89 years and therefore likely also future H1N1 variants. This is highly unexpected since traditional protein split vaccines only protects against the strain it is designed from and thus has to be produced from the actual circulating H1N1 strains sometimes as frequent as every year. Thus, a DNA vaccine encoding the HA and NA of 1918 H1N1 was not expected to protect against such a divergent strain as the present day H1N1, but it does.
Other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A illustrates the mean serum specific IgG antibody response (ELISA) to influenza A of A/New Caledonia/20/99(H1N1) days after viral challenge. Six ferrets were in each group.
FIG. 1B illustrates the number of viral RNA copies (real time RT/PCR) in nasal wash in days after challenge. Six ferrets were in each group.
FIGS. 2A-2D provide results of 1918 pandemic H1N1 DNA vaccinated ferrets challenged with 2007 H1N1.
FIG. 2A shows fever at day 2 post challenge.
FIG. 2B shows body weight loss by day 4 post challenge.
FIG. 2C shows virus titer in nasal washings at day 7 post challenge.
FIG. 2D shows clinical score for illness based on a scoring table for sneezing, nasal discharge and activity level.
FIG. 3 is a bar chart showing 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).



DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses the use of the naked DNA and/or RNA molecule encoding hemagglutinin (HA) from pandemic influenza, e.g. the 1918 H1N1 and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus, as a vaccine component against present day and coming H1, H2, H3 containing influenza A infections in humans and/or swine. The naked DNA and/or RNA molecule encoding neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza virus.
The naked DNA and/or RNA molecule encoding neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza virus can optionally be included as vaccine components against present day and coming H1, H2, H3, N1, N2 containing influenza A infections in humans and/or swine.
The vaccine of the invention is believed to offer many of the advantages a DNA vaccine can provide over conventional vaccines. More particularly, it may 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. However, DNA vaccines have the ability to induce a cytotoxic cellular response as well as a humoral antibody response. Therefore, these vaccines may better mimic the natural response to viral infection than inactivated vaccines in respect to specificity and antibodies isotypes.
The components in the vaccine are naked DNA and/or RNA coding for the hemagglutinin and/or the neuraminidase and/or matrix protein (M) and/or the nucleoprotein (NP) from pandemic influenza strains, preferably with a mixture of such proteins from several pandemic strains.
In a preferred embodiment of the invention the DNA and/or RNA codons are humanized e.g. the DNA sequence for hemagglutinin and neuraminidase and Matrix and Nucleoprotein is changed so the sequence coding for said proteins is changed to be optimally expressed in mammalian cells.
The invention also discloses the vaccines against present day human and swine influenza A infection comprising the above-mentioned naked DNA and/or RNA coding hemagglutinin and/or neuraminidase and/or a matrix protein and/or the hemagglutinin protein and/or DNA or RNA from the pandemic influenza, e.g., the 1918 H1N1 and/or the 1957 H2N2 and/or the 1968 H3N2 influenza A virus, preferably with a mixture from various influenza strains.
In another embodiment the vaccine comprises naked DNA and/or RNA coding HA and/or NA from 1918 H1N1 strain plus HA and NA from 1957 H2N2 plus HA from 1968 H3N2 virus strains as DNA vaccines and/or proteins. The vaccine is intended to protect humans against circulating H1, H2, and H3 influenza A strains.
In another embodiment the vaccine comprises naked DNA and/or RNA molecules encoding HA and/or 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, H7 and/or H2 containing influenza A strains.
In another embodiment the vaccine comprises naked DNA and/or RNA molecules encoding 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 (MV) strain (A/buzzard/Denmark/6370/06(H5N1)), A/duck/Denmark/53-147-8/08 (H7N1) and A/widegeon/Denmark/66174/G18/04 (H2N3). The vaccine is intended to broadly protect birds and humans and swine against any H5 containing influenza A strains.
Above mentioned vaccines can be used both prophylactically and therapeutically.
Definitions
Hemagglutinin
The name hemagglutinin 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 disulfide linked 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 comprises 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 post translationally 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 a-(2,6) linkage to galactose, while avian influenza viruses (AIV) preferentially bind to a-(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. The main function of the NA 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 [5-acetamido-4-guanidino-6-(1,2,3-trihydroxypropyl)-5,6-dihydro-4H-pyran-2-carboxylic acid] and oseltamivir [(3R,4R,5S)-4-acetylamino-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid ethyl ester]. 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 protein binds to the cytoplasmic tails of HA, NA and M2. NA stimulates 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 and disrupting the M1-RNP interactions. The M2 ion channel is a 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 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 hemagglutinin (HA) and neuraminidase (NA) genes of the 1918 H1N1 strain did not possess known genetic indicators for high virulence that could have explained the severity 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 Vaccines:
DNA vaccines are here defined as naked DNA or RNA, DNA or RNA in solution for direct intramuscular or subcutaneous injection with or without electroporation or coupled to particles, e.g., gold beads for gene gun administration. The DNA can be linear containing only a promoter, the influenza genes and polyadenylation signal or this expression cassette in an expression plasmid.
The administration of DNA vaccine can be by saline or buffered saline injection of naked DNA or RNA, or injection of DNA plasmid or linear gene expressing DNA fragments coupled to particles, or inoculated by gene gun.
The two most common types of DNA vaccine administration are saline injection of naked DNA and gene gun DNA inoculations (DNA coated on solid gold beads administrated with helium pressure). Saline intra muscular injections of DNA preferentially generate 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 consists of a vector with a gene of interest cloned into a bacterial plasmid engineered for optimal expression in eukaryotic cells. In one embodiment, a vaccine vector includes an origin of replication allowing for production in bacteria, a bacterial antibiotic resistance gene allowing for plasmid selection in bacterial culture, a strong constitutive promoter for optimal expression in mammalian cells (eukaryotic promoters such as those 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 can be included to improve expression of genes. Many bacterial DNA vaccine vectors contain unmethylated cytidinephosphate-guanosine (CpG) dinucleotide motifs that may elicit strong innate immune responses in the host.
In one embodiment, a eukaryotic expression vector contains a constitutive eukaryotic promoter, an intron, a polylinker which allows for convenient insertion of a vaccine gene, and a polyadenylation signal is used. Suitable expression vectors are known the art and may be used as backbones engineered to contain the elements described herein. For example, commercially available vectors (e.g., the WRG7079 vector available form PowderJect Vaccines, Madison, WI) and/or vectors described in the literature (e.g., Corbet et al, 2000 (5)) can be used as a backbone to contain the elements described herein. For example, in one embodiment, an expression vector of the invention is designed to contain one or more of the secretory signal from an influenza virus (e.g., the influenza A 1918 HA or NA secretory signals).
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.
Viral DNA Vaccines:
DNA can be delivered by a viral vector such as Adenovirus, Modified vaccinia virus Ankara (MVA), Vaccinia, Adeno-associated virus (AAV), Alphavirus etc. Viral DNA vaccines are not a part of the present study and are not encompassed by this invention.
Codon Optimization:
Codon optimization is the complete exchange of the virus codons to those of highly expressed human genes and therefore also mammalian genes that include swine. Codon optimization does 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 are called “humanized” genes and are frequently used in DNA vaccines to enhance expression.
The DNA or RNA sequence for hemagglutinin and neuraminidase and Matrix and Nucleoprotein is changed so the sequence coding for said proteins is changed to be optimally expressed in humans.
In one embodiment, the invention provides the use of the 1918 HA and/or NA codon-optimized genes in a DNA vaccine against all seasonal circulating H1N1 influenza A strains including the A/New Caledonia/20/99(H1N1) like virus.







