influenza nucleic acid molecules and vaccines made from the same

专利摘要:
  INFLUENZA NUCLEIC ACID MOLECULES AND VACCINES MADE FROM THE SAME.Nucleic acid sequences encoding new hemagglutinin HA amino acid consensus sequences are provided here, as well as genetic constructs / vectors and vaccines that express the sequences. Methods are still provided here to generate an immune response against one or more Influenza A serotypes using the vaccines that are provided.
公开号:BR112012018587A2
申请号:R112012018587-0
申请日:2011-01-26
公开日:2020-09-01
发明作者:David B. Weiner；Jian Yan；Matthew P. Morrow
申请人:The Trustees Of The University Of Pennsylvania；
IPC主号:

专利说明:

INFLUENZA NUCLEIC ACID MOLECULES AND VACCINES MADE THE SAME
FIELD OF THE INVENTION The present invention relates to improved influenza viral vaccines: improved methods for inducing influenza immune responses, improved methods for diagnosing vaccinated versus infected mammalian influenza hosts and for prophylactically and / or therapeutically immunizing individuals against influenza.
BACKGROUND OF THE INVENTION Influenza, commonly referred to as influenza, is an infectious disease caused by the RNA virus of the family Orthomyxoviridae. Influenza or influenza viruses infect birds and mammals. Three of the five genera of Orthomyxoviridae are influenza viruses: Influenza * A, Influenza B and Influenza C. Of these, Influenza A is the most common.
Influenza is typically transmitted through air in aerosols produced by coughing or sneezing and by direct contact with body fluids containing the virus or contaminated surfaces. Seasonal influenza epidemics occur worldwide and result in hundreds of thousands of deaths annually. In a few years, pandemics occur and cause millions of deaths. In addition, animals, especially poultry and pigs, are also susceptible to annual epidemics and occasional pandemics that cause a large number of animal deaths and monetary losses.
Structurally, influenza viruses are similar, containing virus particles, usually spherical or filamentous, about 80-120 nm consisting of a similar molecular component. A central nucleus comprising viral proteins and viral RNA is covered by a viral envelope composed of two different glycoproteins and a lipid coating derived from the cell in which the viral particle is produced. Two other different glycoproteins are anchored within the viral envelope and include portions that protrude outward onto the surface.
The influenza virus RNA genome is normally supplied as eight different single-stranded negative sense RNA segments that together make up the eleven viral genes in the genome that encode the eleven proteins (HA, NA, NP, MI, M2, NS1, NEP, PA , PB1, PB! -F2, PB2). The eight RNA segments are: 1) HA, which encodes hemagglutinins (about 500 molecules of hemagglutinin are needed to create a virion); 2) NA, which encodes neuraminidase (about 100 neuraminidase molecules are needed to create a virion); 3) NP, which encodes nucleoprotein; 4) M, which encodes two matrix proteins (M1 and M2) using different phases of readings from the same RNA structural segment (about 3000 molecules of matrix protein are needed to create a virion); 5) NS, which encodes two distinct non-structural proteins (NS1 and NEP) using different phases of readings from the same RNA segment; 6) PA, which encodes an RNA polymerase; 7) PB1, which encodes an RNA polymerase and protein PB1- F2 (induces apoptosis) using different reading regions from the same RNA segment; and 8) PB2, which encodes an RNA polymerase.
Of these eleven proteins, hemagglutinin (HA) and neuraminidase (NA) are two —glycoproteins anchored in the viral envelope and present on the outer surface of the viral particles.
These proteins serve as immunogens for immune responses against influenza.
HA, which is a lectin that mediates the binding of the virus to target cells and entry of the viral genome into the target cell, is expressed as a single gene product, HAO, and subsequently processed by host proteases to produce two subunits, HA] and HA2 , which together form a complex on the surface of influenza virus particles.
NA is involved in the release of newly produced mature viral particles produced in infected cells.
There are sixteen known HA serotypes and nine known NA serotypes for influenza A viruses.
The identity of the different serotypes present in a viral particle is typically used to describe a virus.
For example, HIN] is an influenza virus with HA serotype H1 and N1 serotype NA; HSNI is an influenza virus with HA serotype H5 and NA serotype N1. Only serotypes H1, H2 and H3, and serotypes N1 and N2 normally infect humans.
Influenza strains are generally species or genus-specific, that is, an influenza virus that can infect pigs (a swine influenza virus) does not normally infect humans or birds; an influenza strain that can infect birds (avian influenza virus) does not infect humans or pigs and an influenza strain that can infect humans (a human influenza virus) does not infect birds and pigs.
Influenza strains, however, can mutate and become infectious from one species to another.
For example, a strain that only infects pigs, a swine influenza, can mutate or recombine to become a strain that can infect humans only or both pigs and humans.
A flu virus commonly referred to as “swine flu” is a strain of the influenza virus, like a HINI strain,
which can infect humans and which was derived from a strain that was previously specific for pigs (ie, a swine flu virus is a human influenza of swine origin, or human influenza derived from swine). A flu virus commonly referred to as “bird flu” is a strain of the influenza virus, such as an HSNI1 strain, that can infect — humans who were derived from a strain that was previously specific to birds (ie, an avian flu virus is a human influenza of avian origin, or human influenza derived from birds). Influenza vaccination is provided for many humans in developed countries and sometimes for animals. The vaccines used are limited in their protection results because the immune responses induced by the vaccines are specific for certain virus subtypes. Different influenza vaccines are developed and administered annually based on international surveillance and scientists' estimates of what types and strains of the virus circulate in a given year. The virus changes significantly by recombination, mutation and rearrangement of the segments. Thus, vaccines given in one year are not considered to be protective against seasonal strains that are widely transmitted in the following year.
A flu shot commonly promoted by US Centers for Disease Control and Prevention generally contains three dead / inactivated influenza viruses: an À (H3N2) virus, an A (HINÍ) virus, and a B virus. Thus, it is apparent that vaccines are limited — subtype forecasts, and the availability of a specific vaccine for that subtype.
Direct administration of nucleic acid sequences to vaccinate against animal and human diseases has been studied and much effort is focused on effective and efficient means of releasing nucleic acid to generate the necessary expression of the desired antigens, resulting in an immunogenic response and, ultimately, this technical.
DNA vaccines have many advantages over the more conceptual methods of traditional vaccination, such as live attenuated viruses and vaccines based on recombinant proteins. DNA vaccines are safe, stable, easily produced, and well tolerated in humans with preclinical trials indicating little evidence of plasmid integration [Martin, T., et al, Plasmid DNA malaria vaccine: the potential for —genomic integration after intramuscular injection. Hum Gene Ther, 1999. 10 (5): p. 759-68; Nichols, W.W ,, et al., Potential DNA vaccine integration into host cell genome. Ann N Y Acad Sci, 1995. 772: p. 30-91. In addition, DNA vaccines are well studied for repeated administration due to the fact that the vaccine's effectiveness is not influenced by pre-existing antibody titers to the vector [Chattergoon, M., J. Boyer, and D.B. Weiner, Genetic immunization: a new era in vaccines and immune therapeutics. FASEB J, 1997. 11 (10): p. 753-63]. However, a major obstacle to clinical adoption of DNA vaccines was a reduction in platform immunogenicity when transferring to larger animals [Liu, M.A. and J.B. Ulmer, Human clinical trials of plasmid DNA vaccines. Adv Genet, 2005. 55: p. 25-40]. Advances in recent technology in DNA vaccine immunogen engineering, such as codon optimization, RNA optimization and addition of leading immunoglobulin sequences have improved the expression and immunogenicity of DNA vaccines [Andre, S., et al. ,, Increascd immune response elicited by DNA — vaccination with a synthetic gp120 sequence with optimized codon usage. J Virol, 1998. 72 (2): p. 1497-503; Dem], L., et al., Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. J Virol, 2001. 75 (22): p. 10991-1001; Laddy, D.J, et al, Immunogenicity of novel consensus-based DNA vaceines against avian influenza. Vaccine, 2007. 25 (16): p. 2984-9; Frelin, L., et al., Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3 / 4A gene. Gene Ther, 2004. 11 (6): p. 522-33], as well as recently developed technology in plasmid delivery systems such as cletroporation [Hirao, L.A., et al., Intradermal / subcutaneous immunization by electroporation improves — plasmid vaccine delivery and potency in pigs and rhesus macaques. Vaccine, 2008. 26 (3): p. 440-8; Luckay, A., et al., Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques. J Virol, 2007. 81 (10): p. 5257-69; Ahlen, G., et al., In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3 / 4A DNA by increased local DNA —uptakc, protein expression, inflammation, and infiltration of CD3 + T cells. J Immunol,
2007. 179 (7): p. 4741-53]. In addition, studies have suggested that the use of consensus immunogens may be able to increase the width of the cellular immune response as compared to native antigens alone [Yan, J., et al., Enhanced cellular immune responses elicited by an engineered HIV- 1 subtype B consensus-based envelope DNA vaccine. Mol —Ther, 2007.15 (2): p. 411-21; Rolland, M ,, et al., Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1 proteins. J Virol, 2007. 81 (16): p.
8507-14]. One method for the release of nucleic acid sequences such as plasmid DNA is the electroporation (EP) technique. The technique has been used in human clinical trials to release anticancer drugs, such as bleomycin, and in many preclinical studies on a large number of animal species. There remains a need for an immunogenic - influenza hemagglutinin consensus protein, for nucleic acid constructs encoding this protein for compositions useful for inducing immune responses against various strains of influenza. There is still a need for effective influenza vaccines that are economical and effective on numerous influenza subtypes for treating individuals.
SUMMARY OF THE INVENTION Nucleic acid molecules comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 1 are provided herein; a fragment of SEQ ID NO: 1; * a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 1; SEQ ID NO: 3; a nucleic acid sequence that is 95% homologous to SEQ ID NO: 3; a fragment of SEQ ID NO: 3; a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 3; SEQ ID NO: 6; a nucleic acid sequence that is 95% homologous to SEQ ID NO: 6; a fragment of SEQ ID NO: 6; a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 6; SEQ ID NO: 9, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 9; a fragment of SEQ ID NO: 9; a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 9; SEQ ID NO: 11, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 11; a fragment of SEQ ID NO: 11; a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 11; SEQ ID NO: 13; a nucleic acid sequence that is 95% homologous to SEQ ID NO: 13; a fragment of SEQ 1D NO: 13; a - nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 13; and SEQ ID NO: 15; a nucleic acid sequence that is 95% homologous to SEQ ID NO: 15; a fragment of SEQ ID NO: 15; a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 15. Compositions are further provided comprising: a) a first nucleic acid sequence selected from the group consisting of one or more of: SEQ ID NO: 1, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 1; a fragment of SEQ ID NO: |; a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 1; SEQ ID NO: 3; a nucleic acid sequence that is 95% homologous to SEQ ID
NO: 3; a fragment of SEQ ID NO: 3; a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 3; SEQ ID NO: 6; a nucleic acid sequence that is 95% homologous to SEQ ID NO: 6; a fragment of SEQ ID NO: 6; a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 6; SEQ ID NO: 9; a nucleic acid sequence that is 95% homologous to SEQ ID NO: 9; a fragment of SEQ ID NO: 9; a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 9; SEQ ID NO: 11; a nucleic acid sequence that is 95% homologous to SEQ ID NO: 11; a fragment of SEQ ID NO: 11; and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 11; SEQ ID NO: 13; a nucleic acid sequence that is 95% homologous to SEQ ID NO: 13; a fragment of SEQ ID NO: 13; a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 13; SEQ ID NO: 15; a nucleic acid sequence that is 95% homologous to SEQ ID NO: 15; a fragment of SEQ ID NO: 15; and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 15; and b) a second nucleic acid sequence — encoding a protein selected from the group consisting of one or more of: influenza A H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, Ni, N2, N3, N4, N5, N6, N7, N8, N9, influenza B hemagglutinin, neuraminidase and fragments thereof.
Some aspects of the invention provide methods of inducing an immune response comprising the step of: administering said nucleic acid molecules and / or compositions to an individual.
Additional aspects of the invention provide methods of protecting an individual from infection. The methods comprise the step of: administering to said individual a prophylactically effective amount of a nucleic acid molecule comprising said - nucleic acid sequence or compositions, wherein the nucleic acid sequence is expressed in cells of said individual and a protective immune response it is induced against a protein encoded by said nucleic acid sequence. In some embodiment, the immune response is a protective immune response against human influenza of swine origin.
In some aspects of the invention, methods are provided to treat an individual who has been infected with Influenza. The methods comprise the step of: administering to said individual a therapeutically effective amount of said nucleic acid molecules and / or composition. In some embodiment, the immune response is a protective immune response against human influenza of swine origin.
