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Definitions

• Embodiments of the present invention report methods, compositions and uses for generating and expressing constructs having enterobacterial-associated peptides.
• enterobacterial-associated peptides include, but are not limited to, plague- associated peptides.
• the present invention discloses making and using constructs including, but not limited to, attenuated or modified vaccinia virus vectors expressing enterobacterial-associated peptides.
• Vaccines to protect against viral infections have been effectively used to reduce the incidence of human disease.
• One of the most successful technologies for viral vaccines is to immunize animals or humans with a weakened or attenuated strain of the virus (a "live, attenuated virus"). Due to limited replication after immunization, the attenuated strain does not cause disease. However, the limited viral replication is sufficient to express the full repertoire of viral antigens and generates potent and long-lasting immune responses to the virus. Thus, upon subsequent exposure to a pathogenic strain of the virus, the immunized individual is protected from disease.
• live, attenuated viral vaccines are among the most successful vaccines used in public health.
• Yersinia is a genus of bacteria in the family of Enterobacteriaceae. Yersinia are facultative anaerobes. Some members of Yersinia are pathogenic in humans. Often, rodents are the natural reservoirs of Yersinia; less frequently other mammals may serve as a host to these bacteria. Infection can occur either through arthopod bite, exposure to blood, aerosol transmission (e.g. Y. pestis), or by, for example, consumption of food products (e.g.
• Yersinia family is rather large, but only two have been linked to water-borne outbreaks of disease, Y.
• Yersinia species are found all over the world in animal reservoirs (e.g., rodent reservoirs for Y. pestis), isolated in well-water, water treatment plants, rivers and lakes.
• Yersinia pestis also referred to as Pasteurella pestis
• Pasteurella pestis is the most famous member of the Yersinia species and is the causative organism of plague.
• Embodiments of the present invention generally relate to methods, compositions and uses for expressing enterobacterial-associated peptides.
• enterobacterial-associated peptides include, but are not limited to, plague-associated peptides.
• Certain embodiments report making and using constructs of the present invention for treating or protecting a subject having been exposed or likely to be exposed to an Enterobacteria.
• constructs may include, but are not limited to, attenuated or modified vaccinia virus vectors expressing enterobacterial-associated peptides.
• compositions having constructs including, but not limited to, attenuated or modified vaccinia virus vectors expressing Yersinia spp-associated peptides, for example, in order to induce an immune response in a subject against the Yersinia spp.
• compositions having constructs with antigens or peptides associated with Yersinia spp. including, but not limited to, Fl, V, truncated V or YopD polypeptides, or combinations thereof.
• Other embodiments may include one or more low- calcium response (V) antigens with a C-terminal truncation.
• C-terminal truncation of low-calcium response (V) antigens may include, but are not limited to, a truncation that suppresses expression of a pro-inflammatory cytokine, truncations that remove immunosuppressive sequences, truncations that are less immunosuppressive than corresponding full-length or unmodified LcrV protein, deletion is of up to 163 contiguous residues of LcrV, internal deletions, internal deletion up to 90 contiguous residues, internal deletion extending into the region spanning amino acids 240 to 325 of LcrV protein, C-terminal deletions of up to 50 contiguous residues, an LcrV protein of at least 275 residues in length or combinations thereof.
• Some embodiments report vaccine compositions capable of reducing or preventing infection in a subject caused by exposure to enterobacteria (e.g. Yersinia spp), including, for example, protection from encapsulated and unencapsulated forms of the organism.
• enterobacteria e.g. Yersinia spp
• constructs of use as vaccine compositions can include one or more secretory signal sequences alone or in combination with one or more translation control region sequences.
• a secretory signal sequence can be one or more signal sequences functional in mammalian cells.
• a secretory signal sequence includes, but is not limited to, tissue plasminogen activator (IP A) leader sequence, the co-factor leader sequence, the pre-proinsulin leader sequence, the invertase ieader sequence, the immunoglobulin A leader sequence, the ovalbumin leader sequence, and the P-globin leader sequence or other proleader sequences known in the art.
• IP A tissue plasminogen activator
• Vaccine compositions disclosed herein can be administered by any method known in the art.
• a vaccine can be administered intradermally,
• compositions can be administered by time-release or other formulations as assessed by a health provider.
• kits for making or using compositions disclosed may include constructs having a modified vaccinia viral vector and one or more enterobacterial-derived antigen.
• kits can include methods for making a construct contemplated herein.
• Fig. 1 represents an exemplary construct of modified vaccinia virus and a
• Figs. 2A-2E represent exemplary electrophoretic separations and analyses of expression patterns from clonal recombinant viruses.