TABLE 1





nucleotide and amino acid sequences of the


codon optimized genes and the proteins they


express.















HA 1918 synthetic gene 0607838, Based on acc. No.:


AF117241: A/South Carolina/1/18


SEQ ID NO: 1: Nucleotide


ATGGAGGCCAGGCTGCTGGTGCTGCTGTGCGCCTTCGCCGCCACCAACGC


CGACACCATCTGCATCGGCTACCACGCCAACAACAGCACCGACACCGTGG


ATACCGTGCTGGAGAAGAACGTGACCGTGACCCACAGCGTGAACCTGCTG


GAGGACAGCCACAACGGCAAGCTGTGCAAGCTGAAGGGAATCGCTCCCCT


GCAGCTGGGCAAGTGCAACATCGCCGGCTGGCTGCTGGGCAACCCCGAGT


GCGACCTGCTGCTGACCGCCAGCAGCTGGTCCTACATCGTGGAGACCAGC


AACAGCGAGAACGGCACCTGCTACCCCGGCGACTTCATCGACTACGAGGA


GCTGCGGGAGCAGCTGTCCAGCGTGAGCAGCTTCGAGAAGTTCGAGATCT


TCCCCAAGACCAGCTCCTGGCCCAACCACGAGACCACCAAGGGCGTGACC


GCCGCCTGTAGCTACGCCGGAGCCAGCAGCTTCTACAGAAACCTGCTGTG


GCTGACCAAGAAGGGCAGCAGCTACCCCAAGCTGTCCAAGAGCTACGTGA


ACAACAAGGGCAAGGAAGTGCTGGTGCTGTGGGGCGTGCACCACCCCCCT


ACCGGCACCGACCAGCAGAGCCTGTACCAGAACGCCGACGCCTACGTGAG


CGTGGGCAGCAGCAAGTACAACAGAAGGTTCACCCCCGAGATCGCCGCCA


GGCCCAAGGTGCGCGACCAGGCCGGCAGGATGAACTACTACTGGACCCTG


CTGGAGCCCGGCGACACCATCACCTTCGAGGCCACCGGCAACCTGATCGC


CCCTTGGTACGCTTCGCCCTGAACAGGGGCAGGGGCAGCGGCATCATCAC


CAGCGACGCCCCCGTGCACGACTGCAACACCAAGTGCCAGACCCCCCACG


GAGCCATCAACAGCAGCCTGCCCTTCCAGAACATCCACCCCGTGACCATC


GGCGAGTGCCCCAAGTACGTGAGAAGCACCAAGCTGAGGATGGCCACCGG


CCTGAGGAACATCCCCAGCATCCAGAGCAGGGGCCTGTTCGGAGCCATCG


CCGGATTCATCGAGGGCGGCTGGACCGGCATGATCGACGGCTGGTACGGC


TACCACCACCAGAACGAGCAGGGCAGCGGCTACGCCGCCGACCAGAAGAG


CACCCAGAACGCCATCGACGGCATCACCAACAAGGTGAACAGCGTGATCG


AGAAGATGAACACCCAGTTCACCGCCGTGGGCAAGGAGTTCAACAACCTG


CAGAGGCGGATCGAGAACCTGAACAAGAAGGTGGACGACGGCTTCCTGGA


CATCTGGACCTACAACGCCGAGCTGCTGGTGCTGCTGGAGAACGAGAGGA


CCCTGGACTTCCACGACAGCAACGTGAGGAACCTGTACGAGAAGGTGAAG


AGCCAGCTGAAGAACAACGCCAAGGAGATCGGCAACGGCTGCTTCGAGTT


CTACCACAAGTGCGACGACGCCTGCATGGAGAGCGTGAGAAACGGCACCT


ACGACTACCCCAAGTACAGCGAGGAGAGCAAGCTGAACCGGGAGGAGATC


GACGGCGTGAAGCTGGAGAGCATGCGCGTGTACCAGATCCTGGCCATCTA


CAGCACCGTGGCCAGCAGCCTGGTGCTGCTGGTGTCCCTGGGAGCCATCA


GCTTTTGGATGTGCAGCAACGGCAGCCTGCAGTGCAGGATCTGCATCTGA


 


SEQ ID NO: 2 Amino acid


MEARLLVLLCAFAATNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLL


EDSHNGKLCKLKGIAPLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETS


NSENGTCYPGDFIDYEELREQLSSVSSFEKFEIFPKTSSWPNHETTKGVT


AACSYAGASSFYRNLLWLTKKGSSYPKLSKSYVNNKGKEVLVLWGVHHPP


TGTDQQSLYQNADAYVSVGSSKYNRRFTPEIAARPKVRDQAGRMNYYWTL


LEPGDTITFEATGNLIAPWYAFALNRGSGSGIITSDAPVHDCNTKCQTPH


GAINSSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFGAI


AGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAIDGITNKVNSVI


EKMNTQFTAVGKEFNNLERRIENLNKKVDDGFLDIWTYNAELLVLLENER


TLDFHDSNVRNLYEKVKSQLKNNAKEIGNGCFEFYHKCDDACMESVRNGT


YDYPKYSEESKLNREEIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAI


SFWMCSNGSLQCRICI.


 


NA 1918 synthetic gene 0607869, Based on acc. No.:


AF250356: A/Brevig mission/1/1918


SEQ ID NO: 3 Nucleotide


ATGAACCCCAACCAGAAGATCATCACCATCGGCAGCATCTGCATGGTGGT


GGGCATCATCAGCCTGATCCTGCAGATCGGCAACATCATCAGCATCTGGG


TGTCCCACAGCATCCAGACCGGCAACCAGAACCACCCCGAGACCTGCAAC


CAGTCCATCATCACCTACGAGAACAACACCTGGGTGAACCAGACCTACGT


GAACATCAGCAACACCAACGTGGTGGCCGGCCAGGACGCCACCTCCGTGA


TCCTGACAGGCAACAGCAGCCTGTGCCCCATCAGCGGCTGGGCCATCTAC


AGCAAGGACAACGGCATCAGGATCGGCAGCAAGGGCGACGTGTTCGTGAT


CAGAGAGCCCTTCATCAGCTGCAGCCACCTGGAATGCAGGACCTTCTTCC


TGACCCAAGGAGCCCTGCTGAACGACAAGCACAGCAACGGCACCGTGAAG


GACAGAAGCCCCTACAGGACCCTGATGAGCTGCCCCGTGGGCGAGGCTCC


CAGCCCCTACAACAGCAGATTCGAGAGCGTGGCCTGCTCCGCCAGCGCCT


GCCACGACGGCATGGGCTGGCTGACCATCGGCATCAGCGGCCCTGACAAC


GGGGCCGTGGCCGTGCTGAAGTACAACGGAATCATCACCGACACCATCAA


GAGCTGGCGGAACAACATCCTGAGGACCCAGGAAAGCGAGTGCGCCTGCG


TGAACGGCAGCTGCTTCACCATCATGACCGACGGCCCCAGCAACGGCCAG


GCCAGCTACAAGATCCTGAAGATCGAGAAGGGCAAGGTGACCAAGAGCAT


CGAGCTGAACGCCCCCAACTACCACTACGAGGAATGCAGCTGCTACCCCG


ACACCGGCAAGGTCATGTGCGTGTGCAGGGACAACTGGCACGGCAGCAAC


AGGCCCTGGGTGTCCTTCGACCAGAACCTGGACTACCAGATCGGATACAT


CTGCAGCGGCGTGTTCGGCGACAACCCCAGGCCCAACGACGGCACCGGCA


GCTGCGGCCCTGTGAGCAGCAACGGGGCCAATGGCATCAAGGGCTTCAGC


TTCAGATACGACAACGGCGTGTGGATCGGCCGCACCAAGAGCACCAGCAG


CAGATCCGGCTTCGAGATGATCTGGGACCCCAACGGCTGGACCGAGACCG


ACAGCAGCTTCAGCGTGAGGCAGGACATCGTGGCCATCACCGACTGGTCC


GGCTACAGCGGCAGCTTCGTGCAGCACCCCGAGCTGACCGGCCTGGACTG


CATGAGGCCCTGTTTCTGGGTGGAGCTGATCAGAGGCCAGCCCAAGGAGA


ACACCATCTGGACCAGCGGCAGCAGCATCAGCTTTTGCGGCGTGAACAGC


GACACCGTGGGCTGGTCCTGGCCCGACGGGGCCGAGCTGCCCTTCAGCAT


CGATAAGTGA


 