Ti43
BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a map of the 2999 base pair structure vector plasmid pVAX1 (Invitrogen, Carlsbad CA). The CMV promoter is located at bases 137-724, The T7 promoter / primer site is at bases 664-683. Several cloning sites are in the “bases696-811”. The bovine GH polyadenylation signal is found in bases 829-1053. The kanamycin resistance gene is at bases 1226-2020. The pUC origin is in the 2320-
2993. Based on the pVvAX1 sequence available from Invitrogen, the following mutations were found in the pVAX1 sequence that were used as the structure for pGX2009: C> G 241 in the CMV promoter C> T 1942 structure, downstream of the hormone polyadenylation signal bovine growth (DGHpolyA) 'A> - 2876 structure, downstream of the kanamycin gene CT 3277 in the origin of replication pUC (Ori) high copy number mutation (see Nucleic Acid Research 1985) G> C 3753 at each end of pUC Ori upstream of RNASeH base pairs 2, 3 and 4 are changed from ACT to CTG in structure, upstream from the CMV promoter.
Figure 2 shows two maps of plasmid pGX2009, which is further referred to as pH1HAOS9. The nucleic acid sequence of plasmid pGX2009 (SEQ ID NO: 5) includes the coding sequence for the consensus protein construct H1 (amino acid SEQ ID NO: 4 encoded by SEQ ID NO: 3) which includes leader IE (amino acid SEQ ID NO: 17) linked to the N terminal of the H1 amino acid consensus sequence (amino acid SEQ —IDNO: 2 encoded by SEQ ID NO: 1) which is linked to its C terminal by the HA Tag (SEQ ID NO: 18). The consensus protein H1l (amino acid SEQ ID NO: 4 encoded by SEQ ID NO: 3) is labeled SwiHum Con HA and H1IHAO9.
Figure 3 shows a plasmid map pGX2006. The nucleic acid sequence of plasmid pGX2006 (SEQ ID NO: 8) includes the coding sequence for the protein - consensus H2 (amino acid SEQ ID NO: 7 encoded by SEQ ID NO: 6) which is marked H2HA.
Figure 4 shows data from hemagglutination inhibition tests performed with serum from immunized ferrets.
Figure 5 shows results of a challenge for ferrets immunized and not immunized with a new HIN1 strain.
DETAILED DESCRIPTION Consensus amino acid sequences for each influenza A H1 and H2 (referred to here as “HI consensus” (SEQ ID NO: 2) and “H2 consensus” (SEQ ID NO: 7), respectively), as well as new synthetic hybrids of consensus H1 influenza A hemagglutinin amino acid sequence (referred to here as “U2 consensus” (SEQ ID NO: 10)) and a consensus influenza B amino acid hemagglutinin (referred to here as “BHA consensus” (SEQ ID NO: 13)) are provided, which can provide protection from mammals against influenza. In addition, proteins are provided 'comprising the consensus H1 amino acid sequence, the consensus H2 amino acid sequence, the consensus U2 amino acid sequence and / or the consensus BHA amino acid sequence. In some respects, nucleic acid sequences are provided that encode proteins comprising the H1 consensus amino acid sequence (eg (SEQ ID NO: 1) or (SEQ ID NO: 3)), the consensus H2 amino acid sequence (eg ( SEQ ID NO: 6)), the U2 consensus amino acid sequence (for example (SEQ ID NO: 9) or (SEQ ID NO: 11)), & / or the consensus BHA amino acid sequence (for example (SEQ ID NO: : 13) or (SEQ ID NO: 15)). While not binding by scientific theory, a vaccine that can be used to induce an immune response (humoral, cellular, or both) widely against various influenza subtypes may comprise one or more of the following: 1) a nucleic acid sequence that encodes a protein comprising the consensus H1 amino acid sequence; 2) a protein comprising the consensus H1 amino acid sequence; 3) a nucleic acid sequence encoding a protein comprising the consensus H2 amino acid sequence; 4) a protein comprising the consensus H2 amino acid sequence; 5) a nucleic acid sequence encoding a protein comprising the consensus U2 amino acid sequence; 6) a protein comprising the U2 consensus amino acid sequence; 7) a nucleic acid sequence encoding a protein comprising the consensus BHA amino acid sequence; and 8) a - protein comprising the BHA consensus amino acid sequence. Immunization methods can be performed and vaccines can be prepared whose use uses and / or combines the two or more of the following components: 1) a nucleic acid sequence that encodes a protein comprising the amino acid sequence
H1 consensus; 2) a protein comprising the consensus H1 amino acid sequence; 3) a nucleic acid sequence encoding a protein comprising the consensus H2 amino acid sequence, 4) a protein comprising the consensus H2 amino acid sequence; 5) a nucleic acid sequence encoding a protein - comprising the U2 consensus amino acid sequence, 6) a protein comprising the U2 consensus amino acid sequence, 7) a nucleic acid sequence encoding a protein comprising the BHA consensus amino acid sequence , and 8) a protein comprising the BHA consensus amino acid sequence. For broader treatments against influenza, immunization methods can be performed and vaccines can be prepared whose use and / or combination of one or more other influenza proteins such as influenza A HI1-H16, influenza A NI-N9, influenza B hemagglutinin, influenza B neuraminidase and / or genes that encode these proteins together with one or more of the following components: 1) a nucleic acid sequence that encodes a protein comprising the consensus Hi amino acid sequence; 2) a protein comprising — consensus H1 amino acid sequence; 3) a nucleic acid sequence encoding a protein comprising the consensus H2 amino acid sequence, 4) a protein comprising the consensus H2 amino acid sequence; 5) a nucleic acid sequence encoding a protein comprising the U2 consensus amino acid sequence, 6) a protein comprising the U2 consensus amino acid sequence, 7) a - nucleic acid sequence encoding a protein comprising the BHA consensus amino acid sequence , and 8) a protein comprising the BHA consensus amino acid sequence.
1. Definitions, The terminology used here is for the purpose of describing particular modalities — they are not intended to be limiting only. As used in the specification and appended claims, the singular forms "one," "one" and "o / a" include plural referents unless the context clearly indicates otherwise. For the indication of numerical ranges here, each intervening number with the same degree of precision is explicitly contemplated. For example, for the 6-9 range, the —numbers7 and 38 are included in addition to 6 and 9, and for the 6.0-7.0 range, the numbers 6.0, 6.1, 6.2, 6 , 3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. The. Adjuvant "Adjuvant" as used herein means any molecule added to the DNA plasmid vaccines described here to increase the immunogenicity of the antigens encoded by DNA plasmids and the nucleic acid coding sequences described below. B.
Antibody "Antibody" as used herein means an antibody of classes IgG, IZM, IgA, 1gD or IgE, or fragments, fragments or derivatives thereof, including Fab, F (ab'2, Fd, and single chain antibodies, diabody, bispecific antibodies, binfunctional antibodies and derivatives thereof.
The antibody can be an antibody isolated from the mammalian serum sample, a polyclonal antibody, purified affinity antibody, or mixtures of mesinos that exhibit binding specificity to a desired epitope or a sequence derived therefrom. ç.
Coding Sequence "Coding sequence" or "encoding nucleic acid" as used herein means nucleic acids (RNA or DNA molecule) comprising a nucleotide sequence that encodes a protein.
The coding sequence may further include initiation and termination signals operatively linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. d.
Complement "Complement" or "complementary" as used here means a nucleic acid can mean a Watson-Crick base pair (eg, A-T / U and C-G) or Hoogsteen between nucleotides or nucleotide analogs of nucleic acid molecules. and.
Consensus or Consensus Sequence "Consensus" or "consensus sequence" as used here means a polypeptide sequence based on analysis of an alignment of various subtypes of a particular influenza antigen.
Nucleic acid sequences that encode a consensus polypeptide sequence can be prepared.
Vaccines comprising proteins that comprise consensus sequences and / or nucleic acid molecules that —code these proteins can be used to induce broad immunity against various subtypes or serotypes of a particular influenza antigen.
Consensus influenza antigens can include hemagglutinin consensus amino acid sequence of influenza A, including for example H1 consensus, H2 consensus, or hemagglutinin consensus influenza B amino acid sequence. F.
Constant Current “Constant current” as used here means a current that is received or experienced by a tissue, or cells that define that tissue, for the duration of an electrical pulse released to the same tissue.
The electrical pulse is released from the electroporation devices described here.
This current remains at a constant amperage in said tissue for the life of an electrical pulse because the electroporation device provided here has a feedback element, preferably containing instantaneous feedback.
The feedback element can measure the resistance of the tissue (or cells) over the duration of the pulse and causes the electroporation device to change its electrical energy output (for example, voltage increase) so that the current in the same tissue remains across the electrical pulse (in the order of microseconds), and from pulse to pulse.
In some embodiments, the feedback element comprises a controller. g.
Current feedback or “Current feedback” or “feedback” can be used interchangeably and means the active response of the electroporation devices provided, which comprise measuring the current in the tissue between electrodes and changing the energy output released by the EP device so to keep the current at a constant level.
This constant level is predefined by a user prior to the initiation of a pulse treatment sequence. The feedback can be achieved by an electroporation component, for example, controller, of the electroporation device, as the electrical circuit in it is able to continuously monitor the current in the tissue between electrodes and compare that monitored current (or current within the tissue) to a predetermined current and continuously produce energy output adjustments to keep the monitored current at predetermined levels.
The feedback loop can be instantaneous since this is analogous closed loop feedback. H.
Decentralized Current “Decentralized Current” as used here means the pattern of electrical currents released from the various electrode needle arrangements of the electroporation devices described here, in which the patterns minimize, or preferably eliminate, the occurrence of heat stress related electroporation in any area of tissue being electroporated.
i.
Electroporation “Electroporation,” “electro-permeabilization,” or “electro-kinetic improvement” (“EP”) as used interchangeably here means the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a biomembrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cell membrane to another. j.
Feedback Mechanism "Feedback Mechanism" as used here means a process performed by software or hardware (or firmware), the process of which receives and compares the impedance of the desired tissue (before, during, and / or after releasing the energy pulse) ' with a present value, preferably current, and adjusts the pulse of the released energy to obtain the predetermined value.
A feedback mechanism can be performed by an analog loop loop. k.
Fragment "Fragment" as used here with respect to nucleic acid sequences means a nucleic acid sequence or a part thereof, which encodes a polypeptide capable of inducing an immune response in a mammal that cross-reacts with a strain-type antigen full-length wild animal, including, for example, a hemagglutinin H1l influenza A, a hemagglutinin H2 influenza A or a hemagglutinin influenza B.
The fragments can be fragments of DNA selected from at least one of several nucleotide sequences encoding the consensus amino acid sequence and constructs comprising said sequences, including SEQ ID NOS: 1, 3, 6, 9, 11 13 and 15. Fragments of DNA can comprise coding sequences for the leader — immunoglobulins such as IgE or IgG sequences.
DNA fragments can be 30 or more nucleotides in length, 45 or more, 60 or more, 75 or more, 90 or more, 120 or more, 150 or more, 180 or more, 210 or more, 240 or more, 270 or more, 300 or more, 360 or more, 420 or more, 480 or more, 540 or more, 600 or more, 660 or more, 720 or more, 780 or more, 840 or more, 900 or more, 960 or more , 1020 or more, 1080 or more, 1140 or more, 1200 or more, 1260 or more, 1320 or more, 1380 or more, 1440 or more, 1500 or more, 1560 or more, 1620 or more, 1680 or more, 1740 1800 or more, 1860 or more, 1820 or more, 1880 or more, 1940 or more, 2000 or more, 2600 or more, 2700 or more, 2800 or more, 2900 or more, 2910 or more, 2920 or more , 2930 or more,
2931 or more, 2932 or more, 2933 or more, 2934 or more, 2935 or more, 2936 or more, 2937 or more, or 2938 or more in length. DNA fragments can be smaller than 10 nucleotides, smaller than 20, smaller than 30, smaller than 40, smaller than 50, smaller than 60, smaller than 75, smaller than 90, smaller than 120, less than 150, less than 180, less than 210, less than 240, less than 270, less than 300, less than 360, less than 420, less than 480, less than 540, less than 600, less than 660, less than 720, less than 780, less than 840, less than 900, less than 960, less than 1020, less than 1080, less than 1140, less than 1200, less than 1260, less than 1320, less than 1380, less than 1440, less than 1500, less than 1560, less than 1620, less than 1680, or less than 1740 nucleotides, smaller than 1800, smaller than 1860, smaller than 1820, smaller than 1880, smaller than 1940, smaller than 2000, smaller than 2600, smaller than q eu 2700, less than 2800, less than 2900, less than 2910, less than 2920, less than 2930, less than 2931, less than 2932, less than 2933, less than 2934, less than than 2935, less than 2936, less than 2937, or less than
2938. "Fragment" with respect to polypeptide sequences means a polypeptide capable of inducing an immune response in a mammal that cross-reacts with a full-length wild-type influenza antigen, including, for | example, a hemagglutinin H1 influenza A, a hemagglutinin H2 influenza A or a hemagglutinin influenza B. The fragment can be a polypeptide fragment selected from at least one of the various polypeptide sequences of the present invention, including —SEQIDNOS: 2,4,7,10,12, 14 and 16. Polypeptide fragments can be analyzed to contact at least one epitope as provided by a publicly available database such as Los Alamos National Laboratory's HA Sequence Database. The HA polypeptide fragments may further comprise amino acid sequences for the leading immunoglobulin sequence such as IgE or IgG. Polypeptide fragments can be 30 or more amino acids in length, 45 or more, 60 or more, 75 or more, 90 or more, 120 or more, 150 or more, 180 or more, 210 or more, 240 or more, 270 or more, 300 or more, 360 or more, 420 or more, 480 or more, 540 or more, 600 or more, 660 or more, or 710 amino acids or more in length. Polypeptide fragments can be less than 10 amino acids, less than 20, less than 30, less than 40, less than 50, less than 60, less than 75, less than 90, less than 120 , less than 150, less than 180, less than 210, less than 240, less than 270, less than 300, less than 360, less than 420, less than 480, less than 540 , less than 600, less than 660, less than 700, less than 701, less than 702, less than 703, less than 704, less than 705, less than 706, less than 707 , less than 708, less than 709, or less than 710 amino acids in length. l.