• Fig. 3A represents a histogram of an immune response in mice to vaccine of construct compositions of some embodiments disclosed herein, pre-boost and pre-challenge.
• Fig. 3B represents an exemplary histogram of an immune response in mice to vaccine of construct compositions of some embodiments disclosed herein, pre-boost and pre-challenge.
• Fig. 4A represents an exemplary plot illustrating survival rates of mice immunized with an exemplary enterobacterial-directed vaccines or control formulations following intranasal challenge with enterobacterium.
• Fig. 4B represents an exemplary plot illustrating survival rates of mice immunized with an exemplary enterobacterial-directed vaccines or control formulations following intraperitoneal challenge with enterobacterium.
• Fig. 5 represents an exemplary assessment of safety of enterobacterial-directed vaccines inoculated in immunocompromised mice.
• Fig. 6 represents an exemplary plot of antibody titers measured and plotted after IM (intramuscular) or ID (intradermal) pre and post boosts of vaccines of some
• Fig. 7 represents a compilation of construct data as Table 3.
• vessel can include, but is not limited to, test tube, mini- or micro- fuge tube, channel, vial, microtiter plate or container.
• subject or “subjects” may include, but are not limited to, mammals such as humans or mammals, domesticated or wild, for example dogs, cats, other household pets (e.g. hamster, guinea pig, mouse, rat), ferrets, rabbits, pigs, horses, cattle, prairie dogs, wild rodents, or zoo animals.
• mammals such as humans or mammals, domesticated or wild, for example dogs, cats, other household pets (e.g. hamster, guinea pig, mouse, rat), ferrets, rabbits, pigs, horses, cattle, prairie dogs, wild rodents, or zoo animals.
• Attenuated virus can mean a virus that demonstrates reduced or no clinical signs of disease when administered to a subject such as a mammal (e.g. human or an animal).
• MSC can mean multiple cloning site.
• dSP can mean divergent vaccinia promoter.
• MVA can mean modified vaccinia Ankara.
• EMCV can mean encephalomyocarditis virus.
• IRES can mean internal ribosome entry site from
• IRES(A7) can mean IRES from encephalomyocarditis virus with 7 adenosine residues in bifurcation loop; source- pCITE-1.
• IRES(A6) can mean IRES from encephalomyocarditis virus mutated to have 6 adenosine residues in bifuraction loop.
• pDIIIgfp can mean MVA del III gfp marker transfer plasmid.
• pi* can mean transfer vector plasmids.
• tPA can mean secretory signal from tissue plaminogen activator.
• sirolid/1 can mean synthetic optimized early late poxvirus promoter.
• H6 can mean the vaccinia gene H6 early/late native poxvirus promoter.
• Fr can mean Y. pestis capsular protein.
• V can mean Y. pestis virulence factor LcrV.
• V307 or “V307” can mean C-terminal LcrV truncation of amino acids 308-326 of Y. pestis. V protein.
• YopD can mean Y. pestis outer protein D.
• del III can mean modified vaccinia Ankara deletion region III.
• GFP can mean enhanced green fluorescent protein
• CEF can mean chicken embryo fibroblasts.
• a vaccine composition may include a recombinant modified vaccinia Ankara (MVA) vector associated with one or more enterobacterial-associated peptides.
• a vaccine composition may include a recombinant modified vaccinia Ankara (MVA) vector associated with one or more enterobacterial-associated peptides where at least one of the enterobacterial-associated peptides includes one or more Fera ' wz ' a-associated peptides.
• one vaccine composition can include recombinant modified vaccinia Ankara (MVA) vector expressing Yersinia pestis antigens.
• MVA modified vaccinia Ankara
• an MVA construct expressing one or more Yersinia pestis-associated antigens may be generated (e.g. V, Fl, LcrV protein or mutants or fragments thereof).
• Plague is primarily a disease of wild rodents transmitted by fleas, but it can also afflict humans, domestic pets, and wild animals. The disease has threatened human and animal populations throughout history. In recent years, it has caused severe epidemics in many parts of the world, resulting in human deaths and severe economic losses. Yersinia pestis is widespread throughout wild rodent populations in the southeastern United States, Southeast Asia, Eastern Europe, central and southern Africa, as well as South America, and human populations in these areas are highly susceptible. In the United States, plague has spread throughout the Western states, causing significant mortality in squirrels, wild mice, and prairie dogs. Domestic cats are also susceptible to Y. pestis infections, and they have been identified as the source of infection in many recent cases of plague in humans.