SEQ ID NO: 4: Amino acid


MNPNQKIITIGSICMVVGIISLILQIGNIISIWVSHSIQTGNQNHPETCN


QSIITYENNTWVNQTYVNISNTNVVAGQDATSVILTGNSSLCPISGWAIY


SKDNGIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKHSNGTVK


DRSPYRTLMSCPVGEAPSPYNSRFESVAWSASACHDGMGWLTIGISGPDN


GAVAVLKYNGIITDTIKSWRNNILRTQFSECACVNGSCFTIMTDGPSNGQ


ASYKILKIEKGKVTKSIELNAPNYHYEECSCYPDTGKVMCVCRDNWHGSN


RPWVSFDQNLDYQIGYICSGVFGDNPRPNDGTGSCGPVSSNGANGIKGFS


FRYDNGVWIGRTKSTSSRSGFEMIWDPNGWTETDSSFSVRQDIVAITDWS


GYSGSFVQHPELTGLDCMRPCFWVELIRGQPKENTIWTSGSSISFCGVNS


DTVGWSWPDGAELPFSIDK.


 


NP 1918 synthetic gene 0607866, Based on acc. No.:


AY44935: A/Brevig mission/1/1918


SEQ ID NO: 5: Nucleotide


ATGGCCAGCCAGGGCACCAAGAGAAGCTACGAGCAGATGGAAACCGACGG


CGAGAGGCAGAACGCCACCGAGATCAGGGCCAGCGTGGGCAGGATGATCG


GCGGCATCGGCAGGTTCTACATCCAGATGTGCACCGAGCTGAAGCTGTCC


GACTACGAGGGCAGGCTGATCCAGAACAGCATCACCATCGAGAGGATGGT


GCTGTCCGCCTTCGACGAGAGAAGAAACAAGTACCTGGAAGAGCACCCCA


GCGCCGGCAAGGACCCCAAGAAAACCGGCGGACCCATCTACAGAAGGATC


GACGGCAAGTGGATGAGAGAGCTGATCCTGTACGACAAGGAGGAAATCAG


AAGGATCTGGCGGCAGGCCAACAACGGCGAGGACGCCACAGCCGGCCTGA


CCCACATGATGATCTGGCACAGCAACCTGAACGACGCCACCTACCAGAGG


ACCAGGGCCCTCGTCAGAACCGGCATGGACCCCCGGATGTGCAGCCTGAT


GCAGGGCAGCACACTGCCCAGAAGAAGCGGAGCTGCTGGAGCCGCCGTGA


AGGGCGTGGGCACCATGGTGATGGAACTGATCAGGATGATCAAGAGGGGC


ATCAACGACAGGAACTTTTGGAGGGGCGAGAACGGCAGAAGGACCAGGAT


CGCCTACGAGAGGATGTGCAACATCCTGAAGGGCAAGTTCCAGACAGCCG


CCCAGAGGGCCATGATGGACCAGGTCCGGGAGAGCAGGAACCCCGGCAAC


GCCGAGATCGAGGACCTGATCTTCCTGGCCAGAAGCGCCCTGATCCTGAG


GGGCAGCGTGGCCCACAAGAGCTGCCTGCCCGCCTGCGTGTACGGACCCG


CCGTGGCCAGCGGCTACGACTTCGAGAGAGAGGGCTACAGCCTGGTCGGC


ATCGACCCCTTCAGGCTGCTGCAGAACTCCCAGGTGTACTCTCTGATCAG


GCCCAACGAGAACCCCGCCCACAAGTCCCAGCTGGTCTGGATGGCCTGCC


ACAGCGCCGCCTTCGAGGATCTGAGAGTGAGCAGCTTCATCAGGGGCACC


AGAGTGGTGCCCAGGGGCAAGCTGTCCACCAGGGGCGTGCAGATCGCCAG


CAACGAGAACATGGAAACCATGGACAGCAGCACCCTGGAACTGAGAAGCA


GGTACTGGGCCATCAGGACCAGAAGCGGCGGCAACACCAACCAGCAGAGG


GCCAGCGCCGGACAGATCAGCGTGCAGCCCACCTTCTCCGTGCAGAGGAA


CCTGCCCTTCGAGAGGGCCACCATCATGGCCGCCTTCACCGGCAACACCG


AGGGCAGGACCAGCGACATGAGGACCGAGATCATCAGAATGATGGAAAGC


GCCAGGCCCGAGGACGTGAGCTTCCAGGGCAGGGGCGTGTTCGAGCTGTC


CGATGAGAAGGCCACCTCCCCCATCGTGCCCAGCTTCGACATGAGCAACG


AGGGCAGCTACTTCTTCGGCGACAACGCCGAGGAATACGACAACTGA


 


SEQ ID NO: 6: Amino acid


MASQGTKRSYEQMETDGERQNATEIRASVGRMIGGIGRFYIQMCTELKLS


DYEGRLIQNSITIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRI


DGKWMRELILYDKEEIRRIWRQANNGEDATAGLTHMMIWHSNLNDATYQR


TRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRMIKRG


INDRNFWRGENGRRTRIAYERMCNILKGKFQTAAQRAMMDQVRESRNPGN


AEIEDLIFLARSALILRGSVAHKSCLPACVYGPAVASGYDFEREGYSLVG


IDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGT


RVVPRGKLSTRGVQIASNENMETMDSSTLELRSRYWAIRTRSGGNTNQQR


ASAGQISVQPTFSVQRNLPFERATIMAAFTGNTEGRTSDMRTEIIRMMES


ARPEDVSFQGRGVFELSDEKATSPIVPSFDMSNEGSYFFGDNAEEYDN.


