Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/145—Orthomyxoviridae, e.g. influenza virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
  • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• H5N1 strains indicate that these highly pathogenic stains are more variable than previously observed. It would therefore be unexpected for vaccines with less than 90% homology to challenge strains to be effective in controlling symptoms or reduce shedding.
• the subject invention provides a method of inducing an immunoprotective response against a strain of Avian Influenza Virus (AIV) in an animal or human which comprises; a) expressing in a plant cell a nucleic acid sequence comprising known HAl variable region polypeptide that has between about 70% to about 90% homology to a challenge strain HAl variable region polypeptide; b) preparing a vaccine composition using the known HAl variable region polypeptide expressed in said plant cell, and; c) administering said vaccine composition to an animal or human such that a protective immune response is induced in said animal or human.
• the invention further provides vectors, host cells, and novel vaccine compositions for practicing the aforementioned methods.
• Such vaccines compositions comprise plant-made, known HAl polypeptide sequences that provide protective immunity below the 90% homology level compared to a challenge strain when administered to an animal or human.
• the avian influenza HA protein of influenza A/turkey/Wisconsin/68 sequence is shown below.
• This HA protein contains 568 amino acids and exhibits 5 distinct domains including: a signal peptide (amino acids 1-16); the variable head region of fragment Hl (amino acids 17-323 [also referred to herein as HAl]); the constant base region of fragment H2 (amino acids 324-527); the transmembrane domain (amino acids 528-557); and the intracellular thioester lipid fragment (amino acids 558-568).
• a signal peptide amino acids 1-16
• the variable head region of fragment Hl amino acids 17-323 [also referred to herein as HAl]
• the constant base region of fragment H2 amino acids 324-527
• the transmembrane domain amino acids 528-557
• intracellular thioester lipid fragment amino acids 558-568.
• the full length avian influenza HA protein (SEQ ID NO: 1):
• Signal peptide SEQ ID NO: 2: MERIVIALAIISVVKG;
• Hl variable head region fragment (SEQ ID NO: 3):
• DQICIGYHANNSTKQ VDTIMEKNVTVTHAQDILEKEHNGKLCSLKGVRPLILKDCSV AGWLLGNPMCDEFLNVPEWSYIVEKDNPTNGLCYPGDFNDYEELKYLMSNTNHFE KIQIIPRNSWSNHDASSGVSSACPYNGRSSFFRNVVWLIKKSNA YPTIKRTYNNTNVE DLLILWGIHHPNDAAEQTELYQNSNTYVSVGTSTLNQRSIPEIATRPKVNGQSGRIEFF WTILRPNDAISFESNGNFIAPEYA YKIVKKGDSAIMRSELEYGNCDTKCQTPVGAINSS MPFHNVHPLTIGECPKYVKSDKLVLATGLRNVPQRETR;
• H2 base constant fragment (SEQ ID NO: 4):
• Transmembrane anchor (SEQ ID NO: 5): QILSIYSTVASSLALAIMV AGLSFWMCS; and
• Intracellular thioester lipid fragment SEQ ID NO: 6
• NGSLQCRICI Intracellular thioester lipid fragment
• the full length HA protein (SEQ ID NO: 7):
• Signal peptide SEQ ID NO: 8: MERIVIALAIISVVKG;
• Hl variable head region fragment (SEQ ID NO: 9):
• H2 base constant fragment (SEQ ID NO: 10):
• plant-made vaccine sub-unit antigens are superior to conventionally prepared sub-unit antigens because they are integrated into the cellular matrix of membrane and carbohydrate components of the plant providing an adjuvant property.
• the plant made antigen will also contain unique plant carbohydrate glycosylation patterns as compared to non-plant made platforms.
• plant glycan structures contribute to an increased spectrum of cross protection and the use of antigens that are not completely purified from the plant cell or plant cell matrix components may also be partially responsible for the ability to protect an individual from infection by an AIV having between about 90% to about 70% homology to the HAl polypeptide disclosed herein. As will be appreciated, any percentage of homology between about 70.0% and about 90.0% homology is expressly contemplated by the subject invention.
• a known HAl variable region polypeptide can have at least 70% and less than Y% homology to a challenge strain HAl variable region polypeptide, wherein Y is selected from 87.0%, 86.5%, 86.0%, 85.5% 85.0%, 84.5%, 84.0%, 83.5%, 83.0%, 82.5%, 82.0%, 81.5%, 81.0%, 80.5%, 80.0%, 79.5%, 79.0%, 78.5%, 78.0%, 77.5%, 77.0%, 76.5%, 76.0%, 75.5%, 75.0%, 74.5%, 74.0%, 73.5%, 73.0%, 72.5%, 72.0%, 71.5% or 71.0%.
• the known HAl variable region polypeptide can have between 70% and 71.0%, 71.5%, 72.0%, 72.5%, 73.0%, 73.5%, 74.0%, 74.5%, 75.0%, 75.5%, 76.0%, 76.5%, 77.0%, 77.5%, 78.0%, 78.5%, 79.0%, 79.5%, 80.0%, 80.5%, 81.0%, 81.5%, 82.0%, 82.5%, 83.0%, 83.5%, 84.0%, 84.5%, 85.0%, 85.5%, 86.0%, 86.5% or 87.0% homology to a challenge strain HAl variable region polypeptide.