Genetic construct As used here, the term "genetic construct" refers to DNA or RNA molecules that comprise a nucleotide sequence that encodes a protein.
The coding sequence includes initiation and termination signals operatively linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to which the nucleic acid molecule is administered.
As used herein, the term "expressable form" refers to gene constructs that contain the necessary regulatory elements operationally linked to the coding sequence that encodes a protein so that when present in the individual's cell, the coding sequence will be expressed. m.
Identical "Identical" or "identity" as used here in the context of two or more nucleic acid or polypeptide sequences, means that the sequences have a specified percentage of residue that are the same over a specified region.
The percentage can be calculated by optimally aligning the two sequences, comparing the - two sequences over the specified region, determining the number of positions in which the identical residue occurs in both sequences to generate the number of combined positions, dividing the number of positions combined by the total number of positions in the specified region, and multiplying the result by 100 to generate the sequence identity percentage.
In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the single sequence residues are included in the denominator, but not in the numerator of the calculation.
When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.
Identity can be performed manually or using a computer sequence algorithm like BLAST or BLAST 2.0. n. Impedance “Impedance” can be used when discussing the feedback mechanism and can be converted to a current value according to Ohm's law, thus allowing comparisons with the current current.
O. Immune Response “Immune response” as used here means the activation of a host immune system, for example, that of a mammal, in response to the introduction of '10 antigen as a consensus antigen from influenza hemagglutinin. The immune response can be in the form of a cellular or humoral response, or both.
P. Nucleic acid "Nucleic Acid" or "oligonucleotide" or "polynucleotide" as used herein means at least two nucleotides covalently joined. The description of a single tape further defines the complementary tape sequence. Thus, a nucleic acid further includes the complementary strand of a described single strand. Many variants of a nucleic acid can be used for the same purpose as a certain nucleic acid. Thus, a nucleic acid still substantially includes identical nucleic acids and complements therefrom. A single strand provides a probe that can hybridize to a target sequence under strict hybridization conditions. Thus, a nucleic acid further includes a probe that hybridizes under stringent hybridization conditions.
Nucleic acids can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyrib- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, hypoxanthine xanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.
q. Operationally linked “Operationally linked” as used here means that the expression of a gene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5 '(upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene that controls the gene from which the promoter is derived.
As is known in the art, the variation in this distance can be accommodated without loss of promoter function. r.
Promoter "Promoter" as used here means a synthetic or naturally derived molecule that is capable of conferring, activating or improving the expression of a nucleic acid in a cell.
A promoter may comprise one or more specific transcriptional regulatory sequences to further improve expression and / or alter its spatial and / or temporal expression.
A promoter can also comprise elements of 'distal or repressive enhancement, which can be located as several thousand pairs': base from the initial transcription site.
A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
A promoter can regulate the expression of a gene component constitutively, or differently with respect to the cell, tissue or organ in which the expression occurs, with respect to the stage of development in which the expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducing agents.
Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, late SV40 promoter, early S V40 promoter, RSV-LTR promoter, CMV IE promoter, early SV40 promoter - late and SV40 promoter the CMV IE promoter. s.
Strict Hybridization Conditions "Strict Hybridization Conditions" as used here means the conditions under which a first nucleic acid sequence (eg probe) will hybridize to a second nucleic acid sequence (eg target), as in a mixture complex — nucleic acids.
Strict conditions are sequence dependent and will be different in different circumstances.
Rigid conditions can be selected at about 5-10ºC lower than the thermal melting point (Tm) for the specific sequence at a defined pH ionic strength.
Tr, can be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium (as the target sequences are present in excess, in Tm: 50% the probes are occupied in equilibrium). Rigid conditions can be those in which the salt concentration is less than about 1.0 M in sodium ion, such as about 0.01-1.0 M sodium phon concentration (or other salts) in pH 7.0 to 8.3 and the temperature is at least about 30ºC for small probes (for example, about 10-50 nucleotides) and at least about 60ºC for large probes (for example, greater than about 50 nucleotides) ). Rigid conditions can still be achieved with the addition of destabilizing agents such as formamide.
For selective or specific hybridization, a positive signal can be at least 2 to 10 times the background hybridization.
Exemplary rigid hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42ºC, or, 5x SSC, 1% SDS, incubating at 65ºC, with washing in 0.2x SSC, and 0, 1% SDS at 65ºC. . t.
Substantially Complementary “Substantially complementary” as used here means that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, 630, 720, 810, 900, 990, 1080, 1170, 1260, 1350, 1440,1530, 1620, 1710, 1800, 1890, 1980, 2070 or more nucleotides or amino acids, or that the two sequences hybridize under strict hybridization conditions. u.
Substantially identical “Substantially identical” as used here means that a first and second strings are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99 Identical% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22,23,24,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, 630, 720, 810, 900, 990, 1080, 1170, 1260, 1350, 1440, 1530, 1620, 1710, 1800, 1890, 1980, 2070 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence. v.
Subtype or Serotype “Subtype” or “serotype”: as used here, interchangeably, and in relation to the influenza virus, it means genetic variants of an influenza virus as that subtype is recognized by an immune system away from a different subtype. w.
Variant "Variant" used here with respect to a nucleic acid means (1) a part or fragment of a referenced nucleotide sequence; (1i) the complement of a referenced nucleotide sequence or part of it; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under conditions rigid to the referenced nucleic acid, complement to it, or a sequence substantially identical thereto. "Variant" with respect to a peptide or polypeptide that differ in amino acid sequence by insertion, deletion, or conserved amino acid substitution, but retain at least one biological activity.
Variant can also mean a protein with an amino acid sequence that is substantially identical to a protein referenced with an amino acid sequence that retains at least one biological activity.
A conservative substitution of an amino acid, that is, replacing an amino acid with a different amino acid with similar properties (for example, hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change.
These minor changes can be identified, in part, considering the hydropathic index of amino acids, as understood in the art.
Kyte et al., J.
Mol.
Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge.
It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function.
In one respect, amino acids containing hydropathic indices of +2 are replaced.
The hydrophilicity of amino acids can also be used to reveal substitutions that could result in proteins retaining biological function A consideration of the hydrophilicity of amino acids in the context of a peptide allows the calculation of the highest average hydrophilicity of that peptide, a useful measure that has been reported for correlating well with antigenicity and immunogenicity.
US patent 4,554,101 incorporated herein by reference.
The substitution of amincacids containing similar values of hydrophilicity can result in peptides retaining biological activity, for example, immunogenicity, as is understood in the art.
Substitutions can be made with amino acids containing hydrophilicity values within +2 of each other.
Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by another particular side chain of that amino acid.
Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of amino acids, and particularly the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.
x. Vector "Vector" as used here means a nucleic acid sequence containing an origin of replication. A vector can be a vector, bacteriophage, artificial bacterial chromosome or artificial yeast chromosome. A vector can be a DNA or RNA vector. A vector can be an extrachromosomal self-replicating vector, and is preferably a DNA plasmid.
2. Influenza Antigen Antigens capable of inducing an immune response in a mammal against one or more influenza serotypes are provided here. The antigen may be able to induce an immune response in a mammal against one or more influenza serotypes, including against one or more pandemic strains, such as 2009 swine HIN1 influenza. The antigen may be able to induce an immune response in a mammal against one or more serotype of influenza, including against one or more strains of human influenza of porcine origin. The antigen can comprise epitopes that can make them particularly effective as immunogens with anti-influenza immune responses can be induced. The antigen can comprise the full-length translation product HAO, HAI subunit, HA2 subunit, a variant thereof, a fragment thereof or a combination thereof. The influenza hemagglutinin antigen can be a consensus sequence derived from several strains of serotype H1 influenza A, the consensus sequence derived from several strains of serotype H2 influenza A, a hybrid sequence containing parts of two different consensus sequences derived from various sets of various strains of serotype H1 influenza A or a consensus sequence derived from various strains of influenza B. the influenza hemagglutinin antigen may be influenza B. The antigen may contain at least one antigenic epitope that may be effective against particular immunogens influenza against which an immune response can be induced. The antigen can provide an entire repertoire of immunogenic sites and epitopes present in an intact influenza virus. The antigen can be a consensus hemagglutinin antigen sequence that can be derived from hemagglutinin antigen sequences from a plurality of serotype influenza A virus strains such as a plurality of serotype H1 influenza A strains or of serotype H2. The antigen can be a hybrid consensus hemagglutinin antigen sequence that can be derived by combining two consensus hemagglutinin antigen sequences or portions thereof.
Each of the two different consensus consensus hemagglutinin antigen sequences can be derived from a different set of a plurality of serotype influenza A virus strains as a plurality of serotype HI influenza A virus strains.
The antigen can be a hemagglutinin antigen consensus sequence that can be derived from hemagglutinin antigen sequences from a plurality of influenza B virus strains.
The consensus hemagglutinin antigen can be a protein comprising SEQ ID NO: 2 (the consensus H1 amino acid sequence) in which amino acids 1-343 correspond to the HA1 subunit of the HAO precursor H1 consensus H1 amino acid sequence 'and amino acids 344-566 correspond to HA2 subunit of HAO sequence of amino acid H1 consensus.
The consensus hemagglutinin antigen can be a protein comprising SEQ ID NO: 7 (the consensus H2 amino acid sequence). The hemagglutinin consensus antigen can be a synthetic hybrid of consensus H1 sequences comprising parts of two different consensus H1 sequences that are derived from a different set of sequences from the others.
An example of a consensus HA antigen that is a synthetic hybrid HI1 protein consensus is a protein comprising SEQ ID NO: 10 (the U2 amino acid sequence). The consensus hemagglutinin antigen may be a hemagglutinin consensus protein derived from hemagglutinin sequences of deinfluenza B strains, as a protein comprising SEQ ID NO: 14 (the consensus amino acid sequence BHA). The hemagglutinin consensus antigen can further comprise one or more additional amino acid clement sequences.
The hemagglutinin consensus antigen can also comprise a leading amino acid IgE or IgG in its N-terminal.
The leading amino acid IgE sequence can be SEQ ID NO: 17. The hemagglutinin consensus antigen can further comprise an immunogenic tag that is an epitope is a single epitope that can be detected by readily available antibodies.
An example of said immunogenic tag is the 9 amino acid influenza HA Tag that can be linked in the C-terminal hemagglutinin consensus.
The HA Tag amino acid sequence can be SEQ ID NO: 18. In some modalities, hemagglutinin consensus antigen may also comprise an N-terminal leading IZE or IgG amino acid sequence in its C-terminal an HA tag. The hemagglutinin consensus antigen may be a hemagglutinin consensus protein that consists of influenza amino acid consensus or fragments and variants thereof. The hemagglutinin consensus antigen can be a hemagglutinin consensus protein that comprises non-influenza protein sequences and influenza protein sequences or fragments and variants thereof.
Examples of an H1 consensus protein include those that may consist of the H1 consensus amino acid sequence (SEQ ID NO: 2) or those that further comprise additional elements such as an IgE leader sequence, or an HA Tag, or both an I8E leader sequence and a Tag THERE IS. An example of a consensus H1 protein that includes both an IgE leader sequence and an HA Tag is SEQ ID NO: 4, which comprises the consensus amino acid H1 coding sequence (SEQ ID NO: 2) linked to the IgE leader amino acid sequence (SEQ ID NO: 17) in its terminal N and linked to the HA Tag (SEQ ID NO: 18) in its terminal C.
* Examples of consensus H2 proteins include those that may consist of a consensus H2 amino acid sequence (SEQ ID NO: 7) or those that are still 15th comprised of an IgE leader sequence, or an HA Tag, or both IgE leader sequence and an HA tag.
Examples of hybrid consensus H1 proteins that may consist of the consensus U2 amino acid sequence (SEQ ID NO: 10) or those that still comprise an IgE leader sequence, or an HA Tag, or both an IZE leader sequence and an HA Tag. An example of the U2 consensus protein is SEQ ID NO: 12, which comprises the U2 consensus amino acid sequence (SEQ ID NO: 10) linked to the leading amino acid IZE sequence (SEQ ID NO: 17) in its N terminal and linked to the Tag HA (SEQ ID NO: 18) in its C terminal.