 


M 1918 synthetic gene 0607868, Based on acc. No.:


AY130766: A/Brevig mission/1/1918


SEQ ID NO: 7: Nucleotide


ATGAGTCTTTTAACCGAGGTCGAAACGTACGTTCTCTCTATCGTCCCGTC


AGGCCCCCTCAAAGCCGAGATCGCGCAGAGACTTGAAGATGTCTTTGCAG


GGAAGAACACCGATCTTGAGGCTCTCATGGAATGGCTAAAGACAAGACCA


ATCCTGTCACCTCTGACTAAGGGGATTTTAGGATTTGTGTTCACGCTCAC


CGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCTTTGTCCAAAATGCCC


TTAATGGGAACGGGGATCCAAATAACATGGACAGAGCAGTTAAACTGTAC


AGGAAGCTTAAGAGGGAGATAACATTCCATGGGGCCAAAGAAGTAGCACT


CAGTTATTCCGCTGGTGCACTTGCCAGTTGTATGGGCCTCATATACAACA


GGATGGGGACTGTGACCACTGAAGTGGCATTTGGCCTGGTATGCGCAACC


TGTGAACAGATTGCTGATTCCCAGCATCGGTCTCACAGGCAAATGGTGAC


AACAACCAATCCACTAATCAGACATGAGAACAGAATGGTACTGGCCAGCA


CTACGGCTAAGGCTATGGAGCAAATGGCTGGATCGAGTGAGCAAGCAGCA


GAGGCCATGGAGGTTGCTAGTCAGGCTAGGCAAATGGTGCAGGCGATGAG


AACCATTGGGACTCATCCTAGCTCCAGTGCTGGTCTGAAAGACGATCTTA


TTGAAAATTTGCAGGCCTACCAGAAACGAATGGGGGTGCAGATGCAACGA


TTCAAGTGATCCTCTCGTTATTGCCGCAAGTATCATTGGGATCTTGCACT


TGATATTGTGGATTCTTGATCGTCTTTTTTTCAAATGCATTTATCGTCGC


CTTAAATACGGTTTGAAAAGAGGGCCTTCTACGGAAGGAGTGCCGGAGTC


TATGAGGGAAGAATATCGAAAGGAACAGCAGAGTGCTGTGGATGTTGACG


ATGGTCATTTTGTCAACATAGAGCTGGAGTAAGGCGCC


 


Amino acid


SEQ ID NO: 8: M1 protein


MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTRP


ILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDRAVKLY


RKLKREITFHGAKEVALSYSAGALASCMGLIYNRMGTVTTEVAFGLVCAT


CEQIADSQHRSHRQMVTTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAA


EAMEVASQARQMVQAMRTIGTHPSSSAGLKDDLIENLQAYQKRMGVQMQR


FK.


 


SEQ ID NO: 9: M2 protein


MSLLTEVETPTRNFWGCRCNDSSDPLVIAASIIGILHLILWILDRLFFKC


IYRRLKYGLKRGPSTEGVPESMREEYRKEQQSAVDVDDGHFVNIELE.









The 1918 HA and NA amino sequences are publicly 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, Kozak sequence, vaccine gene inclusive of 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 Caledonia/20/99(H1N1) virus challenge in DNA vaccinated ferrets (Mustela Putorius Furo). This is highly surprising since the two viruses 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 Caledonia). 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 unusually 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 adjuvants 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 provide 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 unusually broad and/or efficient protection obtained using a pandemic influenza A strain instead of the present day circulating strains may be due to special features in the sequence of the first new pathogenic and spreading virus. These features may gradually wane by accumulation of sequence changes during years of adaptation to the human and swine population.
If the protective feature is contained in the encoded amino acid sequence of the HA and/or NA 1918 and not the nucleotide sequence then the HA and/or NA protein(s) from 1918 may be used alone as an alternative to DNA or in combination with the DNA vaccine for immunization or vaccinations.
The use of the DNA vaccine components may serve as an adjuvant for the protein components and thus the protein and the DNA can be preferentially administered together as a mixed vaccine.
As a more universal DNA and/or protein vaccine against contemporary influenza in humans and/or swine a mixture may be used of HA and NA from the 1918 H1N1 pandemic strain plus HA and/or NA from the 1957 H2N2 pandemic strain plus HA from the 1968 H3N2 pandemic strain, where the N2 component is similar to the NA of the preferred earlier 1957 H2N2 strain.







TABLE 2





nucleotide and amino acid sequences of the codon


optimized genes and the proteins they express


(not codon optimized).















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


SEQ ID NO: 10: Nucleotide


ATAATTCTATTAATCATGAAGACCATCATTGCTTTGAGCTACATTTTCTG


TCTGGCTCTCGGCCAAGACCTTCCAGGAAATGACAACAGCACAGCAACGC


TGTGCCTGGGACATCATGCGGTGCCAAACGGAACACTAGTGAAAACAATC


ACAGATGATCAGATTGAAGTGACTAATGCTACTGAGCTAGTTCAGAGCTC


CTCAACGGGGAAAATATGCAACAATCCTCATCGAATCCTTGATGGAATAG


ACTGCACACTGATAGATGCTCTATTGGGGGACCCTCATTGTGATGTTTTT


CAAAATGAGACATGGGACCTTTTCGTTGAACGCAGCAAAGCTTTCAGCAA


CTGTTACCCTTATGATGTGCCAGATTATGCCTCCCTTAGGTCACTAGTTG


CCTCGTCAGGCACTCTGGAGTTTATCACTGAGGGTTTCACTTGGACTGGG


GTCACTCAGAATGGGGGAAGCAATGCTTGCAAAAGGGGACCTGGTAGCGG


TTTTTTCAGTAGACTGAACTGGTTGACCAAATCAGGAAGCACATATCCAG


TGCTGAACGTGACTATGCCAAACAATGACAATTTTGACAAACTATACATT


TGGGGGGTTCACCACCCGAGCACGAACCAAGAACAAACCAGCCTGTATGT


TCAAGCATCAGGGAGAGTCACAGTCTCTACCAGGAGAAGCCAGCAAACTA


TAATCCCGAATATCGAGTCCAGACCCTGGGTAAGGGGTCTGTCTAGTAGA


ATAAGCATCTATTGGACAATACTTAAGCCGGGAGACGTACTGGTAATTAA


TAGTAATGGGAACCTAATCGCTCCTCGGGGTTATTTCAAAATGCGCACTG


GGAAAAGCTCAATAATGAGGTCAGATGCACCTATTGATACCTGTATTTCT


GAATGCATCACTCCAAATGGAAGCATTCCCAATGACAAGCCCTTTCAAAA


CGTAAACAAGATCACATATGGAGCATGCCCCAAGTATGTTAAGCAAAACA


CCCTGAAGTTGGCAACAGGGATGCGGAATGTACCAGAGAAACAAACTAGA


GGCCTATTCGGCGCAATAGCAGGTTTCATAGAAAATGGTTGGGAGGGAAT


GATAGACGGTTGGTACGGTTTCAGGCATCAAAATTCTGAGGGCACAGGAC


AAGCAGCAGATCTTAAAAGCACTCAAGCAGCCATCGACCAAATCAATGGG


AAATTGAACAGGGTAATCGAGAAGACGAACGAGAAATTCCATCAAATCGA


AAAGGAATTCTCAGAAGTAGAAGGGAGAATTCAGGACCTCGAGAAATACG


TTGAAGACACTAAAATAGATCTCTGGTCTTACAATGCGGAGCTTCTTGTC


GCTCTGGAGAATCAACATACAATTGACCTGACTGACTCGGAAATGAACAA


GCTGTTTGAAAAAACAAGGAGGCAACTGAGGGAAAATGCTGAAGACATGG


GCAATGGTTGCTTCAAAATATACCACAAATGTGACAACGCTTGCATAGAG


TCAATCAGAAATGGGACTTATGACCATGATGTATACAGAGACGAAGCATT


AAACAACCGGTTTCAGATCAAAGGTGTTGAACTGAAGTCTGGATACAAAG


ACTGGATCCTGTGGATTTCCTTTGCCATATCATGCTTTTTGCTTTGTGTT


GTTTTGCTGGGGTTCATCATGTGGGCCTGCCAGAGAGGCAACATTAGGTG


CAACATTTGCATTTGAGTGTATTAGTAATTA


 


SEQ ID NO: 11: Amino acid


MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKT


ITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDV


FQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWT


GVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLY


IWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIESRPWVRGLSS


RISLYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCI


SECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQT


RGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQIN


GKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELL


VALENQHTIDLTDSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCDNACI


ESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLC


VVLLGFIMWACQRGNIRCNICI


 