• Y. pestis is considered a potential candidate for biowarfare. It is believed that Mongols first used plague as a crude bioweapon in the 1300s during the siege of Kaffa as plague victims were launched over the wall of the city. During World War II, a secret branch of the Japanese army is reported to have dropped plague-infected fleas over China causing several outbreaks in humans. In following years, research programs in biological weapons in the US and Soviet Union successfully developed methods for aerosolization of plague. More recently, evidence that scientists from the former Soviet Union worked with plague serves notice that Y. pestis is still considered a feasible bioweapon. In 1970, the World Health Organization estimated that intentional release of 50 kg of the plague bacterium, Y. pestis, over a city of 5 million people could result in as many as 150,000 clinical cases and 36,000 deaths. A 2001 U.S. Congressional Office of
• compositions having constructs directed against Yersini spp may be directed to the prevention or reduced incidence of plague.
• Some embodiments of the present invention may include secreted V antigen which plays for example, a role in delivery of other Yersinia outer proteins (Yops) and stimulates secretion of IL-10 (an antiinflammatory cytokine) associated with the suppression of TNF-a and IFN- ⁇ , a hallmark of plague.
• Yops Yersinia outer proteins
• IL-10 an antiinflammatory cytokine
• Antigens of use for vaccines against Yersinia spp infection may include LcrV (low- calcium- response V or V antigen) or other plasmid-encoded, virulence proteins (e.g. Yops, or Yersinia outer proteins) which are essential for survival in mammalian hosts.
• Yops and LcrV are secreted by a type III mechanism (Ysc), and Yops are unidirectionally targeted into the cytosol of associated eukaryotic cells in a tissue culture infection model.
• LcrV is required for Yops targeting, and recent findings have revealed that it can localize to the bacterial surface.
• some of the coding sequences of use in some embodiments of the present invention can include coding sequences for Y. pestis antigens capsular protein Fl, full length virulence factor LcrV (V), a truncated form of LcrV (V307), other carboxyterminal truncations of LcrV and effector protein YopD.
• V antigens may include one or more low-calcium response (V) antigens with a C-terminal truncation.
• C-terminal truncation of low-calcium response (V) antigens may include, but are not limited to, a truncation that suppresses expression of a pro-inflammatory cytokine, truncations that remove
• deletion is of up to 163 contiguous residues of LcrV, internal deletions, internal deletion up to 90 contiguous residues, internal deletion extending into the region spanning amino acids 240 to 325 of LcrV protein, C- terminal deletions of up to 50 contiguous residues, an LcrV protein of at least 275 residues in length (e.g. Schneedind et. al U.S. Patent Application No. 11/293,024 filed Dec. 02, 2005, incorporated herein by reference in its entirety for all purposes).
• a modified LcrV protein may suppress expression of a pro-inflammatory cytokine to a lesser extent than a corresponding unmodified LcrV protein.
• the proinflammatory cytokine may be TNF-a or other known pro -inflammatory cytokines known in the art.
• a deletion can include residues 271 to 300 of LcrV protein (rVIO). It is contemplated that the same or similar amino acids corresponding to this region of LcrV from Yersinia pestis may be deleted in other LcrV proteins. In one example, amino acids 271 -300 in Yersinia pestis correspond to 280-309 in Y. enter ocolitica.
• enterobacteria-derived proteins or peptides can be of use in vaccine constructs contemplated herein for administration to a subject to reduce incidence of or prevent a condition.
• Certain embodiments report compositions having constructs directed against any pathogenic enterobacterium.
• vaccine compositions may be directed to the prevention or reduced incidence of an infection in a subject caused by exposure or suspected exposure to a pathogenic enterobacteria.
• enterobacteria can include, but are not limited to Salmonella spp., Shigella spp, Escherichia coli strains or other pathogenic enterobacteria.
• Poxviruses are viruses that can, as a family, infect both vertebrate and invertebrate animals.
• Orthopox include, but are not limited to, variola virus, vaccinia virus, cowpox virus, monkeypox virus, and smallpox.
• Parapox include, but are not limited to, orf virus, pseudocowpox, bovine papular stomatitis virus; Yatapox: tanapox virus, yaba monkey tumor virus.
• Molluscipox include, but are not limited to, molluscum contagiosum virus (MCV). Some of the more common oixviruses are vaccinia and molluscum contagiousum, but monkeypox infections seem to be on the rise.
• MCV molluscum contagiosum virus
• Poxvirus family vaccinia virus
• Vaccinia virus has been used to successfully vaccinate against smallpox virus.