Examples of hemagglutinin consensus proteins for influenza B include those that - may consist of the consensus amino acid sequence BHA (SEQ ID NO: 14) or may comprise an IgE leader sequence, or an HA Tag, or both an IZE leader sequence and an HA Tag. An example of the BHA consensus protein is SEQ ID NO: 16 which comprises the BHA consensus amino acid sequence (SEQ ID NO: 14) linked to the leading amino acid IgE sequence (SEQ TD NO: 17) at its N terminal and linked to the HA Tag (SEQ ID NO: 18) in your Terminal.
The hemagglutinin consensus protein can be encoded by a hemagglutinin consensus nucleic acid, a variant thereof or a fragment thereof. Unlike consensus hemagglutinin protein which can be a consensus sequence derived from a plurality of different hemagglutinin sequences from different strains and variants, hemagglutinin nucleic acid refers to a nucleic acid sequence that encodes a consensus protein sequence and the coding sequence used may differ from those used to encode the particular amino acid sequence in the plurality of different hemagglutinin sequences from which the hemagglutinin consensus protein sequence is derived.
The consensus nucleic acid sequence can be codon optimized and / or RNA optimized.
The hemagglutinin nucleic acid sequence may comprise a Kozak sequence in the 5th untranslated region.
The nucleic acid hemagglutinin consensus sequence may comprise i 10 —nucleic acid sequences that encode a leader sequence.
The coding sequence of a leading N terminal is 5 th of the hemagglutinin coding sequence.
The N-terminal leader can facilitate secretion.
The N-terminal leader can be an IgE leader or an IgG leader.
The hemagglutinin consensus nucleic acid sequence may comprise nucleic acid sequences that encode an immunogenic tag.
The immunogenic tag can be at the C terminal of the protein and the coding sequence is 3rd of the HA coding sequence.
The immunogenic tag provides a single epitope for which antibodies are readily available so that said antibodies can be used in assays to detect and confirm protein expression.
The immunogenic tag can be an H tag at the C-terminus of the protein.
Consensus hemagglutinin nucleic acid may have a polynucleotide sequence that encodes a protein that comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 14. A hemagglutinin consensus nucleic acid encoding SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 14 can be SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9 or SEQ ID NO: 13, respectively.
The hemagglutinin consensus nucleic acid can further comprise a polynucleotide sequence that encodes the IgE leading amino acid sequence, or the polynucleotide sequence that encodes an HA Tag amino acid sequence, or both.
SEQ ID NO: 17 is an IgE polypeptide leader sequence.
SEQ ID NO: 18 is a tag HA polypeptide sequence.
Examples of consensus nucleic acids — hemagglutinin that still comprise polynucleotide sequences that encode an IBGE leader sequence and an HA Tag include nucleic acid molecules that encode proteins that comprise the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 12 or SEQ ID NO: 16. A hemagglutinin consensus nucleic acid encoding SEQ ID
NO: 4, SEQ ID NO: 12 or SEQ ID NO: 16 can be SEQ ID NO: 3, SEQ ID NO: 11 or SEQ ID NO: 15, respectively,
3. Genetic Constructs and Plasmids Genetic constructs that can comprise a sequence — nucleic acid encoding the hemagglutinin antigen are provided here. The genetic construct may be present in the cell as an extrachromosomal functioning molecule comprising the nucleic acid that encodes the hemagglutinin antigen. The genetic construct comprising the nucleic acid encoding the hemagglutinin antigen can be a linear minichromosome including centromere, telomeres or plasmids or cosmids. '10 The genetic construct may furthermore be part of a recombinant viral vector genome, including recombinant adenoviruses, virus-associated recombinant adenoviruses and recombinant vaccine. The genetic construct may be part of the genetic material in live attenuated microorganisms or recombinant microbial vectors. that live in the cells.
The genetic constructs can comprise regulatory elements for gene expression of the hemagglutinin nucleic acid. The regulatory elements can be a promoter, an enhancer, an initiation codon, a stop codon or a polyadenylation signal.
The compositions may comprise a first nucleic acid sequence that encodes the hemagglutinin consensus antigen selected from the group consisting of one or more of: hemagglutinin H1 influenza A consensus antigen, hemagglutinin H2 consensus antigen influenza A, hemagglutinin U2 antigen consensus A, and hemagglutinin U2 consensus BHA protein influenza B, and may also comprise one or more additional nucleic acid sequences encoding one or more proteins selected from the group consisting of: influenza A hemagglutinin proteins H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, influenza A neuraminidase N1, N2, N3, N4, N5, N6, N7, N8, N9, influenza B hemagglutinin (BHA) and neuraminidase from influenza B (BNA). The first and additional nucleic acid sequences can be present in the same nucleic acid molecule or different “nucleic acid molecules. The first and additional nucleic acid sequences may be under the control of regulatory elements that function in a human cell. Additional encoding sequences can encode one or more H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, NI, N2, N3, N4, N5,
N6, N7, N8, N9, BHA and BNA from one or more strains of influenza, or be a consensus derived from a plurality of strains containing the serotype, or be a hybrid that includes sequences from two or more sequences of consensus.
The nucleic acid sequence can generate a genetic construct that can be a vector.
The vector may be able to express a hemagglutinin consensus antigen in a mammalian cell in an amount effective to induce an immune response in the mammal.
The vector can be recombinant.
The vector can comprise heterologous nucleic acid encoding the hemagglutininan consensus antigen. The vector can be a plasmid.
The vector may also be useful for transfecting cells with nucleic acid that encodes a hemagglutinin consensus antigen, which the transformed host cell is' cultured and maintained in conditions where expression of the hemagglutinin consensus antigen occurs.
The vector may comprise heterologous mycolic acid encoding a hemagglutinin consensus antigen and may further comprise a start codon, which may be upstream of the hemagglutinin consensus coding sequence, and a stop codon, which may be downstream of the coding sequence hemagglutinin consensus.
The initiation and termination codon can be in a region with the hemagglutinin consensus coding sequence.
The vector may further comprise a promoter that is operably linked to the hemagglutinin consensus coding sequence.
The promoter operably linked to the hemagglutinin consensus coding sequence may be a simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the virus long-term repeat bovine immunodeficiency (BIV), a Moloney virus promoter, a leukosis avian virus (ALV) promoter, a decitomegalovirus (CMV) promoter as the CMV intermediate early promoter, a virus promoter Epstein Barr (EBV), or a Rous sarcoma virus (RSV) promoter. The promoter can be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein.
The promoter may also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic.
Examples of said promoters are described in patent application US 20040175727, the contents of which are incorporated herein in their entirety.
The vector may further comprise a polyadenitation signal, which may be downstream of the HA coding sequence. The polyadenylation signal can be an SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (ADG) polyadenylation signal, human growth hormone (DG) polyadenylation signal, or human B-globin polyadenylation signal . The SV40 polyadenylation signal —can be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, CA).
The vector may further comprise an enhancer upstream of the hemagglutinin consensus coding. The enhancer may be necessary for expression of DNA. The enhancer can be a human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, HA, RSV or EBV. Improvements in polynucleotide function are described in U.S. Patent Nos. 5,593,972,
5,962,428, and WOS4 / 016737, the contents of each are fully incorporated by reference.
The vector may further comprise a mammalian origin of replication to maintain the vector extrachromosomally and produce multiple copies of the vector in a cell. The vector can be pVAXI! (Figure 1), pCEP4 or pREP4 from Invitrogen (San Diego, CA), which can comprise the Epstein Barr virus origin of replication and EBNA-1 nuclear antigen coding region, which can produce episomal replication in high copies without integration. The vector can be pvAX1 with changes such as those described in the paragraph referring to Figure 1 in the section above Brief Description of the Figures. The structure of the vector can be —pAVO242. The vector can be a defective replication vector for adenovirus type 5 (Ad5S).
The vector may further comprise a regulatory sequence, which may be well suited for gene expression in a mammalian or human cell in which the vector is administered. The hemagglutinin consensus coding sequence can comprise a codon, which can allow for more efficient transcription of the coding sequence in the host cell.
The vector can be pSE420 (Invitrogen, San Diego, Calif.), Which can be used for protein production in Escherichia coli (E.coli). The vector can also be pYES2 (Invitrogen, San Diego, Calif.), Which can be used for the production of protein in yeast Saccharomyces cerevisiae strains. The vector can still be from the expression system - from the complete baculovirus MAXBAC'MY (Invitrogen, San Diego, Calif.), Which can be used for the production of protein in insect cells. The vector can also be peDNA 1 or pcDNA3 (Invitrogen, San Diego, Calif.), Which can be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells. The vector can be vectors or expression systems to produce protein by routine techniques and readily available starting materials including Sambrook et al., Molecular Cloning an Laboratory Manual, Second Ed., ColdSpringHarbor (1989), which is fully incorporated by reference.
The vector can be pGX2009 or pGX2006, which can be used to express the hemagglutinin consensus antigen. The pGX2009 vector (4739 bp, Figure 2; SEQ ID NO: 5) is a modified plasmid pVvAX1 with a nucleic acid sequence encoding a H1l consensus protein (SEQ ID NO: 4 amino acid encoded by SEQ ID NO: 3) comprising an IgE leader sequence (amino acid SEQ ID NO: 12 encoded by SEQ ID NO: 11) linked to an H consensus] amino acid sequence (amino acid SEQ ID NO: 2 encoded by SEQ ID NO: 1). The pPGX2006 vector (4628 bp; Figure 3, SEQ ID NO: 8) is a plasmid pVAX1 with a nucleic acid sequence encoding an H2 consensus protein (amino acid SEQ ID NO: 7 encoded by SEQ ID NO: 6).
The genetic constructs and components disclosed here that include hemagglutinin consensus coding sequence can be used to express other influenza proteins such as influenza A HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12 , H13, H14, H15, H16, N1, N2, N3, N4, Node, N6, N7, N8, N9, influenza B hemagglutinin or neuraminidase protein through which coding sequence for influenza A proteins H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, NJ, N2, N3, N4, N5, N6, N7, N8, N9, influenza B hemagglutinin or neuraminidase protein they are included in place of the hemagglutinin consensus coding sequence.
4. Pharmaceutical Compositions Pharmaceutical compositions according to the present invention comprising about 1 nanogram to about 10 mg of DNA are provided here. In some embodiments, pharmaceutical compositions according to the present invention comprise of: 1) at least 10, 15, 20.25, 30.35, 40.45, 50.55, 60.65, 70.75, 80, 85, 90.95 or 100 nanograms, or at least 1, 5, 10, 15, 20.25, 30.35, 40.45, 50.55, 60.65, 70.75, 80.85, 90, 95,100, 105, 110, 115, 120,125, 130,135, 140,145, 150,155, 160,165, 170,175, 180,185, 190,195, 200, 205, 210, 215, 220,225, 230,235, 240,245, 250,255, 260,265, 270,275, 280,285, 290,295, 300, 305, 310, 315, 320,325, 330,335, 340,345, 350,355, 360,365, 370,375, 380,385, 390,395, 400, 405, 410, 415, 420,425, 430,435, 440,445, 450,455, 460,465, 470,475, 480,485, 490,495, 500, 605, 610, 615, 620,625, 630,635, 640,645, 650,655, 660,665, 670,675, 680,685, 690,695, 700, 705, 710,
715, 720,725, 730,735, 740,745, 750,755, 760,765, 770,775, 780,785, 790,795, 800, 805, 810, 815, 820,825, 830,835, 840,845, 850,855, 860,865, 870,875, 880,885, 890,895. 900, 905, 910, 915, 920,925, 930,935, 940,945, 950,955, 960,965, 970,975, 980,985, 990,995 or 1000 micrograms, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,950ul0mgoumais; e2) up to and including 15, 20,25, 30,35, 40,45, 50,55, 60,65, 70,75, 80,85, 90.95 or 100 nanograms, or up to and including 1, 5, 10, 15, 20.25, 30.35, 40.45, 50.55, 60.65, 70.75, 80.85, 90.95,100 , 105, 110, 115, 120,125, 130,135, 140,145, 150,155, 160,165, 170,175, 180,185, 190,195, 200, 205, 210, 215, 220,225, 230,235, 240,245, 250,255, 260,265, 270,275, 280,285, 290,295, 300, 305 , 310, 315, 320,325, 330,335, '10 340,345, 350,355, 360,365, 370,375, 380,385, 390,395, 400, 405, 410, 415, 420,425, 430,435, 440,445, 450,455, 460,465, 470,475, 480,485, 490,495, 500, 605 , 610, 615, 620,625, 630,635, 640,645, 650,655, 660,665, 670,675, 680,685, 690,695, 700, 705, 710, 715, 720,725, 730,735, 740,745, 750,755, 760,765, 770,775, 780,785, 790,795, 800, 805 , 815, 820,825, 830,835, 840,845, 850,85 5, 860,865, 870,875, 880,885, 890,895. 900, —905,910,915,920,925, 930,935, 940,945, 950,955, 960,965, 970,975, 980,985, 990,995, or 1000 micrograms, or up to and including 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 , 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg.