NA H3N2 Acc. No.: AB295606: A/Aichi/2/1968(H3N2)


SEQ ID NO: 12: Nucleotide


GAAAATGAATCCAAATCAAAAGATAATAACAATTGGCTCTGTCTCTCTCA


CCATTGCAACAGTATGCTTCCTCATGCAGATTGCCATCCTGGTAACTACT


GTAACATTGCATTTTAAGCAATATGAGTGCGACTCCCCCGCGAGCAACCA


AGTAATGCCGTGTGAACCAATAATAATAGAAAGGAACATAACAGAGATAG


TGTATTTGAATAACACCACCATAGAGAAAGAGATATGCCCCAAAGTAGTG


GAATACAGAAATTGGTCAAAGCCGCAATGTCAAATTACAGGATTTGCACC


TTTTTCTAAGGACAATTCAATCCGGCTTTCTGCTGGTGGGGACATTTGGG


TGACGAGAGAACCTTATGTGTCATGCGATCATGGCAAGTGTTATCAATTT


GCACTCGGGCAGGGGACCACACTAGACAACAAACATTCAAATGACACAAT


ACATGATAGAATCCCTCATCGAACCCTATTAATGAATGAGTTGGGTGTTC


CATTTCATTTAGGAACCAGGCAAGTGTGTATAGCATGGTCCAGCTCAAGT


TGTCACGATGGAAAAGCATGGCTGCATGTTTGTATCACTGGGGATGACAA


AAATGCAACTGCTAGCTTCATTTATGACGGGAGGCTTGTGGACAGTATTG


GTTCATGGTCTCAAAATATCCTCAGAACCCACGAGTCGGAATGCGTTTGT


ATCAATGGGACTTGCACACTAGTAATGACTGATGGAAGTGCTTCAGGAAG


AGCCGATACTAGAATACTATTCATTGAAGAGGGGAAAATTGTCCATATTA


GCCCATTGTCAGGAAGTGCTCAGCATGTAGAAGAGTGTTCCTGTTATCCT


AGATATCCTGGCGTCAGATGTATCTGCAGAGACAACTGGAAAGGCTCTAA


TAGGCCCGTCGTAGACATAAATATGGAAGATTATAGCATTGATTCCAGTT


ATGTGTGCTCAGGGCTTGTTGGCGACACACCTAGAAACGACGACAGATCT


AGCAATAGCAATTGCAGGAATCCTAATAATGAGAGAGGGAATCAAGGAGT


GAAAGGCTGGGCCTTTGACAATGGAGATGACGTGTGGATGGGAAGAACGA


TCAGCAAGGATTTACGCTCAGGTTATGAAACTTTCAAAGTCATTGGTGGT


TGGTCCACACCTAATTCCAAATCGCAGATCAATAGACAAGTCATAGTTGA


CAGCGATAATCGGTCAGGTTACTCTGGTATTTTCTCTGTTGAGGGCAAAA


GCTGCATCAATAGGTGCTTTTATGTGGAGTTGATAAGGGGAAGGAAACAG


GAGACTAGAGTGTGGTGGACCTCAAACAGTATTGTTGTGTTTTGTGGCAC


TTCAGGTACCTATGGAACAGGCTCATGGCCTGATGGGGCGAACATCAATT


TCATGCCTATATAAGCTTTCGCAATTTTAGA


 


SEQ ID NO: 13: Amino acid


MNPNQKIITIGSVSLTIATVCFLMQIAILVTTVTLHFKQYECDS


PASNQVMPCEPIIIERNITEIVYLNNTTIEKEICPKVVEYRNWSKPQCQI


TGFAPFSKDNSIRLSAGGDIWVTREPYVSCDHGKCYQFALGQGTTLDNKH


SNDTIHDRIPHRTLLMNELGVPFHLGTRQVCIAWSSSSCHDGKAWLHVCI


TGDDKNATASEIYDGRLVDSIGSWSQNILRTQESECVCINGTCTVVMTDG


SASGRADTRILFIEEGKIVHISPLSGSAQHVEECSCYPRYPGVRCICRDN


WKGSNRPVVDINMEDYDIDSSYVCSGLVGDTPRNDDRSSNSNCRNPNNER


GNQGVKGWAFDNGDDVWMGRTISKDLRSGYETFKVIGGWSTPNSKSQINR


QVIVDSDNRSGYSGIFSVEGKSCINRCFYVELIRGRKQETRVWWTSNSIV


VFCGTSGTYGTGSWPDGANINFMPI


 


HA H2N2 Acc. No.: CY022013: A/Albany/20/1957(H2N2)


SEQ ID NO: 14: Nucleotide


ATAGACAACCAAAAGCAAAACAATGGCCATCATTTATCTCATTCTCCTGT


TCACAGCAGTGAGAGGGGACCAGATATGCATTGGATACCATGCCAATAAT


TCCACAGAGAAGGTCGACACAATTCTAGAGCGGAACGTCACTGTGACTCA


TGCCAAGGACATTCTTGAGAAGACCCATAACGGAAAGTTATGCAAACTAA


ACGGAATCCCTCCACTTGAACTAGGGGACTGTAGCATTGCCGGATGGCTC


CTTGGAAATCCAGAATGTGATAGGCTTCTAAGTGTGCCAGAATGGTCCTA


TATAATGGAGAAAGAAAACCCGAGAGACGGTTTGTGTTATCCAGGCAGCT


TCAATGATTATGAAGAATTGAAACATCTCCTCAGCAGCGTGAAACATTTC


GAGAAAGTAAAGATTCTGCCCAAAGATAGATGGACACAGCATACAACAAC


TGGAGGTTCACGGGCCTGCGCGGTGTCTGGTAATCCATCATTCTTCAGGA


ACATGATCTGGCTGACAAAGAAAGGATCAAATTATCCGGTTGCCAAAGGA


TCGTACAACAATACAAGCGGAGAACAAATGCTAATAATTTGGGGGGTGCA


CCATCCCAATGATGAGACAGAACAAAGAACATTGTACCAGAATGTGGGAA


CCTATGTTTCCGTAGGCACATCAACATTGAACAAAAGGTCAACCCCAGAC


ATAGCAACAAGGCCTAAAGTGAATGGACTAGGAAGTAGAATGGAATTCTC


TTGGACCCTATTGGATATGTGGGACACCATAAATTTTGAGAGTACTGGTA


ATCTAATTGCACCAGAGTATGGATTCAAAATATCGAAAAGAGGTAGTTCA


GGGATCATGAAAACAGAAGGAACACTTGGGAACTGTGAGACCAAATGCCA


AACTCCTTTGGGAGCAATAAATACAACATTGCCTTTTCACAATGTCCACC


CACTGACAATAGGTGAGTGCCCCAAATATGTAAAATCGGAGAAGTTGGTC


TTAGCAACAGGACTAAGGAATGTTCCCCAGATTGAATCAAGAGGATTGTT


TGGGGCAATAGCTGGTTTTATAGAAGGAGGATGGCAAGGAATGGTTGATG


GTTGGTATGGATACCATCACAGCAATGACCAGGGATCAGGGTATGCAGCG


GACAAAGAATCCACTCAAAAGGCATTTGATGGAATCACCAACAAGGTAAA


TTCTGTGATTGAAAAGATGAACACCCAATTTGAAGCTGTTGGGAAAGAAT


TCAGTAACTTAGAGAGAAGACTGGAGAACTTGAACAAAAAGATGGAAGAC


GGGTTTCTAGATGTGTGGACATACAATGCTGAGCTTCTAGTTCTGATGGA


AAATGAGAGGACACTTGACTTTCATGATTCTAATGTCAAGAATCTGTATG


ATAAAGTCAGAATGCAGCTGAGAGACAACGTCAAAGAACTAGGAAATGGA


TGTTTTGAATTTTATCACAAATGTGATGATGAATGCATGAATAGTGTGAA


AAACGGGACGTATGATTATCCCAAGTATGAAGAAGAGTCTAAACTAAATA


GAAATGAAATCAAAGGGGTAAAATTGAGCAGCATGGGGGTTTATCAAATC


CTTGCCATTTATGCTACAGTAGCAGGTTCTCTGTCACTGGCAATCATGAT


GGCTGGGATCTCTTTCTGGATGTGCTCCAACGGGTCTCTGCAGTGCAGGA


TCTGCATATGATTATAAGTCATTTTATAATTAA


 