• Vaccinia virus is also used as an effective tool for foreign protein expression to elicit strong host immune response.
• Vaccinia virus enters cells mainly by cell fusion, although currently the receptor is not known.
• Virus contains three classes of genes, early, intermediate and late, transcribed by viral RNA polymerase and associated transcription factors. Diseases caused by poxviruses have been known about for centuries.
• Orthopoxviruses may include using modified or attenuated orthopoxviruses in vaccine compositions.
• Orthopoxvirus is a genus of the
• Poxviridae family that includes many agents isolated from mammals, including, but not limited to, vaccinia, monkeypox, cowpox, camelpox, seal poxvirus, buffalo poxvirus, raccoon poxvirus, skunk poxvirus, vole poxvirus and ectromelia viruses.
• Members of Poxviridae have large linear double-stranded DNA, with genome sizes ranging from 130 to 300 kbp.
• One of the members of the genus is variola virus, which causes smallpox. Smallpoxwas previously eradicated using another orthopoxvirus, the vaccinia virus, as a vaccine.
• VSA Modified vaccinia virus Ankara
• compositions and methods of use of recombinant vaccinia viruses derived from attenuated poxviruses e.g., modified vaccinia virus Ankara (MVA), NYVAC, LC16m8 or CVI-78
• attenuated poxviruses e.g., modified vaccinia virus Ankara (MVA), NYVAC, LC16m8 or CVI-78
• VVA modified vaccinia virus Ankara
• LC16m8 LC16m8 or CVI-78
• predetermined genes may be inserted at the site of a naturally occurring deletion in the MVA genome.
• recombinant MVA viruses can be used, for example, for the production of polypeptides (e.g. antigens) or for encoding antigens of use for vaccine compositions capable of inducing an immune response in a subject administered the vaccine compositions.
• polypeptides e.g. antigens
• antigens of use for vaccine compositions capable of inducing an immune response in a subject administered the vaccine compositions.
• modified or attenuated poxviruses e.g. modified vaccinia Ankara (MVA), NYVAC, LC16m8, or CVI-78
• a subject e.g., a cortavia, a cortavia, or a cortavia.
• MVA human immunodeficiency virus
• MVA human immunodeficiency virus
• recombinant MVA vaccine candidates have been shown to induce protective humoral and cellular immunity against diseases caused by viruses, bacteria, parasites, or tumors from which antigens or peptides were derived. Additional features that make MVA a suitable vector include its ability to induce protective immune responses when administered by different routes and its genetic and physical stability properties.
• a translation control sequence may include an optional enhancer, for example, a translation control sequence.
• a translation control sequence may include an internal ribosomal entry site (IRES) (e.g. EMCV-IRES).
• IRESs can be classified into four groups: Group 1 (Cricket paralysis virus (CrPV), Plautia stali intestine virus (PSIV) and Taura syndrome virus (TSV)); Group 2 (Hepatitis C virus, (HCV), classical swine fever virus (CSFV) and porcine teschovirus 1 (PTV-1)); Group 3 (encephalo myocarditis virus (EMCV), foot-and-mouth-disease virus (FMDV) and Theiler's Murine Encephalomyelitis virus (TMEV)); and Group 4 (poliovirus (PV) and rhinovirus (RV)).
• CrPV CrPV
• TSV Taura syndrome virus
• HCV Hepatit
• viral untranslated regions found 5' to viral coding sequences can be used to direct translation. Any translation control sequence of use in viral constructs known in the art is contemplated.
• a viral internal ribosome entry site may be used to increase expression of plague antigens contemplated herein.
• An IRES sequence can be positioned after a stop codon in a messenger RNA molecule and a ribosome can re-attach to the mRNA and a second protein can be translated from the same RNA.
• the IRES sequence can be used to express multiple antigens, for instance multiple enterobacterial antigens or an enterobacterial antigen and an antigen from another pathogenic virus or bacterium.
• the second protein is a selectable marker
• this marker will increase the probability that the gene of interest (placed between the promoter and the IRES) will be expressed by detection of the selectable marker.
• a number of bicistronic vectors have been produced based on these concepts. Use of any of bicistronic vectors known in the art are contemplated herein.
• embodiments of the present invention may include constructs having one or more secretory signal sequences.
• Secretory signals of use can include, but are not limited to, a mammalian secretory signal sequence. Translation control sequences and/or secretory signals were demonstrated to increase efficacy of some vaccines.
• one or more secretory signal sequences may include a proleader sequence.