In some embodiments, pharmaceutical compositions according to the present invention comprise about 5 nanograms to about 10 mg of DNA.
In some embodiments, pharmaceutical compositions according to the present invention comprise about 25 nanograms to about 5 mg of DNA.
In some embodiments, the pharmaceutical compositions contain about 50 nanograms to about 1 mg of DNA. In some embodiments, the pharmaceutical compositions contain about 0.1 to about 500 micrograms of DNA.
In some embodiments, pharmaceutical compositions contain about | about 350 micrograms of DNA.
In some embodiments, pharmaceutical compositions contain about 5 to about 250 micrograms of DNA.
In some embodiments, the pharmaceutical compositions contain about 10 to about 200 micrograms of DNA.
In some embodiments, the pharmaceutical compositions contain about 15 to about 150 micrograms of DNA.
In some embodiments, the pharmaceutical compositions contain about 20 to about 100 micrograms of DNA.
In some embodiments, the pharmaceutical compositions contain about 25 to about 75 micrograms of DNA.
In some embodiments, the pharmaceutical compositions contain about 30 to about 50 micrograms of DNA.
In some embodiments, the pharmaceutical compositions contain about 35 to about 40 micrograms of DNA.
In some embodiments, the pharmaceutical compositions contain about 100 to about 200 micrograms of DNA. In some embodiments, the pharmaceutical compositions comprise about 10 micrograms to about 100 micrograms of DNA. In some embodiments, the pharmaceutical compositions comprise about 20 micrograms to about 80 micrograms of DNA. In some embodiments, the pharmaceutical compositions comprise about 25 micrograms to about 60 micrograms of DNA. In some embodiments, the pharmaceutical compositions comprise about 30 nanograms to about 50 micrograms of DNA. In some embodiments, the pharmaceutical compositions comprise about 35 nanograms to about 45 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about | about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 100 to about 200 micrograms of DNA.
The pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used. In cases where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, free of pyrogen and free of particles. An isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation.
Preferably, the pharmaceutical composition is a vaccine and more — preferably a DNA vaccine.
Vaccines capable of generating an immune response in a mammal against or more influenza serotypes are provided here. The vaccine can comprise the genetic construct as discussed above. The vaccine can comprise a plurality of vectors each targeting one or more Influenza A serotypes such as HI-H16 Influenza B — hemagglutinin or combinations thereof. The vaccine may comprise one or more nucleic acid sequences encoding one or more hemagglutinin consensus antigens. When the vaccine comprises more than one consensus hemagglutinin nucleic acid sequence, all said sequences may be present in a single nucleic acid molecule or each said sequence may be present in a different nucleic acid molecule. Alternatively, vaccines that comprise more than one consensus sequence of hemagglutinin nmucleic acids can comprise nucleic acid molecules with a unique nucleic acid hemagglutinin sequence and — nucleic acid molecules with more than one hemagglutinin nucleic acid sequence. In addition, vaccines comprising one or more hemagglutinin consensus nucleic acid sequences may further comprise coding sequence for one or more proteins selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, N1, N2, N3, N4, N5, N6, N7, N8, No. and influenza B neuramiinysis. 'In some modalities, vaccines may comprise proteins. Some vaccines may comprise one or more more consensual hemagglutinin antigens such as Hl, H2, U2 and BHA. Vaccines may comprise one or more other proteins selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, Hl2, 13, Hl4, H15, H16, Nl, N2 , N3, N4, Node, N6, N7, N8, NO and influenza B neuraminidase. Vaccines may comprise one or more antigen or more hemagglutinin consensus in combination with one or more other proteins selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, HIS, H16, NI, N2, N3, N4, Node, Node, N7, N8, N9, influenza B hemagglutinin and neuraminidase.
The vaccine can be a DNA vaccine. The DNA vaccine may comprise a plurality of the same or different plasmids comprising one or more hemagglutinin nucleic acid consensus sequences. The DNA vaccine can comprise one or more nucleic acid sequences that encode one or more hemagglutinin consensus antigens. When the DNA vaccine comprises more than one hemagglutinin nucleic acid consensus sequence, all said sequences may be present on a single plasmid, or said each sequence may be present on different plasmids, or some plasmids may comprise a single hemagglutinin consensus sequence of nucleic acids while other plasmids have more than one hemagglutinin nucleic acid consensus sequence. In addition, DNA vaccines may further comprise one or more consensus coding sequences for one or more proteins selected from the group consisting of influenza A H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11 , H12, H13, H14, H15, H16, NI, N2, N3, N4, Node, N6, N7, N8, N9, influenza B hemagglutinin and neuramidase. Said additional coding sequence can be on the same or different plasmid from each other and from plasmids comprising one or more hemagglutinin nucleic acid consensus sequences. In some embodiments, vaccines may comprise nucleic acid sequences that encode influenza antigens in combination with influenza antigens.
In some embodiments, the nucleic acid sequence encodes one or more hemagglutinin consensus antigens such as H1, H2, U2 and BHA. In some embodiments, the nucleic acid sequence encodes one or more other proteins selected from the group consisting of influenza A H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H1l4 , H15, Hl6, NI, N2, N3, N4, N5, N6, N7, N8, N9, influenza B hemagglutinin and —neuramidase. In some embodiments, vaccines comprise one or more hemagglutinin consensus antigens such as H1l, H2, U2 and BHA. In some embodiments, vaccines comprise one or more other proteins selected from the group consisting of influenza A H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, HIS, H16, NI, N2, N3, N4, N5, N6, N7, N8, N9, influenza B hemagglutinin and neuramidase.
In some embodiments, vaccines comprise a combination of three or more hemagglutinin nucleic acid consensus sequences including those that encode one or more of H1, H2, U2 and BHA. In some embodiments, vaccines comprise a combination of three or more hemagglutinin nucleic acid sequences including those encoding U2 consensus, BHA consensus and one H3 —hemagglutinin. In some embodiments, vaccines comprise a combination of three or more hemagglutinin nucleic acid sequences including those that encode BHA consensus, Hi hemagglutinin and H3 hemagglutinin In some embodiments, vaccines comprise one or more nucleic acid sequences that encode one or more influenza antigens disclosed in US 12 / 375,518, which is incorporated herein by reference and / or US 12 / 269,824, which is incorporated herein by reference. In some embodiments, the vaccines comprise a SEQ ID NO: 19 nucleic acid sequence encoding SEQ ID NO: 20 (which is a H1 hemagglutinin disclosed in US 12 / 375,518 as SEQ ID NO: 36 and SEQ ID NO: 37 respectively in this ) and / or nucleic acid sequence SEQ ID NO: 21 encoding SEQ ID NO: 22 (which is an H1 hemagglutinin disclosed in US 12 / 269,824 as SEQ ID NO: 9 and SEQ ID NO: 10 respectively). In some embodiments, the vaccines comprise a SEQ ID NO: 23 nucleic acid sequence encoding SEQ ID NO: 24 (which is an H3 hemagglutinin disclosed in US 12 / 269,824 as SEQ ID NO: 11 and SEQ ID NO: 12 respectively in this .
In some embodiments, vaccines comprise a combination of three or more hemagglutinin consensus proteins including one or more of H1, H2, U2 and BHA. In some embodiments, vaccines comprise a combination of three or more - hemagglutinin proteins including U2 consensus, BHA consensus and H3 hemagglutinin. In some embodiments, vaccines comprise a combination of three or more hemagglutinin proteins including BHA consensus, Ht hemagglutinin and H3 hemagglutinin. In some embodiments, the vaccines comprise one or more antigens of US 12 / 375,518 and / or US 12 / 269,824. In some embodiments, the vaccines comprise SEQ ID NO: 20 NT and / or SEQ ID NO: 22 and / or SEQ ID NO: 24.
In some embodiments, vaccines comprise a combination of 1) hemagglutinin U2 consensus protein and / or a nucleic acid sequence encoding the hemagglutinin U2 consensus protein, 2) the hemagglutinin BHA consensus protein and / or a nucleic acid sequence that encodes the hemagglutinin BHA consensus protein, and 3) an H3 hemagglutinin protein disclosed in SEQ ID NO: 24.
In some embodiments, vaccines comprise a combination of 1) hemagglutinin BHA consensus protein and / or a nucleic acid sequence encoding the hemagglutinin BHA consensus protein, 2) an Ht hemagglutinin protein containing SEQ ID NO: 20 and / or SEQ ID NO: 22 and / or a H1 homagglutinin protein encoding SEQ ID NOI9 nucleic acid sequence and / or SEQ ID NO: 21, and 3) a H3 hemagglutinin protein containing SEQ ID NO: 24 and / or a H3 hemagglutinin protein that encodes nucleic acid sequence SEQ ID NO: 23 in this).
DNA vaccines are disclosed in US Patents 5,593,972, 5,739,118, 5,817,637,
5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, and 5,676,594, which are incorporated - hereof by reference. The DNA vaccine. it can also comprise elements or reagents that inhibit this integration into the chromosome. The vaccine may be an hemagglutinin antigen RNA. The RNA vaccine can be introduced into the cell.
The vaccine can be a recombinant vaccine comprising the genetic construct or antigen described above. The vaccine may further comprise one or more hemagglutinin antigen - consensus in the form of one or more protein subunits, one or more dead influenza particles comprising one or more hemagglutinin consensus antigens, or one or more attenuated influenza particles comprising one or more more hemagglutinin consensus antigens. The attenuated vaccine can be live attenuated vaccine, dead vaccine and vaccine that uses recombinant vectors to release foreign genes that encode one or more hemagglutinin consensus antigens, as well as subunit and glycoprotein vaccines. Examples of live attenuated vaccines, those using recombinant vectors to release foreign antigens, subunit vaccines, and glycoprotein vaccines are described in US patents: 4,510,245; 4,797,368; 4,722,848;
4,790,987; 4,920,209; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424;
5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668; 5,387,744; 5,389,368;
5,424,065; 5,451,499; 5,453.3 64; 5,462,734; 5,470,734; 5,474,935; 5,482,713; 5,591,439;
5,643,579; 5,650,309; 5,698,202; 5,955,088; 6,034,298; 6,042,836; 6,156,319 and 6,589,529, —which are each incorporated herein by reference. 'The vaccine may comprise vectors and / or proteins targeting influenza A serotypes from particular regions in the world, for example, Asia. The vaccine can also be targeted against swine influenza A serotypes that now infect humans. The vaccine may comprise vectors and / or proteins targeting Influenza B from particular regions in the world. The vaccine can also be targeted against Influenza B that infects humans. The vaccine can comprise one or more vectors and / or one or more proteins targeting one or more strains of Influenza A and / or B. The vaccine provided can be used to induce immune responses including therapeutic or prophylactic immune responses. Antibodies and / or killer T cells can be generated that are targeted to the hemagglutinin consensus antigen, and still widely through various influenza virus subtypes. Said antibodies and cells can still be isolated. The vaccine may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules such as vehicles, adjuvants, carriers, or diluents. The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surface active agents, such as immune stimulating complexes (ISCOMS), incomplete Freunds adjuvant, LPS analogue including monophosphoryl A lipid, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other agents that facilitate known transfection. The transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. The agent that facilitates transfection is poly-L-glutamate, and more preferably, poly-L-glutamate is present in the vaccine at a concentration of less than 6 mg / ml. The agent that facilitates transfection may also include surface active agents such as immune stimulating complexes (ISCOMS), incomplete Freunds adjuvant, LPS analogue including monophosphoryl A lipid, S -muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid can also be used in conjunction with the genetic construct. In some embodiments, DNA vector vaccines may also include an agent that facilitates transfection such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, such as a DNA-liposome mixture (see, for example, W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other agents that facilitate transfection. Preferably, the agent that facilitates transfection is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. The concentrations of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.
The pharmaceutically acceptable excipient can be an adjuvant. The adjuvant can be other genes that are expressed in an alternative plasmid or are released in combination with the above plasmid in the vaccine. The adjuvant can be selected from the group consisting of: a-interferon (IFN-o), B-interferon (LFN-B), y-interferon, platelet-derived growth factor (PDGF), TNFa, TNFB, GM -CSF, epidermal growth factor (EGF), chemokine that attracts cutaneous T cell (CTACK), chemokine expression in epithelial thymus (TECK), mucosal associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80 , CD86 including IL-15 containing the deleted signal sequence c - optionally including the signal peptide from IZE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFa, TNFB, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, 11-18, or a combination thereof.
Other genes that may be useful adjuvants include those that encode: MCP-1, MIP-la, MIP-Ip, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GIyCAM- 1, MadCAM- 1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL, mutant forms IL.-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL- 1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DRA, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, e-jun, Sp-1, Ap-1, Ap -2, p38, powderSRel, MyD88, TIRAK, TRAFG, TKkB, Inactive NIK, SAP K, SAP-1, INK, interferon response genes, NFKB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4 , RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP and functional fragments thereof.