• HAl polypeptides according to the subject invention can be completely or partially purified from their expression systems as described in US 2004/0268442 and WO 2004/098533, both of which are hereby incorporated by reference in their entirety.
• partially purified HAl polypeptides can exist in a composition that includes various parts or portions of the plant cell expression system in which the polypeptide was made.
• a composition comprising the purified HAl polypeptide identified herein can include plant cell components (e.g., cell walls, the cellular matrix of plant cell membranes and carbohydrates, etc.) or plant cell matrix components.
• Recombinant plant-made antigens in isolated plant homogenates may contain various plant constituents including but not limited to cell wall material, small carbohydrates, membranes, lipid components, proteins, nucleic acids as well as small biosynthetic intermediates and secondary metabolites.
• Such plant-made vaccine preparations stimulate an immune response with generally higher titers than the same antigens purified to homogeneity or prepared in conventional systems.
• the unexpected higher response in invoking seroconversion by the formulated enriched plant-cell-produced antigen, as compared to the formulated crude material or conventionally-prepared vaccine antigens is believed to be due to a unique presentation of the antigen to immune system cells as well as the improved stability of the antigen during processing, formulation and storage.
• the synergistic or adjuvant-like effect of the plant matrix or components on the activity of the antigen is a property that is unique to the plant expression platform. This improved property provides for the ability to administer lower doses of antigen and provides better protection from disease challenge.
• APCs professional antigen presenting cells
• This preferential targeting comes about through the interaction of the plant matrix and/or the plant made antigen, specifically plant glycosolated antigens, directly with mannose receptors and related C-type lectin receptors on APCs.
• APCs are able to process and present these antigens to other components of the immune system through other receptors on their surface (e.g. Class II major histocompatablity complex) and can drive both humoral and cell mediated immune responses.
• Th cells class 1 and 2 T helper (Th) cells
• APCs T helper cells
• MR or related lectin receptors Targeted interaction with these APCs through their MR or related lectin receptors is responsible for the robust cellular and humoral immune responses.
• Cellular immunity associated with CD8+ Tcell immunity has only been achieved by vaccinating with live attenuated pathogens. This immunity is an important requirement for the control of intracellular pathogens like Leshmania spp and viruses. (D. M. Pardoll, Nat Med. 1998. 4, 525-531.)
• variable regions of the myriad of known HA polypeptides will differ from the amino acid positions of the HAl region of SEQ ID NO: 1 and SEQ ID NO: 7; however, such regions can be readily discerned by those skilled in the art
• inactive HA is matured into HAl and HA2 outside the cell by one or more trypsin-like, arginine-specific endopro tease secreted by the bronchial epithelial cells.
• tryptase Clara One identified protease involved in this process is tryptase Clara.
• the extent of infection into host organism is determined by HA. Influenza viruses bud from the apical surface of polarized epithelial cells (e.g. bronchial epithelial cells) into lumen of lungs and are therefore usually pneumotropic.
• the HAl fragment binds to sialic acid-containing receptors on the cell surface, bringing about the attachment of the virus particle to the cell. It also plays a major role in the determination of host range restriction and virulence.
• the HAl fragment is a class I viral fusion protein and is responsible for penetration of the virus into the cell cytoplasm by mediating the fusion of the membrane of the endocytosed virus particle with the endosomal membrane. Low pH in endosomes induce an irreversible conformational change in HA2, releasing the fusion hydrophobic peptide.
• trimers are required to form a competent fusion pore.
• heterologous avian influenza virus or "'heterologous avian influenza virus strain(s)” are to be construed as avian influenza viruses (or strains) that express a heterologous or related HAl polypeptide that exhibits between about 70% to about 90% sequence homology to the amino acid sequence of the plant-made sub-unit vaccine antigen.
• a comparison of the amino acid sequence homology is performed. This homology analysis is performed by comparing the homology of the variable head fragment of Hl using the sequence from amino acid 17 to 323 by BLAST analysis.
• the blast analysis is performed as follows: The NCBI non redundant protein database is downloaded from the ftp site (ftp.ncbi.nih.gov/blast/db/FASTA) as nr.gz. to a computer running under either Linux (Red Hat Enterprise Linux 3.2 ) or UNIX (Solaris 8).
• the resulting analysis reports those sequences which have a statistically significant homology to the reference sequence.
• the statistical significance is dependent on the setting of parameters such as the scoring matrix used and the expected value selected as well as the cost to open and extend a gap as well as the penalties for mismatches.
• the parameters used were the default parameters of the BLAST program.
• Polypeptides comprising known HAl fragments described herein can also be fused to one or more heterologous polypeptide sequences (e.g., tags that facilitate purification of the polypeptides of the invention (see, for example, U.S. Patent No. 6,342,362, hereby incorporated by reference in its entirety; Altendorf et al. [1999- WWW, 2000] "Structure and Function of the F 0 Complex of the ATP Synthase from Escherichia CoIi " J. of Experimental
• heterologous sequences include transcribed, untranslated sequences that may play a role in transcription and mRNA processing, such as ribosome binding and stability of mRNA.