• a tPA pre- proleader sequence may be used, where a leader sequence includes, but is not limited to, tissue plasminogen activator (tPA) leader sequence, oc-factor leader sequence, pre-pro insulin leader sequence, invertase leader sequence,
• immunoglobulin A leader sequence ovalbumin leader sequence, and P-globin leader sequence or other proleader sequences known in the art or a combination thereof.
• designing a construct, such that a protein is expressed it may be necessary to incorporate into a first nucleic acid region a DNA sequence encoding a signal sequence, for example, in cleavable form, where the expressed protein is desired to be secreted.
• a signal sequence can be a peptide that is present on proteins destined either to be secreted or to be membrane bound. These signal sequences are normally located at the N- terminus of the protein and are generally cleaved from the mature protein. The signal sequence generally interacts with the signal recognition particle and directs the ribosome to the endoplasmic reticulum where co-translational insertion takes place.
• signal sequence is cleavable
• it is generally removed by for example, a signal peptidase.
• the choice of signal sequence which is to be utilized may depend on the requirements of the particular situation and can be determined by the person of skill in the art.
• tPA may be used to facilitate secretion of a peptide, protein or construct of interest.
• a membrane protein is desired, both a 5' cleavable signal sequence at the amino end of the protein and a non- cleavable membrane anchor at the 3 ' (carboxy) end of the protein may be needed. These could be provided within the vector or one or both could be encoded by the DNA of the protein of interest,
• compositions including one or more constructs include, but are not limited to, compositions including one or more constructs.
• a construct may be designed to produce proteins or peptides that are cytoplasmically retained, secreted or membrane bound. Deciding what form a protein or peptide of interest may need to take can depend on functional requirements.
• anchored cell surface expression of a protein of interest provides a convenient means for screening for molecules that interact with the protein of interest such as antibodies, antagonists, agonists or the like particularly to the extent that the protein is expressed on the membrane of an adherent ceil type.
• Still further embodiments concern membrane anchored forms of proteins or peptide that may be suitable for administration to a subject, for example, for generating monoclonal antibodies to the protein or peptide.
• a host cell may provide adjuvant properties, for example, antigenic differences from a recipient subject, notably in major histocompatibility complexes (MHC).
• MHC major histocompatibility complexes
• secreted proteins can be suitable where a protein or peptide is to be harvested and purified.
• a nucleic acid molecule encoding a signal sequence to the extent that one is utilized may be positioned in the construct at any suitable location which can be determined as a matter of routine procedure by the person of skill in the art.
• a signal sequence may be positioned immediately 5 ' to the nucleic acid sequence encoding a peptide, protein or construct of interest (such that it can be expressed as an immediately adjacent fusion with the protein of interest) but 3' to a promoter such that expression of a signal sequence is placed under control of the promoter.
• a nucleic acid sequence encoding a signal sequence can form part of a first nucleic acid region of a construct.
• additional selection markers may be used, for example, one may insert any number of selection markers which may be designed, for example, to facilitate the use of the vectors in a variety of ways, such as purification of a molecule of interest.
• GST glutathione S -transferase
• a GST-fusion protein can be purified, by virtue of the GST tag, using glutathione agarose beads.
• Embodiments of the present invention should be understood to extend to constructs encoding a. secretable GST-molecule fusion.
• a fusion tag could be used which is itself a fusion between 360 bp of protein A (allowing purification of the secreted product) and beta lactamase (a bacterial enzyme which allows testing of supernatants by a simple colour reaction). Beta lactamase facilitates selection of an assay for a molecule of interest in the absence of an assay for molecule of interest.
• the protein A/beta lactamase fusion can be separated from the molecule of interest by a cleavage site to facilitate cleavage, so that after the molecule is purified, the tag can be easily removed. Any other selection marker known in the art may be used.
• fusion tags that could be included to facilitate purification of a molecule or construct of interest include, but are not limited to, staphylococcal protein A, streptococcal protein G, hexahistidine, calmodulin-binding peptides and maltose-binding protein (e.g. the latter is also useful to help ensure correct folding of a molecule of interest).
• Another selectable marker may include an antibiotic resistance gene.
• Other embodiments may include an antibiotic resistance gene. These genes have previously been utilized in the context of bicistronic vectors as the selection marker or HAT-based selectable bicistronic vector may be used.
• Electrophoresis may be used to separate molecules (e.g. large molecules such as proteins or nucleic acids) based on their size and electrical charge. There are many variations of electrophoresis known in the art. A solution through which the molecules move may be free, usually in capillary tubes, or it may be embedded in a matrix. Common matrices include polyacrylamide gels, agarose gels, and filter paper.