SEQ ID NO: 15: Amino acid


MAIIYLILLFTAVRGDQICIGYHANNSTEKVDTILERNVTVTHA


KDILEKTHNGKLCKLNGIPPLELGDCSIAGWLLGNPECDRLLSVPEWSYI


MEKENPRDGLCYPGSFNDYEELKHLLSSVKHFEKVKILPKDRWTQHTTTG


GSRACAVSGNPSFFRNMIWLTKKGSNYPVAKGSYNNTSGEQMLIIWGVHH


PNDETEQRTLYQNVGTYVSVGTSTLNKRSTPDIATRPKVNGLGSRMEFSW


TLLDMWDTINFESTGNLIAPEYGFKISKRGSSGIMKTEGTLGNCETKCQT


PLGAINTTLPFHNVHPLTIGECPKYVKSEKLVLATGLRNVPQIESRGLFG


AIAGFIEGGWQGMVDGWYGYHHSNDQGSGYAADKESTQKAFDGITNKVNS


VIEKMNTQFEAVGKEFSNLERRLENLNKKMEDGFLDVWTYNAELLVLMEN


ERTLDFHDSNVKNLYDKVRMQLRDNVKELGNGCFEFYHKCDDECMNSVKN


GTYDYPKYEEESKLNRNEEKGVKLSSMGVYQILAIYATVAGSLSLAIMMA


GISFWMCSNGSLQCRICI


 


NA H2N2 Acc. No.: CY022015: A/Albany/20/1957(H2N2)


SEQ ID NO: 16: Nucleotide


TGAAAATGAATCCAAATCAAAAGATAATAACAATTGGCTCTGTCTCTCTC


ACCATTGCAACAGTATGCTTCCTCATGCAGATTGCCATCCTGGCAACTAC


TGTGACATTGCATTTTAAACAACATGAGTGCGACTCCCCCGCGAGCAACC


AAGTAATGCCATGTGAACCAATAATAATAGAAAGGAACATAACAGAGATA


GTGTATTTGAATAACACCACCATAGAGAAAGAGATTTGCCCCGAAGTAGT


GGAATACAGAAATTGGTCAAAGCCGCAATGTCAAATTACAGGATTTGCAC


CTTTTTCTAAGGACAATTCAATCCGGCTTTCTGCTGGTGGGGACATTTGG


GTGACGAGAGAACCTTATGTGTCATGCGATCCTGGCAAGTGTTATCAATT


TGCACTCGGGCAAGGGACCACACTAGACAACAAACATTCAAATGGCACAA


TACATGATAGAATCCCTCACCGAACCCTATTAATGAATGAGTTGGGTGTT


CCATTTCATTTAGGAACCAAACAAGTGTGTGTAGCATGGTCCAGCTCAAG


TTGTCACGATGGAAAAGCATGGTTGCATGTTTGTGTCACTGGGGATGATA


GAAATGCGACTGCCAGCTTCATTTATGACGGGAGGCTTGTGGACAGTATT


GGTTCATGGTCTCAAAATATCCTCAGGACCCAGGAGTCGGAATGCGTTTG


TATCAATGGGACTTGCACAGTAGTAATGACTGATGGAAGTGCATCAGGAA


GAGCCGATACTAGAATACTATTCATTAAAGAGGGCAAAATTGTCCATATC


AGCCCATTGTCAGGAAGTGCTCAGCATATAGAGGAGTGTTCCTGTTACCC


TCGATATCCTGACGTCAGATGTATCTGCAGAGACAACTGGAAAGGCTCTA


ATAGGCCCGTTATAGACATAAATATGGAAGATTATAGCATTGATTCCAGT


TATGTGTGCTCAGGGCTTGTTGGCGACACACCCAGGAACGACGACAGCTC


TAGCAATAGCAATTGCAGGGATCCTAACAATGAGAGAGGGAATCCAGGAG


TGAAAGGCTGGGCCTTTGACAATGGAGATGATGTATGGATGGGAAGAACA


ATCAACAAAGATTCACGCTCAGGTTATGAAACTTTCAAAGTCATTGGTGG


TTGGTCCACACCTAATTCCAAATCGCAGGTCAATAGACAGGTCATAGTTG


ACAACAATAATTGGTCTGGTTACTCTGGTATTTTCTCTGTTGAGGGCAAA


AGCTGCATCAATAGGTGCTTTTATGTGGAGTTGATAAGGGGAAGGCCACA


GGAGACTAGAGTATGGTGGACCTCAAACAGTATTGTTGTGTTTTGTGGCA


CTTCAGGTACTTATGGAACAGGCTCATGGCCTGATGGGGCGAACATCAAT


TTCATGCCTATATAAGCTTTCGCAATTTTAGAAAA


 