• the heterologous sequences may alternatively comprise additional coding sequences that provide additional functionalities.
• a nucleotide sequence encoding a polypeptide may be fused to a tag sequence, such as a sequence encoding a peptide that facilitates purification or detection of the fused polypeptide.
• the tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN), or in any of a number of additional, commercially available vectors.
• hexa-histidine provides for the convenient purification of the fusion protein (see, Gentz et al, 1989, Proc. Natl. Acad. Sci. USA Feb; 86(3):821-4, the disclosure of which is incorporated by reference in its entirety).
• the polypeptides of the present invention may also be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHl, CH2, CH3, any combination thereof including both entire domains and portions thereof) resulting in chimeric polypeptides.
• immunoglobulins IgA, IgE, IgG, IgM
• CHl CH2, CH3, any combination thereof including both entire domains and portions thereof
• HA polypeptides described and used herein can be fused to heterologous polypeptide sequences that have adjuvant activity (a polypeptide adjuvant).
• a polypeptide adjuvant include heat shock proteins (hsp) (see, for example, U.S. Patent No. 6,524,825, the disclosure of which is hereby incorporated by reference in its entirety).
• Adjuvants or immunostimulatory components useful in the preparation of the aforementioned compositions include, and are not limited to, aluminum salts, mineral oils,
• Mycobacterial products e.g., Freund's complete or incomplete adjuvants
• vehicles such as a mixture of the plant glycoside saponin, cholesterol and phosphatidylcholine that provides a vehicle for presentation of several copies of the protein on a cage-like structure.
• an adjuvant is a substance that accentuates, increases, moderates or enhances the immune response to an immunogen or antigen.
• Adjuvants typically enhance both the humor and cellular immune response but an increased response to either in the absence of the other qualifies to define an adjuvant.
• adjuvants and their uses are well known to immunologists and are typically employed to enhance the immune response when doses of immunogen are limited, when the immunogen is poorly immunogenic, or when the route of administration is sub-optimal.
• the term 'adjuvating amount' is that quantity of adjuvant capable of enhancing the immune response to a given immunogen or antigen.
• the mass that equals an 'adjuvating amount' will vary and is dependant on a variety of factors including, but not limited to, the characteristics of the immunogen, the quantity of immunogen administered, the host species, the route of administration, and the protocol for administering the immunogen.
• the 'adjuvating amount' can readily be quantified by routine experimentation given a particular set of circumstances.
• Vaccination and vaccinating is defined as a means for providing protection against a pathogen by inoculating a host with an immunogenic preparation, an immunoprotcctive particle, or an immunogenic preparation of a pathogenic agent, or a non-virulent form or part thereof, such that the host immune system is stimulated and prevents or attenuates subsequent unwanted pathology associated with the host reactions to subsequent exposures of the pathogen.
• vaccination with the compositions of the invention results in a reduction in mortality or death and/or viral shedding from the respiratory tract.
• Administering or administer is defined as the introduction of a substance into the body of an animal, including a human, and includes oral, nasal, ocular, rectal, vaginal and parenteral routes.
• Compositions may be administered individually or in combination with other therapeutic agents via any route of administration, including but not limited to subcutaneous (SQ), intramuscular (IM), intravenous (IV), intraperitoneal (IP), intradermal (ID), via the nasal, ocular or oral mucosa (IN), or orally.
• the subject invention also provides methods for using known isolated, recombinant, and/or purified polynucleotide sequences comprising: a) a polynucleotide sequence encoding a known HAl variable region polypeptide that has between about 70% to about 90% homology to a challenge strain HAl variable region polypeptide; b) a polynucleotide that is complementary to the polynucleotides set forth in (a); c) a genetic construct comprising a polynucleotide sequence as set forth in (a) or
• nucleotide sequence can be used interchangeably and are understood to mean, according to the present invention, either a double-stranded DNA, a single-stranded DNA or products of transcription of the said DNAs (e.g. , RNA molecules). It should also be understood that the present invention does not relate to polynucleotide sequences in their natural environment or natural state.
• nucleic acid, polynucleotide, or nucleotide sequences of the invention can be isolated, purified (or partially purified), by separation methods including, but not limited to, ion-exchange chromatography, molecular size exclusion chromatography, or by genetic engineering methods such as amplification, subtractive hybridization, cloning, subcloning or chemical synthesis, or combinations of these genetic engineering methods.
• Both protein and nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art and available at publicly accessible databases (e.g., see world wide web sites: ebi.ac.uk/fasta33/index.html (European Biotechnology Institute); or ncbi.nlm.nih.gov/BLAST/ (National Center for Biotechnology Information).
• sequence comparison algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. ScL USA, 85(8):2444-2448; Altschul et al, 1990, J. MoI.
• a “complementary" polynucleotide sequence generally refers to a sequence arising from the hydrogen bonding between a particular purine and a particular pyrimidine in double-stranded nucleic acid molecules (DNA-DNA, DNA-RNA, or RNA- RNA). The major specific pairings are guanine with cytosine and adenine with thymine or uracil.