• Proteins, peptides and/or antibodies or fragments thereof may be purified, partially purified, detected or analyzed by any means known in the art.
• methods for separating and analyzing molecules may be used such as gel electrophoresis or column chromatography methods.
• assays for antibodies or antibody fragments may include, but are not limited to, EL1SA assays, chemiluminescence assays, flow cytometry and other techniques known in the ait.
• constructs having proteins or peptides may be linked to a secondary binding iigand or to an enzyme (an enzyme tag) that will generate a fluorescent, a luminescent, or a colored product upon contact with a substrate.
• an enzyme an enzyme tag
• suitable enzymes include !uciferase, green fluorescent protein, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase. The use and identification of such labels is well known to those of skill in the art.
• labels or molecules capable of detecting peptides, antigens, constructs, antibodies or antibody fragments may include using aptamers. Methods for making and using aptamers are well known in the art and these methods and uses are contemplated herein .
• Some embodiments can include methods for detecting and/or making polyclonal or monoclonal antibodies produced by a subject exposed to vaccine compositions disclosed in some embodiments of the present invention.
• antibodies or antibody fragments produced capable of inducing passive immunity to a subject may be isolated, analyzed and/or produced as a whole antibody or fragment thereof, or a polyclonal or a monoclonal antibody. Any means for producing or analyzing these antibodies known in the art are contemplated.
• Nucleic acid sequences used as a template for amplification can be isolated from viruses, bacteria, cells or cellular components contained in the biological sample, according to standard methodologies.
• a nucleic acid sequence may be genomic DNA or fractionated or whole ceil RNA. Where RNA is used, it may be desired to convert the RNA to a complementary cDNA. In one embodiment, the RNA is whole cell RNA and is used directly as the template for amplification. Any method known in the art for amplifying nucleic acid molecules are contemplated (e.g. PGR. LCR, Qheta eplicase).
• Genes or gene segments can be expressed in any number of different recombinant DNA expression systems to generate large amounts of the polypeptide product, which can then be purified and used in methods and compositions reported herein. Any method known in the art for generating and using constructs is contemplated. In certain embodiments, genes or gene fragments encoding one or more polypeptide mays be inserted into an expression vector by standard cloning or subcloning techniques known in the art.
• a gene or gene fragment encoding a polypeptide may be inserted into an expression vector by standard subcloning techniques.
• An expression vector may be used which produces the recombinant polypeptide as a fusion protein, allowing rapid affinity purification of a peptide or protein.
• fusion protein expression systems are the glutathione S-iransferase system (Pharmacia, Piscataway, NJ), the maltose binding protein system (NEB, Beverley, MA), the FLAG system (IBL New Haven, CT), and the 6xHis system (Qiagen, Chatsworth, CA).
• Aqueous compositions of some embodiments herein can include an effective amount of a therapeutic protein, peptide, construct, epitopic core region, stimulator, inhibitor, and the like, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
• Aqueous compositions of vectors expressing any of the foregoing are also contemplated.
• pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
• Aqueous compositions of some embodiments herein can include an effective amount of a therapeutic protein, peptide, construct, an effective amount of the compound, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
• a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
• preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
• the biological material should be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate.
• the active compounds or constructs will then generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, intranasal or even intraperitoneal routes. Any route used for vaccination or boost of a subject can be used.
• the preparation of an aqueous composition that contains an active component or ingredient will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for use in preparing solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
• compositions suitable for injectable use can include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
• a therapeutic agent can be formulated into a composition in a neutral or salt form.
• Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
• Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
• a carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
• the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions can be prepared by incorporating the active compounds or constructs in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the preparation of more, or highly, concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
• solutions can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
• the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but slow release capsules or microparticles and microspheres and the like can also be employed.
• aqueous solutions for parenteral administration in an aqueous solution
• the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
• aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
• sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
• one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion.
• unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the construct composition or boost compositions calculated to produce desired responses, discussed above, in association with its administration, e.g., the appropriate route and treatment regimen.
• the quantity to be administered depends on the subject to be treated, the state of the subject and the protection desired.
• the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
• a subject may be administered a construct composition disclosed herein on a daily or weekly basis for a time period or on a monthly, bi-yearly or yearly basis depending on need or exposure to a pathogenic organism or to a condition in the subject (e.g. cancer).
• the active therapeutic agents may be formulated within a mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so.
• active agents e.g. constructs
• parenteral administration such as intravenous, intradermal or intramuscular injection
• other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; time release capsules; biodegradable and any other form currently used.