The vaccine may further comprise a genetic vaccine facilitating agent as described in US 021,579 filed on April 1, 1994, which is fully incorporated by reference.
5. Delivery Methods A method for delivering pharmaceutical formulations, preferably vaccines, is provided here to provide hemagglutinin antigen genetic constructs and proteins that comprise epitopes that make the same immunogens particularly effective against which an immune response to viral infections can be induced. , The vaccine delivery method, or vaccination, can be provided to induce a therapeutic and / or prophylactic immune response. The vaccination process can generate an immune response in the mammal against a plurality of influenza subtypes, including a HINI serotype, the 2009 swine originating from HINI], or other seasonal and / or pandemic varieties. The vaccine can be released to an individual to modulate the activity of the mammalian immune system and improve the immune response. The release of the vaccine can be the transfection of the HA antigen as a nucleic acid molecule that is expressed in the cell and released on the cell surface where the immune system is recognized and induces a cellular, humoral, or cellular and humoral response. The vaccine release can be used to induce or elicit an immune response in mammals against a plurality of influenza viruses by administering the vaccine to mammals as discussed here.
Upon release of the vaccine to the mammal, and then the vector in the mammalian cells, the transfected cells will express and secrete the corresponding influenza protein, including at least one of the consensus antigens, and preferably H1, H2, U2, and BHA These secreted proteins , or synthetic antigens, will be recognized as foreign by the immune system, which will mount an immune response that includes: antibodies made against antigens, and T cell response especially against the antigen. In some instances, a mammal vaccinated with the vaccines discussed here will have an immune system initiated and when challenged with a viral strain of influenza, the immune system initiator will allow rapid clearance of subsequent influenza viruses, through humoral, cellular, or both . The vaccine can be administered to an individual to modulate the activity of the individual immune system thereby increasing the immune response.
The vaccine can be released in the form of a DNA vaccine and methods of delivering DNA vaccines are described in US patents 4,945,050 and 5,036,006, both of which are fully incorporated by reference.
The vaccine can be administered to a mammal to induce an immune response in a mammal. The mammal can be human, non-human primate, cow, pig, sheep, goat, antelope, bison, buffalo, cattle, deer, hedgehogs, elephants, llama, alpaca, mice, rats, or chickens, and preferably humans, cows, pigs , or chicken. The. Combination treatments Pharmaceutical compositions, preferably vaccines, can be administered in combination with one or more other influenza proteins or genes encoding influenza A H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H1l5, Hl6, NI, N2, N3, N4, N5, N6, N7, N8, N9, influenza B hemagglutinin and neuramidase. The vaccine can be administered in combination with proteins or genes encoding adjuvants, which may include: a-interferon (IFN-a), B-interferon (IFN-B), y-interferon, IL-12, IL-15, IL -28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFa, TNFB, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL -6, IL-10, IL-12, IL-18, MCP-1, MIP-la, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GI, YCAM -1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF , IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DRA, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1 , Ap-1, Ap-2, p38, postRel, MyD88, IRAK, TRAFS, IkB, Inactive NIK, SAP K, SAP-1, JNK, Response benes interferon, NFKB, Bax, TRAIL, TRAILrec, TRAILTecDRCS, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, TAP2, or functional fragments thereof.
B. Routes of administration: The vaccine can be administered by different routes including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal — intratecal, and intraarticular or combinations thereof. For veterinary use, the composition can be administered as an acceptable formulation according to normal veterinary practice. The veterinarian can readily determine the dose regimen and route of administration that is most appropriate for a particular animal. The vaccine can be administered by traditional syringes, needle-free injection devices, "microprojectile bombing guns", or other physical methods such as electroporation ("EP"), "hydrodynamic method", or ultrasound.
The vaccine vector can be released to the mammal by several known technologies including DNA injection (still referred to as DNA vaccination) with and without electroporation in vivo, mediated by liposome, facilitated by nanoparticle, vectors - recombinant as recombinant adenovirus, associated recombinant adenovirus with virus and recombinant vaccinia. The HA antigen can be released through DNA injection and in vivo electroporation.
ç. Electroporation The administration of the vaccine via electroporation of the vaccine plasmids can be accomplished using an electroporation device that can be configured to give a desired tissue of a mammal an energy pulse effective to cause reversible pores in cell membranes, and preferably the pulse of energy is a constant current similar to a current input predefined by a user. The electroporation device may comprise an electroporation component and an electrode or handle assembly. The electroporation component can include and incorporate one or more of the various elements of electroporation devices, including: controller, current wave generator, impedance tester, waveform recorder, input element, status reporting element, communication port, memory component, power source, and power switch. Electroporation can be achieved using an in vivo electroporation device, for example, CELLECTRAG & EP system (VGX Pharmaceuticals, Blue Bell, PA) or Elgen electroporator (Genetronics, San Diego, CA) to facilitate transfection of cells by the plasmid.
The electroporation component can function as an element of the electroporation devices, and the other elements are separate elements (or components) in communication with the electroporation device. The electroporation component may function as more than one element of the electroporation devices, which may be in communication with yet other elements of the electroporation devices separate from the cloporoporation component. The elements of the electroporation devices that exist as parts of an electrochemical or mechanical device may not be limited as the elements can function as one device or as separate elements in communication with another. The electroporation component may be able to release the energy pulse that produces constant current in the desired tissue, and includes a feedback mechanism. The electrode assembly may include an electrode array containing a plurality of electrodes in a spatial arrangement, in which the electrode assembly receives the energy pulse from the electroporation component and releases it to the desired tissue through the electrodes. At least one of a plurality of electrodes is neutral during the release of the energy pulse and measures impedance in the desired tissue and communicates the impedance to the electroporation component. A feedback mechanism can receive the measured impedance and can adjust the energy pulse released by the electroporation component to keep the current constant.
A plurality of electrodes can release the energy pulse in a decentralized pattern. The plurality of electrodes can release the energy pulse in the decentralized pattern through the control of electrodes under a programmed sequence, and the programmed sequence is entered by a user to the electroporation component. The programmed sequence can comprise a plurality of pulses released in sequence, in which each pulse of plurality of pulses is released by at least two active electrodes with a neutral electromagnet that measures impedance, and in which a subsequent pulse of the plurality of - pulses is released by a different one of at least two active electrodes with a neutral electrode that measures impedance.
The feedback mechanism can be performed by hardware or software. The feedback mechanism can be performed by an analog loop loop. Feedback occurs every 50 us, 20 us, 10 us or 1 us, but it is —preferably real-time or instantaneous feedback (ie, substantially instantaneous as determined by available techniques to determine response time). The neutral electrode can measure the impedance in the desired tissue and communicates the impedance to the feedback mechanism and the feedback mechanism responds to the impedance and adjusts the energy pulse to keep the current constant at a value similar to the current current. The feedback mechanism can maintain constant current continuously and instantly during the release of the energy pulse.
Examples of celetroporation devices and methods that can facilitate the release of DNA vaccines of the present invention, include those described in US patent 7,245,963 by Draghia-Akli, et al., US Publication 2005/0052630 submitted by Smith, et al. , whose contents are incorporated herein by reference in their entirety. Other electroporation devices and electroporation methods that can be used for - DNA vaccines include those provided in US 11/874072 co-pending and co-owned patent applications, filed on October 17, 2007, which claim the benefit 35 USC 119 (e) of provisional application US 60 / 852,149, filed on October 17, 2006, and 60 / 978,982, filed on October 10, 2007, all of which are incorporated in their entirety.
US patent 7,245,963 to Draghia-Akli, et al. describe modular clethode systems and their uses to facilitate the introduction of a biomolecule into the cells of a selected tissue in a body or plant. Modular electrode systems can comprise a plurality of needle-free electrodes; a hypodermic needle; an electrical connector that provides a conductive connection from a programmable constant current pulse controller for the plurality of needle electrodes; and a source of energy. An operator can reach the plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue in a body or plant. The biomolecules are then released through the hypodermic needle into the selected tissue. The programmable constant current pulse controller is activated and constant current electrical pulse is applied to the plurality of needle electrodes. The applied electric current constant pulse facilitates the introduction of the biomolecule into the cell between the plurality of electrodes. The entire contents of US patent 7,245,963 are hereby incorporated by reference.
US Patent Publication 2005/0052630 submitted by Smith, et al. describe an electroporation device that can be used to effectively facilitate the introduction of a biomolecule into cells of a selected tissue in a body or plant. The electroporation device comprises an electro-kinetic device ("EKD device") whose operation is specified by software or firmware. The device
EKD produces a series of programmable constant current pulse patterns between electrodes in an array based on user control and input of pulse parameters, and allows the storage and acquisition of current waveform data. The electroporation device further comprises a replaceable electrode disk containing an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The complete content of US Patent Publication 2005/0052630 is incorporated herein by reference.
The electrode arrangements and methods described in US patent 7,245,963 and US Publication 2005/0052630 can be adapted for deep penetration not only into tissues such as muscle, but also into other tissues or organs. Due to the configuration of the electrode arrangement, the injection needle (to release the biomolecule of choice) is still inserted completely into the target organ, and the injection is administered perpendicular to the target tissue, in the area that is pre-outlined by electrodes. The electrodes described in US patent 7,245,963 and US Patent Publication 2005/005263 are preferably 20 mm long and 21 caliber.
Additionally, in some modalities that incorporate electroporation devices and their uses, the electroporation devices are those described in the following patents: US patent 5,273,525 published on December 28, 1993, US patent 6,110,161 published on 29 August 2000, 6,261,281 —published on July 17, 2001, and 6,958,060 published on October 25, 2005, and US patent 6,939,862 published on September 6, 2005. In addition, the patents covering the subject matter provided in US patent 6,697,669 published on February 24, 2004, which relates to the release of DNA using any of a variety of devices, and US patent 7,328,064 published on February 5, 2008, extracted from the method of DNA injection are covered here. The aforementioned patents are incorporated by reference in their entirety.
d. Vaccine preparation method Methods are provided here for preparing the DNA plasmids comprising the DNA vaccines discussed here. DNA plasmids, after the final sub-cloning step in the mammalian expression plasmid, can be used to inoculate a cell culture in a large-scale fermentation tank, using methods known in the art.
The DNA plasmids for use with the EP devices of the present invention can be formulated or produced using a combination of known devices and techniques, but are preferably produced using optimized plasmid production techniques that are described in a US copying license provisionally licensed US 60 / 939,792, which was deposited on May 23, 2007. In some examples, the DNA plasmids used in these studies can be formulated in concentrations greater than or equal to 10 mg / mL. Manufacturing techniques still include or incorporate various devices and protocols that are commonly known to those skilled in the art, in addition to those described in US 60/939792, including those described in a licensed patent, US Patent 7,238,522, published July 3,
2007. The above referenced applications and patents US 60 / 939,792 and US Patent 7,238,522, respectively, are incorporated herein in their entirety.
EXAMPLES The present invention is further illustrated in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are provided by way of illustration only. From the above discussion and these Examples, a person skilled in the art can verify the essential characteristics of this invention, and without departing from the spirit and scope of it, can generate various changes and modifications of the invention to adapt it to various uses and conditions. Thus, various modifications of the invention in addition to those shown and described here will be apparent to those skilled in the art from the above description. Said modifications are still within the scope of the attached claims.
Example 1 PGX2009 (pHIHAO9) - Plasmid encoding hemagglutinin antigen 2009 Influenza HINÍ (Swine Flu) The structure of pGX2009 (H1IHAO09) is the modified expression vector pVAX1 (Invitrogen, Carlsbady CA) under the control of the immediate early promoter of cytomegalovirus ( CMV). The original pVAXI1 was purchased from Invitrogen (catalog number V260-20) and kept at -20ºC. As noted above, sequence analysis revealed differences between the pVvAX sequence] used as the pGX2009 structure and the - pVAX sequence! available from Invitrogen. The differences are set out above.
The plasmid pGX2009, still referred to as pHIHA09, comprises a nucleic acid sequence that encodes a 2009 HINI influenza (swine flu) consensus hemagglutinin molecule. The 79 primary sequences used to generate the consensus sequence were selected from the Influenza Sequence Database.
The accession numbers for nucleotide sequences encoding the amino acid sequence for the various hemagglutinin H1 influenza proteins as well as the amino acid sequence encoded for the nucleotide sequences are in the GenBank database corresponding to the following accession numbers.
Accession numbers not in parentheses reveal the nucleotide sequences and additional list of amino acid sequence encoded by them.
The accession numbers in parentheses are for entries of the corresponding amino acid sequence in the GenBank protein database.