• a “complementary" polynucleotide sequence may also be referred to as an "antisense” polynucleotide sequence or an "antisense sequence”.
• Sequence homology and sequence identity can also be determined by hybridization studies under high stringency, intermediate stringency, and/or low stringency. Various degrees of stringency of hybridization can be employed. The more severe the conditions are, the greater the complementarity required for duplex formation. Severity of conditions can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under low, intermediate, or high stringency conditions by techniques well known in the art, as described, for example, in Keller, G. H., M.M. Manak (1987) DNA Probes, Stockton Press, New York, NY., pp. 169-170.
• hybridization of immobilized DNA on Southern blots with 32 P-labeled gene-specific probes can be performed by standard methods (Maniatis et ah, 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In general, hybridization and subsequent washes can be carried out under intermediate to high stringency conditions that allow for detection of target sequences with homology to the exemplified polynucleotide sequence.
• hybridization can be carried out overnight at 20-25° C below the melting temperature (T 111 ) of the DNA hybrid in 6X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz et al, 1983, Methods of Enzymology, R. Wu, L. Grossman and K. Moldave, eds. Academic Press, New York, 100:266-285).
• Tm 81.5°C+16.6 Log[Na + ]+0.41(%G+C)-0.61(%formamide)-600/length of duplex in base pairs.
• Washes are typically carried out as follows: (1) twice at room temperature for 15 minutes in IX SSPE, 0.1% SDS (low stringency wash);
• T m melting temperature
• Washes can be carried out as follows: (1) twice at room temperature for 15 minutes IX SSPE, 0.1% SDS (low stringency wash);
• salt and/or temperature can be altered to change stringency.
• a labeled DNA fragment >70 or so bases in length the following conditions can be used:
• 0.1X SSPE 65°C.
• Filters are hybridized for 48 h at 65°C, the preferred hybridization temperature, in pre-hybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 x 10 6 cpm of 32 P-labeled probe.
• the hybridization step can be performed at 65°C in the presence of SSC buffer, IX SSC corresponding to 0.15M NaCl and 0.05 M Na citrate.
• filter washes can be done at 37°C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1X SSC at 50 0 C for 45 min.
• filter washes can be performed in a solution containing 2X SSC and 0.1% SDS, or 0.5X SSC and 0.1% SDS, or 0.1X SSC and 0.1% SDS at 68°C for 15 minute intervals.
• the hybridized probes are detectable by autoradiography.
• Other conditions of high stringency which may be used are well known in the art and as cited in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N. Y., pp. 9.47-9.57; and Ausubel et al, 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y. are incorporated herein in their entirety.
• the probe sequences of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.
• Bal3 l exonuclease can be conveniently used for time-controlled limited digestion of DNA (commonly referred to as "erase-a-base” procedures). See, for example, Maniatis et al , 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Wei et al, 1983, J. Biol. Chem., 258:13006-13512.
• the subject invention also provides genetic constructs comprising: a) a polynucleotide sequence encoding a polypeptide comprising (or consisting of) a known HAl variable region polypeptide that has between about 70% to about 90% homology to a challenge strain HAl variable region polypeptide.
• Genetic constructs of the subject invention will also contain additional regulatory elements such as promoters and enhancers and, optionally, selectable markers.
• vectors or expression cassettes containing genetic constructs as set forth herein or polynucleotides encoding the polypeptides, set forth supra, operably linked to regulatory elements.
• the vectors and expression cassettes may contain additional transcriptional control sequences as well.
• the vectors and expression cassettes may further comprise selectable markers.
• the expression cassette may contain at least one additional gene, operably linked to control elements, to be co-transformed into the organism.
• the additional gene(s) and control element(s) can be provided on multiple expression cassettes.
• Such expression cassettes are provided with a plurality of restriction sites for insertion of the sequences of the invention to be under the transcriptional regulation of the regulatory regions.
• the expression cassette(s) may additionally contain selectable marker genes operably linked to control elements.
• the expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of the invention, and a transcriptional and translational termination regions.
• the transcriptional initiation region, the promoter may be native or analogous, or foreign or heterologous, to the host cell. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence.
• “foreign” is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced.
• a chimeric gene comprises a coding sequence operably linked to a transcriptional initiation region that is heterologous to the coding sequence.
• Vectors of this invention can also comprise elements necessary to allow the expression and/or the secretion of the said nucleotide sequences in a given host cell.
• the vector can contain a promoter, signals for initiation and for termination of translation, as well as appropriate regions for regulation of transcription.
• the vectors can be stably maintained in the host cell and can, optionally, contain signal sequences directing the secretion of translated protein. These different elements are chosen according to the host cell used.
• Vectors can integrate into the host genome or, optionally, be autonomously-replicating vectors.
• the subject invention also provides for the expression of a polypeptide encoded by a polynucleotide sequence disclosed herein comprising the culture of a host cell transformed with a polynucleotide of the subject invention under conditions that allow for the expression of the polypeptide and, optionally, recovering the expressed polypeptide.