SEQ ID NO: 17: Amino acid


MNPNQKIITIGSVSLTIATVCFLMQIAILATTVTLHFKQHECDS


PASNQVMPCEPIIIERNITEIVYLNNTTIEKEICPEVVEYRNWSKPQCQI


TGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPGKCYQFALGQGTTLDNKH


SNGTIHDRIPHRTLLMNELGVPFHLGTKQVCVAWSSSSCHDGKAWLHVCV


TGDDRNATASFIYDGRLVDSIGSWSQNILRTQESECVCINGTCTVVMTDG


SASGRADTRILFIKEGKIVHISPLSGSAQHIEECSCYPRYPDVRCICRDN


WKGSNRPVIDINMEDYSIDSSYVCSGLVGDTPRNDDSSSNSNCRDPNNER


GNPGVKGWAFDNGDDVWMGRTINKDSRSGYETFKVIGGWSTPNSKSQVNR


QVIVDNNNWSGYSGIFSVEGKSCINRCFYVELIRGRPQETRVWWTSNSIV


VFCGTSGTYGTGSWPDGANINFMPI









In one embodiment, a method of treating or preventing infection with a H1, H2, H3 containing influenza A in a subject is provided, comprising delivering to the subject a naked DNA and/or RNA molecule encoding hemagglutinin (HA) from pandemic influenza selected from one or more of the 1918 H1N1, the 1957 H2N2, and the 1968 H3N2 influenza A virus. In a further embodiment, the method comprises administering a vaccine for N1, N2 containing influenza A in humans or swine, the method further comprising delivering to the subject a naked DNA and/or RNA molecule encoding neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from a pandemic influenza virus therapeutically or prophylactically.
In another embodiment, the codons of the DNA or RNA may be humanized using codons of highly expressed human proteins. In yet another embodiment, an adjuvant is delivered to the subject. In still a further embodiment, a DNA vaccine is administered by saline injection of naked DNA and/or RNA, inoculated by gene gun or is delivered coupled to particles.
Also provided is a vaccine for human use containing a naked DNA and/or RNA molecule encoding hemagglutinin (HA) from a pandemic influenza selected from the 1918 H1N1, the 1957 H2N2, and the 1968 H3N2 influenza A virus. In a further embodiment, the vaccine also comprises a naked DNA and/or RNA molecule encoding neuraminidase (NA) and/or matrix protein (M) and/or the nucleoprotein (NP) from a pandemic influenza virus.
In another embodiment, the vaccine's antigenic component consists of the HA and one or more of a neuraminidase (NA), a matrix (M) protein, and/or nucleoprotein (NP). The vaccine's antigenic component may also consist of naked DNA or RNA molecule coding for the HA and one or more of a neuraminidase (NA) protein, matrix protein, and/or nucleoprotein (NP). In still a further embodiment, the DNA or RNA codons may be humanized using codons of highly expressed human proteins. In yet another embodiment, the vaccine contains an adjuvant.
In another embodiment, a vaccine described herein may be administered therapeutically to already infected humans or swine.
In one embodiment, a method of treating or preventing infection with H5, H7 or H2 containing influenza A in a subject is provided, wherein a naked DNA and/or RNA molecule encoding hemagglutinin (HA) and/or neuraminidase (NA) from an influenza strain selected from the group consisting of a 2001 H5N7 low pathogenic Avian influenza virus (AIV) strain (A/Mallard/Denmark/64650/03(H5N7)) and a March 2006 Denmark H5N1 high pathogenic AIV strain (A/buzzard/Denmark/6370/06(H5N1)) is delivered to a subject. In a further embodiment, the naked DNA and/or RNA molecule encoding HA or NA are delivered in a composition containing the HA and/or NA proteins. In another embodiment, the HA or NA are delivered in a composition containing the naked DNA and/or RNA molecule encoding the HA and/or NA proteins. In still another embodiment, the DNA or RNA codons are humanized.
In another embodiment, a vaccine for preventing infection with H5, H7 or H2 containing influenza A infections in humans or swine is provided, the vaccine containing a naked DNA and/or RNA molecule encoding hemagglutinin (HA) and/or neuraminidase (NA) from an influenza strain selected from the group consisting of 2001 H5N7 low pathogenic Avian influenza virus (AIV) strain (A/Mallard/Denmark/64650/03(H5N7)), March 2006 Denmark H5N1 high pathogenic AIV strain (A/buzzard/Denmark/6370/06(H5N1)), (A/duck/Denmark/53-147-8/08 (H7N1)) and (A/widegeon/Denmark/66174/G18/04 (H2N3)). In a further embodiment, the vaccine contains naked DNA and/or RNA molecules encoding the HA or NA proteins. In another embodiment, the DNA or RNA codons are humanized. In still another embodiment, the vaccine further contains an adjuvant.
The following examples are illustrative of the compositions and methods of the invention. It will be readily understood by one of skill in the art that the specific conditions described herein can be varied without departing from the scope of the present invention. It will be further understood that other compositions not specifically illustrated are within the scope of the invention as defined herein.
EXAMPLES
Example 1
Construction of Expression Vectors
The 1918 pandemic H1N1 genes were designed from nucleotide sequences published in GenBank (HA: A/South Carolina/1/18 AF117241, and NA, NP and M: A/Brevig Mission/1/18 AF250356, AY744035 and AY130766, respectively). The genes were made synthetically and designed to include the appropriate restriction enzymes and Kozak sequence (GCCACC), −1 base upstream from the start codon, for efficient cloning and transcription in the WRG7079 expression vector (PowderJect, Madison, Wis.). The genes were synthesised using only codons from highly expressed human genes (5) (codon optimised). By this the nucleotide codons are altered (humanised), but the encoded amino acids are identical to those encoded by the viral RNA. The genes were further cloned individually into the WRG7079 expression vector. Key elements in the expression vector are a kanamycin resistance gene, cytomegalovirus immediate-early promoter, 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. The same vector was also applied for expression of 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. The WRG7079 was further modified to remove an unwanted app109 nucleotide sequence from the SIV nef gene.
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:18), HA NC R: 5′-tcggcgcctcagatgcatattctacactgc-3′ (SEQ ID NO:19), NA NC F: 5′-caacgcgtgccaccatgaatccaaatc-3′ (SEQ ID NO:20), NA NC R: 5′-tcg gcgccctacttgtcaatggtgaa cggc-3′ (SEQ ID NO:21)). 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 DH5a bacteria. Endotoxin free DNA purification of the vaccine clones were prepared by EndoFree Plasmid Giga Kit (QIAGEN). All inserts and vaccine clones were control sequenced.
Example 2
Immunisations
A total of 24 ferrets (Mustela Putorius Furo), approximately seven months old, were divided in four groups by using a chip-tag identification for dogs (E-vet, pet-id, Haderslev, Denmark), six animals in each group. All animals were kept together and fed a standard diet with food and water ad libitum. The animals were housed according to the Danish Animal Experimentation Act and kept at level II biosecurity facilities at the Faculty of Life Sciences, Copenhagen. The acclimatisation period was nine days.
Four groups of six ferrets were vaccinated as follows; (1) HA (codon optimised gene) and NA (codon optimised gene) 1918 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 Caledonia/20/99(H1N1) plasmid DNA vaccinated (positive vaccine control). All ferrets received four standard gene gun shots onto shaved abdomen. HA and NA DNA mixed vaccines were given in two shots and NP and M DNA mixed vaccines were given in two shots. Therefore groups 1 and 4 receiving only HA and NA DNA vaccine were additionally shot twice with empty plasmid DNA, ensuring that all animals had received the same amount of DNA and the same number of shots. The ferrets were gene gun (Helios, Bio-Rad, Hercules, Calif.) inoculated (400 psi compressed helium) on shaved abdominal skin, using 2 ng plasmid DNA-coated gold particles (1.6 μm-sized particles), 80-95% coating efficiency each shot. Each ferret received four shots, three times biweekly. Ferrets were challenged ten days after the third immunisation by 1×107 50% egg infectious dose (EID50) of A/New Caledonia/20/99(H1N1) virus in 100 μl PBS administrated into the nostrils with a syringe. Blood serum was collected at day −2,3,5 and 7 post-challenge from vena jugularis of anesthetised animals (tiletamine/zolazepam (zoletil-mix for cats)). Animals were terminated with pentobarbital.
Example 3
Quantitative Real Time RT-PCR Assay for Influenza A
At the day of blood serum collection the nostrils of each ferret were flushed with 1 ml PBS and the flushing were frozen down immediately for real-time RT-PCR analysis. Two hundred micro litres of nasal wash were extracted on an automated MagNA Pure LC Instrument applying the MagNa Pure LC Total Nucleic Acid Isolation Kit (Roche diagnostics, Basel, Switzerland). The extracted material was eluated in 200 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 with known concentration.
Example 4
Serum Antibody Determined by ELISA
ELISA plates (96 wells) were coated with 100 μl, split influenza vaccine (Vaxigrip, Sanofi Pasteur, Belgium) diluted 1:100 in 35 mM NaHCO3 pH 9.6 and 15 mM Na2CO3 overnight 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 5
Results
Ferrets were negative for influenza specific antibodies seven days before start of immunisations as measured by ELISA.
High IgG specific serum antibodies (to A/New Caledonia/20/99(H1N1) in ELISA) were observed at day seven post-challenge in ferrets vaccinated with both HA+NA 1918 (two plasmids) and HA+NA+NP+M 1918 DNA vaccines (four plasmids) (FIG. 1). Ferrets vaccinated with HA+NA DNA A/New Caledonia/20/99(H1N1) induced lower specific serum antibody titre on day seven. It is possible that 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 an A/New Caledonia/20/99(H1N1) like virus (FIG. 1). Partial protection was observed in ferrets vaccinated with HA+NA A/New Caledonia/20/99(H1N1) DNA plasmids.
The data clearly show that DNA gene gun immunisations based on genes from the 1918 H1N1 pandemic strain induce strong specific antibody response and protect ferrets completely against infection with a H1N1 strain that has drifted by 89 years. No negative or positive effects on the humoral immune response or protection was observed by including the NP and M genes in the HA+NA DNA vaccination since the protection from infection already was already 100%.
The A/South Carolina/1/18 and A/New Caledonia/20/99 are 21.2% different in the HA1 protein and possess eight substitutions at residues involved in antigenic sites 3 (1918 to New Caledonia); Cb S83P, Sa T128V and K160N, Sb S156G, Q193H and D196N, Cal N207S and A224E.
DNA vaccines do have the ability of immune stimulatory mechanisms. This might be one reason why such good cross reactivity and protection was induced 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 the 21.2% observed in the present studies.
Example 6
1918 Pandemic H1N1 DNA Vaccinated Ferrets were Challenged with 2007 H1N1
Vaccine production and vaccinations and assays were carried out as described above.
A total of 10 ferrets (Mustela Putorius Euro), approximately seven months old, were divided in two groups by using a chip-tag identification for dogs (E-vet, pet-id, Haderslev, Denmark), five animals in each group. All animals were kept together and fed a standard diet with food and water ad libitum. The animals were housed according to the Danish Animal Experimentation Act and kept at level II biosecurity facilities at the Faculty of Life Sciences, Copenhagen. The acclimatisation period was one day. Two groups of five ferrets were vaccinated as follows; (1) HA (codon optimised gene) and NA (codon optimised gene) 1918 H1N1 plasmid DNA vaccinated, (2) non-vaccinated, naïve animals. HA and NA DNA mixed vaccines were given in four 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. Vaccinated ferrets received four shots, three times biweekly. Ferrets were challenged ten days after third immunisation by 1×107 50% egg infectious dose (EID50) of A/New Caledonia/20/99(H1N1) virus in 1000 μl PBS administrated into the nostrils with a syringe. Blood serum was collected at day −48, 0, 5, 7 and 12 post-challenge from vena jugularis of anesthetised animals (tiletamine/zolazepam (zoletil-mix for cats)). Animals were terminated with pentobarbital. The 1918 DNA vaccinated ferrets had a lower temperature rise than the unvaccinated group (p=0.2) at the day of maximal temperature rise, day 2 post challenge (FIG. 2A). No difference in weight loss between the vaccinated and the unvaccinated animals was observed at the day of maximal weight loss, day 4 post challenge (FIG. 2B). Vaccinated animals displayed fewer influenza symptoms than unvaccinated animals measured by sneezing, nasal discharge and activity level (p=0.065) (FIG. 2C.). Ferrets in both groups had high virus load post infection measured by quantitative real-time RT-PCR, however by day 7 post infection the 1918 DNA vaccinated ferrets better cleared their virus infection than the unvaccinated ferrets (p=0.63) (FIG. 2D).
Example 7
Challenge with New Caledonia H1N1 in Ferrets
Traditional protein H1N1 New Caledonia vaccine plus/minus DDA/TDB adjuvants versus 1918 H1N1 HA plus NA DNA vaccine (versus empty DNA vaccine vector) using two DNA immunizations (instead or the usual 3 DNA immunizations)
Traditional protein H1N1 split vaccine (two immunizations) versus 1918 H1N1 HA plus NA codon optimized DNA vaccine versus codon optimized and non-codon-optimized New Caledonia H1N1 HA and NA versus codon optimized M and NP from 1918 H1N1 virus (versus empty DNA vaccine vector) using three immunizations.
Ferrets are challenged with H1N1 New Caledonia-like virus intra nasally and virus quantitated in basal washings by real-time RT/PCR assay. Ferret antibodies will be examined for ELISA antibodies and HI antibody reactions to H1N1, H2N2, H3N2, H5N7, and/or H5N1.
Example 8
Mouse Antibody Experiments
Codon optimized versus non-codon optimized HA and NA DNA vaccines from New Caledonia 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 9
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. Codon optimized from 1918 H1N1, H5N7 and H3N2 strain versus non-codon optimized HA DNA vaccines from 1918 H1N1 strain is expressed in mammalian cell lines in vitro and hemadsorption is measured. This shows that the H1 is functionally expressed better when codons are optimized (FIG. 3).
Example 10
Cytokine Induction Experiments
Codon optimized versus non-codon optimized HA and NA DNA vaccines from New Caledonia 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 Caledonia HA and NA to examine special cytokine induction by the 1918 genes.
Example 11
1918 HA and NA Protein Vaccine Experiments
Proteins are produced by the DNA vaccine plasmids and used as a protein vaccine in mice or ferrets as compared to DNA vaccination and to traditional protein split vaccine to measure the immune induction of 1918 proteins versus DNA vaccine.
Example 12
Mouse DNA Vaccine Delivery Experiments
Codon optimized HA and/or NA DNA vaccines from 1918 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 13
Swine DNA Vaccine Delivery Experiments
Codon optimized HA and/or NA DNA vaccines from 1918 H1N1 strain is inoculated in pigs as expression plasmids and challenge with a present day New Caledonia-like H1N1 strain and protection against disease and immune induction are measured.
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All publications cited in this specification are incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