• Nasal solutions can be aqueous solutions designed to be administered to the nasal passages in drops or sprays.
• Nasal solutions can be prepared so that they are similar in many respects to nasal secretions.
• the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 6.5.
• antimicrobial preservatives similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if required, may be included in the formulation.
• Various commercial nasal preparations are known and can include, for example, antibiotics and antihistamines and are used for asthma prophylaxis.
• Additional formulations which are suitable for other modes of administration can include suppositories and pessaries.
• a rectal pessary or suppository may also be used.
• traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
• Oral formulations can include excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
• oral pharmaceutical compositions will comprise an inert diluent or assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
• the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• Such compositions and preparations should contain at least 0.1% of active compound.
• the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%.
• the amount of active compounds in such compositions is such that a suitable dosage can be obtained.
• the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
• a binder as gum tragacanth, acacia, cornstarch, or gelatin
• excipients such as dicalcium phosphate
• a disintegrating agent such as corn starch, potato starch, alginic acid and the like
• a lubricant such as magnesium stearate
• a sweetening agent such as sucrose, lactose or saccharin may be added or a flavor
• tablets, pills, or capsules may be coated with shellac, sugar or both.
• a syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
• kits of use with methods and compositions described herein concern kits of use with methods and compositions described herein. Some embodiments concern kits having vaccine compositions of use to prevent or treat subjects having or exposed to an enterobacteria. Kits can be portable, for example, able to be transported and used in remote areas. Other kits may be of use in a health facility to treat a subject having been exposed to an enterobacteria or suspected of being at risk of exposure to an enterobacteria (e.g. Yersinia spp). [00092] Other embodiments can concern kits for making and using molecular constructs described herein. In certain embodiments, compositions can include constructs having attenuated or modified MVA and Yersinia spp.- associated antigens (e.g. V307). Other constructs can also include at least one secretory signal sequence. Yet other embodiments can have a construct that includes translation control sequences (e.g. IRES, UTRs). Other reagents for making and using constructs are contemplated.
• Kits can also include a suitable container, for example, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the constructs, vaccine compositions and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow- molded plastic containers into which the desired vials are retained.
• one or more additional agents such as other anti-viral agents, anti-fungal or anti-bacterial agents may be needed for compositions described, for example, for compositions of use as a vaccine.
• Dose ranges used during vaccination can vary depending on the nature of the live attenuated vaccine and viral vector used. For recombinant poxviruses these doses can range between 10 5 -10 7 PFUs. In certain embodiments of the present invention, immunogenic doses can be as low as 10 2 pfu. Frequency of vaccination can vary depending on the nature of the vaccine and also the route of administration used. One regimen can include a primary immunization (prime) followed up by a boost administration four to six weeks post-prime immunization. In certain embodiments of the present invention, improvements in antigen translation and expression can permit fewer and/or lower doses to be administered to a subject.
• compositions disclosed herein may be administered by any means known in the art.
• compositions may include administration to a subject intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneous ly, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, via a catheter, via a lavage, in a creme, or in a lipid composition.
• compositions can be administered by one route for one boost and another route for a second or additional boost of a composition, as can be pre-determined for a condition or prevention of an infection.
• one boost can be administered intramuscularly and another boost can be administered intradermally or a combinations thereof, depending on a subject's circumstances, as well as dose and frequency determinations for a particular composition.
• master and working seed stocks may be prepared under GMP conditions in qualified primary CEFs or by other methods.
• Cells may be plated on large surface area flasks, grown to near confluence and infected at selected MOI and vaccine virus purified.
• Cells may be harvested and intracellular virus released by mechanical disruption, cell debris removed by large-pore depth filtration and host cell DNA digested with endonuclease.
• Virus particles may be subsequently purified and concentrated by tangential- flow filtration, followed by diafiltration.
• the resulting concentrated bulk vaccine may be formulated by dilution with a buffer containing stabilizers, filled into vials, and lyophilized. Compositions and formulations may be stored for later use. For use, lyophilized vaccine may be reconstituted by addition of diluent.
• Poxviruses are known for their stability.
• the ability to lyophilize vaccinia for long term, room temperature storage and distribution was one of the key attributes that permitted widespread use of the vaccine and eradication of smallpox.
• the Fl capsular antigen was observed as two lower molecular weight forms consistent with the predicted protein (approximately 18 kilodaltons, kd; predicted protein) and a higher molecular weight form at approximately 23 kd (Fig.2A).