• the disclosed polynucleotide sequences can also be regulated by a second nucleic acid sequence so that the protein or peptide is expressed in a host transformed with the recombinant DNA molecule.
• expression of a protein or peptide may be controlled by any promoter/enhancer element known in the art.
• Promoters which may be used to control expression include, but are not limited to, the CMV-IE promoter, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al , 1980, Cell, 22:787-797), the herpes simplex thymidine kinase promoter (Wagner et al, 1981, Proc. Natl. Acad. Sci.
• promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al, 1984, Nature, 310:115-120); promoter elements from yeast or fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, and/or the alkaline phosphatase promoter.
• the vectors according to the invention are, for example, vectors of plasmid or viral origin.
• a vector is used that comprises a promoter operably linked to a protein or peptide-encoding nucleic acid sequence contained within the disclosed polynucleotide sequences, one or more origins of replication, and, optionally, one or more selectable markers (e.g. , an antibiotic resistance gene).
• Expression vectors comprise regulatory sequences that control gene expression, including gene expression in a desired host cell.
• Exemplary vectors for the expression of the polypeptides of the invention include the pET-type plasmid vectors (Promega) or pBAD plasmid vectors (Invitrogen) or those provided in the examples below.
• the vectors according to the invention are useful for transforming host cells so as to clone or express the polynucleotide sequences of the invention.
• Polypeptides useful in the production of the aforementioned compositions or immunization protocols can be derived or obtained from a transgenic plant cell that has been genetically engineered to express a polypeptide comprising (or consisting oi) known HAl variable region polypeptide that has between about 70% to about 90% homology to a challenge strain HAl variable region polypeptide.
• Transgenic plant is herein defined as a plant cell culture, plant cell line, plant tissue culture, lower plant, bryophyte, monocot plant, dicot plant, or progeny thereof derived from a transformed plant cell or protoplast, wherein the genome of the transformed plant contains foreign DNA, introduced by laboratory techniques, not originally present in a native, non- transgenic plant cell of the same species.
• the terms "transgenic plant” and “transformed plant” have sometimes been used in the art as synonymous terms to define a plant whose DNA contains an exogenous DNA molecule.
• Transgenic plants and transforming plants further includes methods and plants whose genome has not been stably transformed or which transiently expresses a recombinant viral vector such as described in US Patent Numbers 5,550,360; 5,846,795; 4,885,248; 5,173,410; 5,602,242; 5,627,060; 5,804,439; WO 05/049839; WO 03/020938; WO 02/101006; WO 02/101060; WO 02/096192; WO 02/088369; WO 02/08386; WO 02/29068; WO 02/46440; and WO 02/068664.
• a recombinant viral vector such as described in US Patent Numbers 5,550,360; 5,846,795; 4,885,248; 5,173,410; 5,602,242; 5,627,060; 5,804,439; WO 05/049839; WO 03/020938; WO 02/101006; WO 02/101060;
• nucleotide sequence in the case of DNA or RNA
• amino acid sequence in the case of a protein or polypeptide
• Any functional or structural differences between sequences having substantial sequence homology will be de minimis; that is they will not affect the ability of the sequence to function as indicated in the present application.
• Sequences that have substantial sequence homology with the sequences disclosed herein are usually variants of the disclosed sequence, such as mutations, but may also be synthetic sequences.
• the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
• Adapters or linkers may be employed for joining the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
• cloning is employed, so as to amplify a vector containing the promoter/gene of interest for subsequent introduction into the desired host cells.
• a wide variety of cloning vectors are available, where the cloning vector includes a replication system functional in E. coli and a marker which allows for selection of the transformed cells.
• Illustrative vectors include pBR322, pUC series, pACYC184, Bluescript series (Stratagenc) etc.
• the sequence may be inserted into the vector at an appropriate restriction site(s), the resulting plasmid used to transform the E. coli host (e.g. , E. coli strains HBlOl, JMlOl and DH5 ⁇ ), the E.
• DNA sequence to be used in the final construct may be restricted and joined to the next sequence, where each of the partial constructs may be cloned in the same or different plasmids.
• Vectors are available or can be readily prepared for transformation of plant cells.
• plasmid or viral vectors should contain all the DNA control sequences necessary for both maintenance and expression of a heterologous DNA sequence in a given host.
• control sequences generally include a leader sequence and a DNA sequence coding for translation start-signal codon, a translation terminator codon, and a DNA sequence coding for a 3' UTR signal controlling messenger RNA processing. Selection of appropriate elements to optimize expression in any particular species is a matter of ordinary skill in the art utilizing the teachings of this disclosure.
• the vectors should desirably have a marker gene that is capable of providing a phenotypical property which allows for identification of host cells containing the vector.
• the activity of the foreign coding sequence inserted into plant cells is dependent upon the influence of endogenous plant DNA adjacent the insert.
• the insertion of heterologous genes appears to be random using any transformation technique; however, technology currently exists for producing plants with site specific recombination of DNA into plant cells (see WO 91/09957). Any method or combination of methods resulting in the expression of the desired sequence or sequences under the control of the promoter is acceptable.