The accession numbers are as follows: GQ323579.1 (ACS72657.1), GQ323564.1 (ACST2654.1), GQ323551.1 (ACST2652.1), GQ323530.1 (ACST26511), GQ323520.1 (ACST72650.1) , GQ323495.1 (ACS72648.1), GQ323489.1 (ACST72647.1), GQ323486.1 (ACS7T2646.1), GQ323483.1 (ACS72645.1), GQ323455.1 (ACS72641.1), GQ323451.1 ( ACS72640.1), GQ323443.1 (ACS72638.1), GQ293077.1 (ACS68822.1), GQ288372.1 (ACSS4301.1), GQ287625.1 (ACSS4262.1), GQ287627.1 (ACSS4263.1), GQ287623.1 15º (ACSS4261.1), GQ287621.1 (ACSS4260.1), GQ286175.1 (ACSS4258.1), GQ283488.1 (ACSS0088.1), GQ280797.1 (ACS45035.1), GQ280624.1 ( ACS45017.1), GQ280121.1 (ACS45189.1), GQ261277.1 (ACS34968.1), GQ253498.1 (ACS27787.1), GQ323470.1 (ACS72643.1), GQ253492.1 (ACS27780.1), FI9SI613.1 (ACQSS359.1), FI971076.1 (ACP52565.1), FJI969540.4 (ACP44189.1), FI969511.1i (ACP44150.1), FJ969509.1 (ACP44147.1), GQ255900.1 (ACS27774 .1), GQ255901.1 (ACS27775.1), FI966974.1 (ACP41953.1), GQ261275.1 (ACS34967.1), FJI966960.1 (ACP41935.1), FJ966952.1 (ACP41926.1), FJI 966082.1 (ACP41105.1), GQ255897.1 (ACS27770.1), CXY041645.1 (ACS27249.1), CY041637.1 (ACS27239.1), CY041629 (ACS27229.1), GQ323446.1 (ACS72639.1), CY041597.1º (ACS27189.1), CYO41581.1 (ACS14726.1), CY040653.1 (ACSI4666.1), CYO041573.1 (ACSI14716.1), CY041565.1 (ACS14706.1), CY041541.1 i ( ACS14676.1), GQ258462.1 (ACS34667.1), CY041557.1 (ACS14696.1), CY041549.1 (ACS14686.1), GQ283484.1 (ACSSO084.1), GQ283493.1 (ACSS0095.1), GQ303340.1 (ACSTI656.1), GQ287619.1 (ACSS4259.1), GQ267839.1 (ACS36632.1), GQ268003.1 (ACS36645.1), CYO041621.1 (ACS27219.1), CY041613.1 (ACS27209 .1), CYO041605.1. (ACS27199.1), FI966959.1 (ACP41934.1), FI966982.1 (ACP41963.1), CY039527.2 (ACQ45338.1), FI981612.1 (ACQSS358.1), FI9SIGIS.1 (ACQSS361.1) , FI982430.1 (ACQS9195.1), FJ998208.1 (ACQ73386.1), GQ259909.1 (ACS34705.1), GQ261272.1 (ACS34966.1), GQ287621.1 (ACSS4260.1), GQ290059.1 ( ACS66821.1), GQ323464.1
(ACS72642.1), GQ323473.1 (ACS72644.1), GQ323509.1 (ACS72649.1), GQ323560.1 (ACS72653.1), GQO323574.1 (ACS7T2655.1), and GQ323576.1 (ACS72656.1 ). The amino acid sequences were downloaded from the NCBI sequence database, and an alignment and consensus sequence generated using Clustal X.
A high-efficiency leader sequence, the IgE leader, was merged in the region upstream from the start codon to facilitate expression.
In order to have a higher level of expression, the codon use of this fusion gene was adapted to the codon bias of Homo Sapiens genes.
In addition, RNA optimization was also carried out: regions of very high (80%) or very low (<30%) GC content and the sequence motifs acting on cis as internal TATA boxes, chi sites and entry sites —ribossomal have been avoided.
The input sequence was synthetically produced in Geneart (Regensburg, Germany). The engineered synthetic gene H1HAO09 was 1818 bp in length (SEQ ID NO: 1) and was cloned into pVAX1 at BamHI and Xhol sites by Geneart (Figure 2). Example 2 Challenge of Ferrets immunized by Influenza pGX2009 with A / Mexico / INDRE4487 / 2009 The challenge experiments were conducted using ferrets, a preferred model for influenza.
Ferrets were immunized using plasmid pGX2009, Animals: 4 groups x 5 animals / group, plus a control group with 4 animals = 24 — total ferrets (males) Duration: 18 weeks (including challenge) Dose: 0.2mg plasmid Summary of the protocol : Ferrets were randomly allocated to DNA vaccine groups.
Animals were immunized on Study Day 0, Day 28, and Day 56. Animals were anesthetized with ketamine / midazolam cocktail, isoflurane or equivalent according to approved anesthesia protocols and IM vaccinated with DNA DNA vaccine combinations.
Groups 1 and 2 were immediately electroporated using an adaptive constant current electroporation device CELLECTRAG (EP) at 0.5 Amp, 52 millisecond pulses, 0.2 seconds between pulses, 4 seconds of trigger delay, 3 total pulses.
Control animals were naive controls (without plasmid, without PE). Ferrets were allowed to recover from anesthesia in their cages and were closely monitored for 24 hours to ensure full recovery.
Food and water were available ad libitum for the duration of the study.
In day
84, animals were challenged by intranasal infection with 1 ml of MX10 (A / Mexico / InDRE4487 / 2009; 5 x 105 PFU / ml). Animals were monitored daily for clinical signs (weight, temperature, etc.), using an established and approved classification sheet.
In 1, 3, 6, 9 c and 15 dpi nasal washes and rectal smears were collected.
The lungs were collected on day 15. The samples were stored in RNAlater for virus loading by real-time PCR, medium for infection virus (TCDI50) and formalin for histology when appropriate.
Figure 4 shows a Hemagglutination Inhibition assay performed with serum from immunized ferrets (3 immunizations). A title of> 1:40 is considered "protective". A dotted line indicates the 1:40 milestone. All animals were below the 1:40 mark after 3 immunizations.
Figure 5 shows results of a challenge for ferrets immunized and not immunized with a new HIN strain! MX10 (A / Mexico / MDRE4487 / 2009). All immunized ferrets survived, while 75% of naive ferrets died within the 15-day period.

权利要求:
Claims (1)
[1]
: : go
1. Isolated nucleic acid molecule, characterized by the fact that it comprises one or more nucleic acid sequences selected from the group consisting of: - a) one selected from the group consisting of: SEQ ID NO: 1, a nucleic acid sequence which is 95% homologous to SEQ ID NO: 1; a fragment of SEQ ID NO: 1 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 1 comprising at least 60 nucleotides; - b) a nucleic acid sequence that is selected from the group consisting of: SEQIDNO: 3, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 3; a fragment of SEQ ID NO: 3 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 3 comprising at least 60 nucleotides; c) a nucleic acid sequence that is selected from the group consisting of: 15º SEQID NO: 6, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 6; a fragment of SEQ ID NO: 6 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 6 comprising at least 60 nucleotides; d) a nucleic acid sequence that is selected from the group consisting of: SEQIDNO: 9, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 9; a fragment of SEQ ID NO: 9 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 9 comprising at least 60 nucleotides; e) a nucleic acid sequence that is selected from the group consisting of: —SEQIDNO: 11, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 11; a fragment of SEQ ID NO: 11 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 11 comprising at least 60 nucleotides; f) a nucleic acid sequence that is selected from the group consisting of: SEQIDNO: 13, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 13; a fragment of SEQ ID NO: 13 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 13 comprising at least 60 nucleotides; and
: 2/11 8) a nucleic acid sequence that is selected from the group consisting of: SEQ ID NO: 15, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 15; a fragment of SEQ ID NO: 15 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 15 - comprising at least 60 nucleotides.
2. Isolated nucleic acid molecule according to claim 1, characterized in that it comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6 , SEQ ID NO: 9, SEQ ID NO: 11, SEQTID NO: 13 and SEQ ID NO: 15.
3. Isolated nucleic acid molecule according to claim 1, characterized in that it comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence that is 95% homologous to SEQ ID NO: 1, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 3, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 6, a 15th nucleic acid sequence that is 95% homologous to SEQ ID NO: 9, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 11, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 13, and a nucleic acid sequence that is 95% homologous to SEQ ID NO: 13 NO: 15.
4, Isolated nucleic acid molecule according to claim 1, characterized in that it comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence that is 98% homologous to SEQ ID NO: 1, a nucleic acid sequence that is 98% homologous to SEQ ID NO: 3, a nucleic acid sequence that is 98% homologous to SEQ ID NO: 6, a nucleic acid sequence that is 98% homologous to SEQ ID NO: 9 , a nucleic acid sequence that is 98% homologous to SEQ ID NO: 11, a nucleic acid sequence that is 98% homologous to SEQIDNO: 13, and a nucleic acid sequence that is 98% homologous to SEQ ID NO: 15 .
,. Isolated nucleic acid molecule according to claim 1, characterized in that it comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 15 and a nucleic acid sequence encoding an IgE leader sequence.
6. Expression vector, characterized by the fact that it comprises a nucleic acid sequence of claim 1 operably linked to regulatory elements.
TF Vector of expression, characterized by the fact that it comprises a
. The nucleic acid sequence of claim 1 operably linked to regulatory elements that are functional in a human cell.
8. Expression vector, according to claim 7, characterized by the fact that said expression vector is a plasmid.
9. Expression vector, according to claim 8, characterized by the fact that said expression vector is pGX2009.
10. Composition, characterized by the fact that it comprises: a) a plurality of one or more nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: i) one selected from the group consisting of: SEQ ID NO: 1, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 1; a fragment of SEQ ID NO: 1 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 1 comprising at least 60 nucleotides; ii) a nucleic acid sequence that is selected from the group consisting of: 15th SEQIDNO: 3, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 3; a fragment of SEQ ID NO: 3 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 3 comprising at least 60 nucleotides; iii) a nucleic acid sequence that is selected from the group consisting of: SEQIDNO: 6, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 6; a fragment of SEQ ID NO: 6 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 6 comprising at least 60 nucleotides; iv) a nucleic acid sequence that is selected from the group consisting of: SEQIDNO: 9, a nucleic acid sequence that is 95% homologous to SEQ TD NO: 9; a fragment of SEQ ID NO: 9 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 9 comprising at least 60 nucleotides; v) a nucleic acid sequence that is selected from the group consisting of: SEQIDNO: 11, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 11; a fragment of SEQ ID NO: 11 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 11 comprising at least 60 nucleotides;
. vii) a nucleic acid sequence that is selected from the group consisting of: SEQ ID NO: 13, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 13; a fragment of SEQ ID NO: 13 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 13 - comprising at least 60 nucleotides; and vii) a nucleic acid sequence that is selected from the group consisting of: SEQ ID NO: 15, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 15; a fragment of SEQ ID NO: 15 comprising at least 60 nucleotides and a nucleic acid sequence that is 95% homologous to a fragment of SEQ ID NO: 15 comprising at least 60 nucleotides; and b) one or more additional nucleic acid sequences encoding one or more proteins selected from the group consisting of one or more of: influenza A hemagglutinin H1, influenza A hemagglutinin H2, influenza A hemagglutinin H3, influenza A H4 influenza A hemagglutinin H5, influenza A hemagglutinin H3, influenza A hemagglutinin H5, influenza A NI ... influenza A hemagglutinin H6, influenza A hemagglutinin H7, influenza A hemagglutinin H5, influenza A hemagglutinin H6, influenza A hemagglutinin H7, hemagglutinin H8, an influenza A hemagglutinin H9, an influenza A hemagglutinin H10, an influenza A hemagglutinin H11, an influenza A hemagglutinin H12, an influenza A hemagglutinin HI4, an influenza A hemagglutinin HIS, an influenza A hemagglutinin HIS, influenza A hehe N1, an influenza A neuraminidase N2, an influenza A neuraminidase N3, an influenza A neuraminidase N4, an influenza A neuraminidase N5, an infl uenza A neuraminidase N6, an influenza A neuraminidase N7, an influenza A neuraminidase N8, an influenza A neuraminidase —N9, an influenza B hemagglutinin and an influenza B neuraminidase.
11. Composition according to claim 10, characterized in that said one or more additional nucleic acid sequences are a plurality of one or more different nucleic acid molecules from the plurality of nucleic acid molecules set out in section a) .
12. "Composition according to claim 10, characterized by the fact that the plurality of nucleic acid molecules established in section a) comprises one or more nucleic acid sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and
: 511 SEQ1ID NO: 15.
13. Composition according to claim 10, characterized in that the plurality of nucleic acid molecules established in section a) comprises one or more nucleic acid sequences selected from the group consisting of: one - nucleic acid sequence that is 95% homologous to SEQ ID NO: 1, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 3, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 6, an acid sequence nucleic acid that is 95% homologous to SEQ ID NO: 9, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 11, a nucleic acid sequence that is 95% homologous to SEQ ID NO: 13, and a sequence nucleic acid that is 95% homologous to SEQ ID NO: 15.