## Claims

1. A method of inducing an immune response to an H1N1 influenza A in a subject, said method comprising delivering to the subject (i) an isolated naked nucleic acid sequence encoding the matrix protein (M) from a pandemic influenza virus and an isolated naked nucleic acid sequence encoding the nucleoprotein (NP) from a pandemic influenza virus, and (ii) an isolated naked nucleic acid sequence encoding hemagglutinin (HA) from a pandemic influenza virus, wherein said M and NP proteins are encoded by the isolated naked nucleic acid sequences of SEQ ID Nos: 7 and 5, respectively, and the M and NP proteins are from a 1918 H1N1 influenza A virus and said HA is from a different pandemic influenza virus than that of said M and NP.

2. The method according to claim 1, wherein the immune response is a cytotoxic cellular response.

3. The method according to claim 1, wherein the immune response is a humoral antibody response.

4. The method according to claim 1, wherein said 1918 H1N1 influenza A virus is strain A/Brevig Mission/1/1918 H1N1 influenza A virus.

5. The method according to claim 1, wherein one or more of the nucleic acid sequences comprises a promoter and a polyadenylation signal sequence.

6. The method according to claim 1, wherein one or more of the nucleic acid sequences is contained in an expression plasmid.

7. The method according to claim 1, wherein the method further comprises delivering to the subject an isolated naked nucleic acid sequence encoding neuraminidase (NA) from a pandemic influenza virus.

8. The method according to claim 1, wherein the codons of one or more of the nucleic acid sequences are humanized using codons of highly expressed human proteins.

9. The method according to claim 1, further comprising delivering an adjuvant to the subject.

10. The method according to claim 1, wherein the one or more nucleic acid sequences is administered by saline injection or by gene gun.

11. The method according to claim 10, wherein the one or more nucleic acid sequences is coupled to particles.

12. The method according to claim 6, wherein the nucleic acid sequence encoding M or the nucleic acid sequence encoding NP, and the nucleic acid sequence encoding HA, are contained in a single expression plasmid.

13. The method according to claim 12, wherein the naked nucleic acid sequence encoding M and the naked nucleic acid sequence encoding NP and the naked nucleic acid sequence encoding HA are contained in a single expression plasmid.

14. The method according to claim 7, wherein said NA and HA are from the same pandemic influenza virus.