• the present invention is not limited to any particular method for transforming plant cells.
• Technology for introducing DNA into plant cells is well-known to those of skill in the art.
• Four basic methods for delivering foreign DNA into plant cells have been described. Chemical methods (Graham and van der Eb, Virology, 54(02):536-539, 1973; Zatloukal, Wagner, Cotten, Phillips, Plank, Steinlein, Curiel, Birnstiel, Ann. N Y. Acad. Sci., 660:136- 153, 1992); Physical methods including microinjection (Capecchi, Cell, 1980, 22(2):479- 488), electroporation (Wong and Neumann, 1982, Biochim. Biophys. Res. Commun.,
• Plant cell wall-degrading enzymes such as pectin-degrading enzymes
• pectin-degrading enzymes are used to render the recipient cells more susceptible to transformation by electroporation than untreated cells.
• friable tissues such as a suspension culture of cells, or embryogenic callus, or immature embryos or other organized tissues directly. It is generally necessary to partially degrade the cell walls of the target plant material to pectin-degrading enzymes or mechanically wounding in a controlled manner. Such treated plant material is ready to receive foreign DNA by electroporation.
• microprojectile bombardment Another method for delivering foreign transforming DNA to plant cells is by microprojectile bombardment.
• microparticles are coated with foreign DNA and delivered into cells by a propelling force.
• micro particles are typically made of tungsten, gold, platinum, and similar metals.
• An advantage of microprojectile bombardment is that neither the isolation of protoplasts (Cristou et al, 1988, Plant Physiol, 87:671 -674,) nor the susceptibility to Agrobacterium infection is required.
• An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen onto a filter surface covered with corn cells cultured in suspension.
• the screen disperses the particles so that they are not delivered to the recipient cells in large aggregates.
• cells in suspension are preferably concentrated on filters or solid culture medium.
• immature embryos or other target cells may be arranged on solid culture medium.
• the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.
• bombardment transformation one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the microprojectiles.
• Bio factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids.
• Agrobacterium-mediated transfer is a widely applicable system for introducing foreign DNA into plant cells because the DNA can be introduced into whole plant tissues, eliminating the need to regenerate an intact plant from a protoplast.
• the use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described in Fraley et al, 1985, Biotechnology, 3:629; Rogers et al, 1987, Meth. in Enzymol, 153:253-277.
• the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements.
• the region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome as described in Spielmann et al, 1986, MoI. Gen. Genet. , 205:34; Jorgensen el al , 1987, MoI Gen. Genet. , 207:471.
• Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations.
• recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various proteins or polypeptides.
• Convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes are suitable for present purposes.
• Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations.
• Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus el al, 1985, MoI. Gen. Genet. , 199: 183; Marcotte et al, 1988, Nature, 335:454).
• Application of these systems to different plant species depends on the ability to regenerate the particular species from protoplasts. Once the plant cells have been transformed, selected and checked for antigen expression, it is possible in some cases to regenerate whole fertile plants. This will greatly depend on the plant species chosen. Methods for regenerating numerous plant species have been reported in the literature and are well known to the skilled artisan.
• plant cell lines that can be cultured and scaled- up rapidly by avoiding the generally lengthy regeneration step.
• plant cell cultures avoids open field production and greatly reduces the chances of gene escape and food contamination.
• Tobacco suspension cell cultures such NT-I and BY-2 (An, G., 1985, Plant Physiol., 79:568-570) are preferred because these lines are particularly suited for handling in culture, are readily transformed, produce stably integrated events and are amenable to cryopreservation.
• NT-I The tobacco suspension cell line, NT-I, is suitable for the practice of the present invention.
• NT-I cells were originally developed from Nicotiana tabacum L.cv. bright yellow 2.
• the NT-I cell line is widely used and readily available; though, any tobacco suspension cell line is consistent with the practice of the invention.
• NT- 1 cells suitable for use in the examples below are available from the American Type Culture Collection under accession number ATCC No. 74840. See also U.S. Patent No 6,140,075, herein incorporated by reference in its entirety.
• the buffer is an aqueous isotonic buffered salt solution at or near a neutral pH value that does not contain harsh detergents that can be used to solubilze membranes.
• Preferred buffers include Dulbecco's Phosphate Buffered Saline and PBS containing 1 mM EDTA.
• cells can be disrupted by sonication.
• the washed cells are placed in buffer in a range of about 0.01 gm/ml to about 5.0 gm/ml, preferably in a range of about 0.1 gm/ml to about 0.5 gm/ml (washed wet weight cells per volume of buffer).
• Many commercially available sonication instruments are consistent with the invention and sonication times range from about 5 to about 20 seconds, preferably about 15 to about 20 seconds.
• the resulting may range in size from a few microns to several hundred microns and expose the HAl polypeptide or immunogenic fragments thereof.
• isolated or “biologically pure” refer to material that is substantially or essentially free from components which normally accompany the material as it is found in its native state.
• isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.