Composition according to claim 10, characterized in that the plurality of nucleic acid molecules established in section a) comprises one or more nucleic acid sequences selected from the group consisting of: a nucleic acid sequence that is 98 % homologous to SEQ ID NO: 1, a nucleic acid sequence that is 98% homologous to SEQ ID NO: 3, a nucleic acid sequence that is 98% homologous to SEQ ID NO: 6, a nucleic acid sequence that is 98% homologous to SEQ ID NO: 9, a nucleic acid sequence that is 98% homologous to SEQ ID NO: 11, a nucleic acid sequence that is 98% homologous to SEQ ID NO: 13, and a nucleic acid sequence which is 98% homologous to SEQ ID NO: 15.
15. Composition according to claim 10, characterized by the fact that the nucleic acid sequences set out in a) and b) are each operationally linked to regulatory elements.
16. Composition according to claim 10, characterized by the fact that the nucleic acid sequences set out in a) and b) are each linked - operationally to regulatory elements that are functional in a human cell.
17. Composition according to claim 10, characterized by the fact that the nucleic acid sequences set out in a) and b) are part of one or more expression vectors.
18. Composition, according to claim 10, characterized by the fact that —which more expression vectors are plasmids.
19. Composition, according to claim 10, characterized by the fact that it comprises pGX2009 and / or pGX2006.
20. Composition according to claim 10, characterized in that it comprises one or more of: a nucleic acid sequence comprising SEQ ID NO: 1; a nucleic acid sequence comprising SEQ ID NO: 6; a nucleic acid sequence comprising SEQ ID NO: 9; Ss is a nucleic acid sequence comprising SEQ ID NO: 13; a nucleic acid sequence encoding an influenza A hemagglutinin H1; and a nucleic acid sequence encoding an influenza A hemagglutinin H3.
21. Composition according to claim 10, characterized in that the nucleic acid sequence encoding an influenza A hemagglutinin H1 comprises SEQ ID NO: 21 and the nucleic acid sequence encoding an influenza A hemagglutinin H3 comprises SEQ 1D NO23.
22. "Composition according to claim 20, characterized in that it comprises: a nucleic acid molecule comprising SEQID NO: S; a nucleic acid molecule comprising SEQ ID NO: 13; and a nucleic acid molecule comprising SEQ ID NO: 23.
23. Composition according to claim 22, characterized in that the nucleic acid molecule comprising SEQ ID NO: 9 is a plasmid; the nucleic acid molecule comprising SEQ ID NO: 13 is a plasmid; and the nucleic acid molecule comprising SEQ ID NO: 23 is a plasmid.
24. Use of an isolated nucleic acid molecule, which comprises one or more nucleic acid sequences according to claim 1, characterized by the manufacture of a medicament to induce an immune response.
25. Use of the composition of claim 10 characterized by the manufacture of a medicament to induce an immune response.
26. The use of the composition of claim 23, characterized by the manufacture of a medicament to induce an immune response.
27. - Use of an isolated nucleic acid molecule, which comprises one or more nucleic acid sequences according to claim 1, characterized by the manufacture of a drug to protect an individual against infection by a strain of human influenza A of origin swine, the nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
. 71 SEQ ID NO: 1, a 95% nucleic acid sequence homologous to SEQ ID NO: 1; a fragment of SEQ ID NO: 1, a nucleic acid sequence 95% homologous to a fragment of SEQ ID NO: 1; SEQ ID NO: 9, a 95% nucleic acid sequence homologous to SEQ ID NO: 9; a fragment of SEQ ID NO: 9, and a 95% nucleic acid sequence homologous to a fragment of SEQ ID NO: 9; wherein the nucleic acid sequence is expressed in cells of said individual and an immune response against said protein is induced which is a protective immune response against human influenza A of porcine origin.
28. - Use of the composition of claim 10 characterized by the manufacture of a medicament for the protection of an individual against infection by a strain of human influenza A of porcine origin, characterized in that the composition comprises: a) a first selected nucleic acid sequence the group consisting of: SEQID NO: 1, a 95% nucleic acid sequence homologous to SEQ ID NO: 1; a fragment of SEQ ID NO: 1, a nucleic acid sequence 95% homologous to a fragment of SEQ ID NO: 1; SEQ1ID NO: 9, a 95% nucleic acid sequence homologous to SEQ ID NO: 9; a fragment of SEQ ID NO: 9, and a 95% nucleic acid sequence homologous to a fragment of SEQ ID NO: 9; and b) one or more additional nucleic acid sequences encoding one or more proteins selected from the group consisting of one or more of: an influenza A hemagglutinin H1, an influenza A hemagglutinin H2, an influenza A hemagglutinin H3, influenza A H4 influenza A hemagglutinin H5, an influenza A hemagglutinin H3, influenza A hemagglutinin H5, influenza A NI ... influenza A hemagglutinin H6, an influenza A hemagglutinin H7, influenza A hemagglutinin HS, influenza A hemagglutinin H6, an influenza A hemagglutinin H7, an influenza A hemagglutinin H7, hemagglutinin H8, an influenza A hemagglutinin H9, an influenza A hemagglutinin H10, an influenza A hemagglutinin H11, an influenza A hemagglutinin H12, influenza A H13 influenza A
It is 8/11 hemagglutinin H14, influenza A hemagglutinin H15, influenza A hemagglutinin H16, an influenza A neuraminidase N1, an influenza A neuraminidase N2, an influenza A neuraminidase N3, an influenza A neuraminidase N4, an influenza A neuraminidase N5, an influenza A neuraminidase N5, an influenza A Neuraminidase N6, influenza A neuraminidase N7, influenza A neuraminidase N8, influenza A neuraminidase N9, influenza B hemagglutinin and influenza B neuraminidase; wherein the first nucleic acid sequence is expressed in said individual cells and an immune response against said first protein is induced which is a protective immune response against human influenza A of porcine origin, the one or more additional nucleic acid sequences are expressed in cells of said individual and immune response against said one or more second proteins is induced.
29. Use according to claim 28, characterized in that the composition comprises one or more of: a nucleic acid sequence comprising SEQ ID NO: 1; a nucleic acid sequence comprising SEQ ID NO: 9; a nucleic acid sequence comprising SEQ ID NO: 13; a nucleic acid sequence encoding influenza A hemagglutinin H1; and a nucleic acid sequence encoding influenza A hemagglutinin H3.
30. Use according to claim 28, characterized in that the - nucleic acid sequence encoding an influenza A hemagglutinin H1 comprises SEQ ID NO: 21 and the nucleic acid sequence encoding an influenza A hemagglutinin H3 comprises SEQ ID NO: 23.
31. Use according to claim 28, characterized in that the composition comprises one or more of: a nucleic acid molecule comprising SEQ ID NO: 9; a nucleic acid molecule comprising SEQ ID NO: 13; and a nucleic acid molecule comprising SEQ ID NO 23.
32. The method of claim 31, wherein the nucleic acid molecule comprising SEQ ID NO: 9 is a plasmid; the nucleic acid molecule comprising SEQ ID NO: 13 is a plasmid; and the nucleic acid molecule comprising SEQ ID NO: 23 is a plasmid.
33. - Use of an isolated nucleic acid molecule, comprising one or more nucleic acid sequences according to claim 1, characterized by
$ / 11 manufacture of a drug for the treatment of an individual who has been infected with a strain of human influenza A of swine origin, the nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, a nucleic acid sequence 95% homologous to SEQ ID NO: 1; a fragment of SEQ ID NO: 1, a nucleic acid sequence 95% homologous to a fragment of SEQ ID NO: 1; SEQ ID NO: 9, a 95% nucleic acid sequence homologous to SEQ ID NO: 9; a fragment of SEQ ID NO: 9, and a 95% nucleic acid sequence homologous to a fragment of SEQ ID NO: 9; wherein the nucleic acid sequence is expressed in the cells of said individual and an immune response against said protein is induced which is a protective immune response against human influenza A of porcine origin.
34. Use of an isolated nucleic acid molecule, which comprises one or more nucleic acid sequences according to claim 10, characterized by the manufacture of a medicament for the treatment of an individual who has been infected with a human influenza A strain of swine origin, characterized by the fact that the composition comprises: a) a first nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, a nucleic acid sequence 95% homologous to SEQ ID NO: 1; a fragment of SEQ ID NO: 1, a nucleic acid sequence 95% homologous to a fragment of SEQTD NO: 1; SEQ ID NO: 9, a 95% nucleic acid sequence homologous to SEQ ID NO: 9; a fragment of SEQ ID NO: 9, and a 95% nucleic acid sequence homologous to a fragment of SEQ ID NO: 9; and b) one or more additional nucleic acid sequences encoding one or more proteins selected from the group consisting of one or more of: an influenza A hemagglutinin H1, an influenza A hemagglutinin H2, an influenza A hemagglutinin H3, influenza A H4 influenza A hemagglutinin H5, an influenza A hemagglutinin H3,
: 10/11 influenza A hemagglutinin H5, influenza A hemagglutinin H6, influenza A hemagglutinin H7, influenza A hemagglutinin H6, influenza A hemagglutinin H6, influenza A hemagglutinin H7, influenza A hemagglutinin H8, Hemagglutinin H9, an influenza A hemagglutinin H10, an influenza A —hemagglutinin HI1, an influenza A hemagglutinin H12, an influenza A H13 influenza A hemagglutinin H14, an influenza A hemagglutinin H15, an influenza A hemagglutinin H1 influenza, an influenza a H1 influenza, a H1 influenza, a H1 influenza A Neuraminidase N2, influenza A neuraminidase N3, influenza A neuraminidase N4, influenza A neuraminidase N5, influenza A neuraminidase N6, influenza A neuraminidase N7, influenza A neuraminidase N8, influenza A neuraminidase N9, influenza B hemagglutinin and an influenza B neuraminidase; wherein the first nucleic acid sequence is expressed in said individual cells and an immune response against said first protein is induced which is a protective immune response against human influenza A of porcine origin, the one or more additional nucleic acid sequences are expressed in the cells of said individual and immune responses against said one or more second proteins are induced.
35. Use according to claim 34, characterized in that the composition comprises one or more of: a nucleic acid sequence comprising SEQ ID NO: 1; a nucleic acid sequence comprising SEQ ID NO: 9; a nucleic acid sequence comprising SEQ ID NO: 13; a nucleic acid sequence encoding influenza A hemagglutinin H1; and a nucleic acid sequence encoding influenza A hemagglutinin H3.
36. - Use according to claim 34, characterized in that - nucleic acid sequence encoding an influenza A hemagglutinin H1 comprises SEQ ID NO: 21 and the nucleic acid sequence encoding influenza A hemagglutinin H3 comprises SEQ ID NO: 23.
37. Use according to claim 34, characterized in that the composition comprises one or more of: a nucleic acid molecule comprising SEQ ID NO: 9; a nucleic acid molecule comprising SEQ ID NO: 13; and a nucleic acid molecule comprising SEQ ID NO: 23.
38. Use according to claim 37, characterized by the fact that
. 11/11 the nucleic acid molecule comprising SEQ ID NO: 9 is a plasmid; the nucleic acid molecule comprising SEQ ID NO: 13 is a plasmid; and the nucleic acid molecule comprising SEQ ID NO: 23 is a plasmid.

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法律状态:
2020-09-08| B15I| Others concerning applications: loss of priority|Free format text: PERDA DA PRIORIDADE US 12/694,238, DE 26/01/2010, CONFORME AS DISPOSICOES PREVISTAS NA LEI 9.279 DE 14/05/1996 (LPI) ART. 167O E NO ART. 29 DA RESOLUCAO INPI-PR 77/2013, POR NAO ATENDER AO DISPOSTO NO ART. 2 DA RESOLUCAO INPI-PR 179/2017, POIS NAO FOI APRESENTADA CESSAO DA REFERIDA PRIORIDADE, QUE POSSUI DEPOSITANTE DIFERENTE DO DEPOSITANTE DA FASE NACIONAL. |

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2020-11-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-02-17| B08F| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]|Free format text: REFERENTE A 10A ANUIDADE. |

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2021-05-11| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-06-01| B08K| Patent lapsed as no evidence of payment of the annual fee has been furnished to inpi [chapter 8.11 patent gazette]|Free format text: EM VIRTUDE DO ARQUIVAMENTO PUBLICADO NA RPI 2615 DE 17-02-2021 E CONSIDERANDO AUSENCIA DE MANIFESTACAO DENTRO DOS PRAZOS LEGAIS, INFORMO QUE CABE SER MANTIDO O ARQUIVAMENTO DO PEDIDO DE PATENTE, CONFORME O DISPOSTO NO ARTIGO 12, DA RESOLUCAO 113/2013. |

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2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US12/694,238|US8298820B2|2010-01-26|2010-01-26|Influenza nucleic acid molecules and vaccines made therefrom|

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PCT/US2011/022642|WO2011094358A1|2010-01-26|2011-01-26|Influenza nucleic acid molecules and vaccines made therefrom|

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