• AIV Avian Influenza Virus
• NT-I plant cells transformed to express H5 antigen were lysed in 100 g aliquots (using BiospecTM bead beaters on ice) in 200 mM Tris pH 8, 5 mM EDTA pH 8, 2 mM dithiothtreitol and 2% Na deoxycholate (Doc). The lysate was stirred overnight at 4 0 C to aide H5 extraction, then clarified by centrifugation followed by filtration (0.45 ⁇ m) then further purified as described below. A portion was quantified for H5, lyophilized, and stored at -20° C. Mab Chromatography.
• Purified bulk H5 antigen was prepared from lysed transformed NT-I plant cells as follows.
• the plant cell lysate prepared above was diluted 4 to 1 with Milli-QTM water, to reduce the Doc concentration to 0.5%.
• the diluted plant cell lysate was passed over an approximately 125 ml H5 Mab affinity column equilibrated with 50 mM Tris pLI 8.
• the column was washed to baseline with 50 mM Tris pLI 8 and bound protein was eluted with 50 mM Tris pH 8, 2M NaSCN.
• the eluted H5 protein was dialyzed against two large volumes of 10.5 mM ammonium bicarbonate (a volatile buffer) prior to lyophilization.
• Antigen Preparation 1.
• mice Lyophilized transformed NT-I cell lysate cake containing approximately 129.5 ⁇ g of H5 antigen was rehydrated using 3.5 ml of sterile water resulting is a plant cell lysate stock solution having concentration of 37 ⁇ g/ml bulk H5 antigen stock solution.
• the bulk H5 antigen stock solution was clarified by centrifugation for 20 minutes at 6000 X G, then sterile filtered through a 0.22 micron filter. This sterile filtered bulk H5 antigen was then held for final experimental vaccine assembly.
• Purified Vaccine Lyophilized vials of Mab chromatography purified H5 antigen were use to prepare purified vaccine. Each of the lyophilized vials contained 500 ⁇ g of H5 antigen. Each vial was rehydrated with 5 ml of sterile water to yield a final concentration of
• NT-I Blank Control was prepared using the same procedure as used for the experimental vaccine (1) above except that untransformed NT-I plant cells were used instead of transformed NT-I cells expressing H5 antigen. Formulation.
• Each of the final vaccines was assembled to a final H5 concentration of 26.7 ng/ml using the following procedure.
• Experimental vaccine antigen, purified vaccine antigen or NT-I blank control lysate was added to a sterile 50 ml conical bottom centrifuge tube.
• the required quantity of sterile filtered Quil A stock (50 mg/ml in sterile water, Brenntag, DK) solution was added to the tube to a final concentration of 40 ug/dose and mixed for one minute using a sterilized rotor stator type homogenizer.
• the required quantity of cholesterol stock solution (18 mg/ml in EtOH) was added to the tube to a final concentration of 10 ug/dose and mixed for one minute using the sterile rotor stator type homogenizer.
• the required volume of a previously prepared and autoclaved mixture of lecithin and acrylic polymer (3:2 lethicinxarbopol) was added to a final concentration of 1 mg/dose and homogenized for one minute.
• the required quantity of sterile water was added to the tube and mixed.
• the assembled vaccine was then aseptically transferred into sterilized serum vials, sealed and labeled.
• the vials of assembled vaccines were stored at 4 0 C until they were shipped as needed to the clinical trial site. Vaccination.
• mice Sixty-five B ALB/c mice (females; 5-6 wks of age) were assigned to Treatment Group 1, Treatment Group 2, Treatment Group 3 or Treatment Group 4 as described in Table 1. On Study Day 0, 14 and 21, mice were vaccinated with a 150ul dose of the prescribed treatment as described in Table 1. Vaccinations were administered subcutaneously. Mice in Group 4 were not vaccinated. Post vaccination analysis.
• mice On day 35, all mice were moved to an ABSL3 facility and acclimated to the new facility for 1 week.
• 5 mice from each of Group 1 , Group 2 and Group 3 were randomly selected and exsanguinated under sedation. Blood was processed into scrum and stored at ⁇ -2O 0 C for serologic analysis. Additionally, on Day 42, the remaining 15 mice from each of Group 1, Group 2 and Group 3 were challenged with 50 uL of approximately 1.5x10 3 TCID 50 Avian Influenza Virus A/Vietnam/I 203/04.
• the 5 mice in Group 4 were mock challenged with Phosphate Buffered Saline (PBS). All challenges were performed under anesthesia with Ketamine.
• PBS Phosphate Buffered Saline
• mice All of the mice in Group 4 (5 mice) were sacrificed and the lungs and brains were removed. Lungs and brains were homogenized and tested for viable virus by TCID 50 quantification.
• mice in Group 1 , Group 2 and Group 3 were monitored for clinical signs of disease until the end of the study Day 56. Serologic Assays.
• Hemagglutination Inhibition (HAI) serologic assay was performed on serum samples collected on Day 42 against inactivated Turkey Wisconsin 68 Strain of AIV prepared in allantoic fluid. The inactivated virus was diluted to yield between 8 to 16 hemagglutination (HA) units per 50ul. The serum samples were two-fold serially diluted in PBS. To the diluted serum samples was added an equal volume of diluted virus. The serum-virus mixture was incubated at room temperature for 60 minutes. A 1% solution of chicken red blood cells (cRBC) was then added to the serum-virus mixture and incubated at 2-7 0 C for 24 hours. The plates were then visually inspected for hemagglutination (positive result) or pelleted cRBC (negative result). The HAI titer represents the inverse dilution of the sera that is able to inhibit the ability of the virus to cause hemagglutination of the cRBC. Serum Neutralization Assay.
• ND 50 Neutralizing dose 50 titers
• EMEM Eagle's Minimum Essential Medium
• Avian Influenza virus Vietnam/1203/04 was added to the diluted serum samples and this mixture was incubated at 37 0 C for 1 hour.
• HBSS Hank's Balanced Salt Solution
• the plates were then incubated at approximately 37 0 C and 5% CO 2 in a humidified incubator for 96 ⁇ 6 hours. Plates were graded for cytopathological effects (CPE) with the aid of a microscope. The ND 50 was reported as the dilution that results in the absence of CPE in 50% of the wells inoculated and was calculated using the Spearman Karber method. Viable Virus Titration from Lung and Brain.
• the brain and lungs from 5 mice in each of Group 1 , 2 and 3 were removed and homogenized in CMF-PBS. The homogenates were aliquoted to microcentrifuge tubes and stored at ⁇ -7O 0 C. Brain and lung homogenate samples were tested for viable virus using TCID50s. Lungs and brain were removed and frozen intact at ⁇ -70°C in 1 ml of phosphate buffered saline (PBS) prepared with 1% antibiotic (Penicillin and Streptomycin). After thawing, lungs and brain were homogenized and the samples were tested for viable virus by tissue culture infectious dose 50 (TCID 50 S). Briefly, serial 5-fold dilutions of each sample were prepared using EMEM.
• PBS phosphate buffered saline
• TCID 50 S tissue culture infectious dose 50
• a dilution series was also prepared for a positive control sample (PC, a positive sample with a known TCID50 titer) and a negative control sample (NC, known to be na ⁇ ve of virus).
• PC positive control sample
• NC negative control sample
• HBSS Hank's Balanced Salt Solution
• CC cell culture control
• Both the positive and negative control plates included a minimum of 5 CC wells each.
• the plates were then incubated at approximately 37 0 C and 5% CO 2 in a humidified incubator for 96 ⁇ 6 hours. Plates were graded for cytopathological effects (CPE) by a technician with the aid of a microscope. In order for the assays to be considered valid there could be no contamination and at least 5 CC wells on each plate needed to be healthy confluent (>80%) monolayers.
• the TCID50 was the dilution that resulted in CPE in 50% of the wells inoculated and was calculated using the Spearman Karber method. Results.
• mice from each of Group 1, Group 2 and Group 3 were bled for serologic testing.
• the serology results (Hemagglutination Inhibition and Serum Neutralization) are presented in Table 2.
• Five out of the five mice from Group 1 (Experimental vaccine) developed antibody against Turkey Wisconsin 68 (homologous to the H5 antigen in the vaccine) as evidenced by Hemmagglutination Inhibition serology (HAI).
• the Geometric Mean Titer (GMT) of Group 1 was 388.
• Three out of the five mice from Group 2 (Purified vaccine) developed HAI titers.
• the GMT of Group 2 was 60.7.
• mice from Group 3 None of the five mice from Group 3 (NT-I blank control) developed HAI antibodies to Turkey Wisconsin 68.
• Three out of the five mice from Group 1 developed antibody against Vietnam/1203/04 (heterologous to the HA in the vaccine) as evidenced by Serum neutralization serology (SN).
• the Geometric Mean Titer (GMT) of Group 1 was 34.0.
• Viable avian influenza virus was isolated from the lungs and brains of 5 mice from each of Group 1, 2, 3 and 4 on Study Day 45 (3 days post challenge). All five mice in Group 4 were sacrificed as discussed above and are therefore not presented Table 4.
• Virus isolation results are presented in Table 3. In all four groups, no viable virus could be isolated from the brain tissue. In Group 1 (Experimental vaccine), 1 of the 5 mice had sterile lung tissue (no virus could be isolated). The GMT of the viable virus from the lung of Group 1 mice was 3.3 IxIO 3 TCIDso/mL. In Group 2 mice (Purified vaccine), viable virus could be isolated from 5 of the 5 mice. The GMT of the viable virus from the lungs of Group 2 mice was 2.19xlO 4 TCID 5 o/ml. Mice in Group 3 (NT-I blank control) had a greater than 1 log higher viable avian influenza virus in lung tissue than Group 1 mice. In Group 3, five out of five mice had positive isolations and the GMT of the group was 8.72xlO 4 TCID 50 /ml. Mice in
• Group 4 (non-vaccinated and challenge with PBS only) had no virus isolation from lung tissues.
• mice in each of Group 1, Group 2 and Group 3 were clinically monitored from the day of challenge (Day 42) until 2 wks post challenge (Day 56).
• Table 4 provides the Day of Death Post Challenge for Group 1, Group 2 and Group 3.

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