• the 23 kd form was more prevalent in the cell supernatant, suggesting that it is
• the capsular Fl protein has one predicted N-glycosylation site and six predicted o-glycosylation sites for vertebrate cells. Thus, it was postulated that the 23 kd form could represent a glycosylated protein. Deglycosylation of CEF-expressed Fl eliminated the 23 kd form and only the 18 kd form remained (Fig. 2E). Higher molecular weight forms of approximately 34 kd and above also were observed in the cell pellets. These forms are consistent with dimers and other multimers; the Fl capsular protein avidly forms multimeric structures upon secretion. These forms were not observed upon extensive denaturation (see for example Fig. 2E). In CEF cells infected with the MVA/IRES/tPA/Fl vims, only low levels of expression of the lower molecular weight forms could be observed in the cell pellets.
• IRES/tPA constmcts The reduced level of expression directed by the IRES/tPA constmcts is in contrast to similar constmcts made in raccoon poxvims, previously described. In that case, IRES/tPA directed higher levels of expression and secretion in infected cells.
• V and V 30 7 antigens were simpler and were similar in both infected CEF and Vero cells (see for example, Figs. 2C and 2D).
• Cells infected with either MVA/V or MVA/ V307 recombinant vimses expressed a single form of approximately 36 and 35 kd, respectively, consistent with predicted sizes (37 and 35 kd).
• Cells infected with MVA/IRES/tPA/V expressed two molecular forms of approximately 36 and 40 kd. The higher molecular weight form was consistent with the size of tPA/V fusion and was preferentially secreted.
• cells infected with the MVA/IRES/tPA/ V307 were simpler and were similar in both infected CEF and Vero cells (see for example, Figs. 2C and 2D).
• Cells infected with either MVA/V or MVA/ V307 recombinant vimses expressed a single form of approximately 36 and 35 kd, respectively, consistent with
• FIGs. 2A-2E illustrates experiments concerning monolayers of CEF or Vero cells transfected with recombinant MVA-plague viruses at MOI of 0.5pfu/cell. After 48 h post transfection, cells were harvested, cellular and supernatant extracts were prepared and subjected to SDS-PAGE followed by western blot analysis as described in the methods.
• A Fl expression in CEF cellular extracts (c) and supernatant (s) fractions.
• B Fl expression in Vero cell extracts (c) and supernatant (s) fractions.
• C V and V307 expression in CEF cell (c) and supernatant (s) fractions.
• FIGs 2A and B MVA/V constructs showed significantly higher expression in CEF (Fig. 2C) as compared to Vero (Fig. 2D). MVA/V and MVA/V 30 7 had similar expression levels in CEF (Fig.2C). Although MVA/IRES/tPA/V307 expression was lower compared to MVA/V or MVA/V 30 7, the V307 protein was more efficiently secreted. (Fig. 2C).
• a construct composition including a truncated version of the low- calcium response V (V307) antigen from Yersinia pestis under translational control of encephalomyocarditis virus (EMVC) internal ribosomal entry site (IRES) and with the tissue plasminogen activator (tPA) secretory signals was administered to mice.
• the construct composition conferred enhanced immunogenicity and consistently conferred significant protection in mice (87.5% - 100%) against intranasal or intraperitoneal challenge with C092 (encapsulated) or Java 9 (non-encapsulated) strains of Y. pestis, respectively.
• MVA construct expressing the full version of V antigen was highly immunogenic it provided significantly less protection (37.5%) against C092 or Java 9 strains, respectively in this experiment.
• An MVA construct expressing the capsular protein (Fl) failed to elicit detectable antibodies but conferred 50% and 25% protection against C092 or Java 9 challenge, respectively.
• All the MVA vectored plague vaccines tested in this study were shown to be completely safe in severe combined immuno-deficient (SCUD) mice.
• SCUD severe combined immuno-deficient mice.
• MVA has been stockpiled for use as a second-generation smallpox vaccine, with superior safety to the original live, attenuated vaccinia strains.
• a recombinant MVA/IRES/tPA/V307 vaccine has the potential to simultaneously provide protection against smallpox and plague.
• MVA expressing a variant of the V antigen in which the V antigen is truncated to remove the segment associated with the suppression of endogenous IL-12, TNF-a and IFN- ⁇ in vivo was tested for immunogenicity and protection. Moreover, effect of combining the IRES translational control sequence with the tPA secretory signal on the immunogenicity and protective capacity of the truncated V antigen was examined. All the MVA constructs expressing the V antigen were found to be immunogenic. When the protective capacities of all generated MVA constructs were tested in a mouse model of pneumonic plague, the
• MVA/IRES/tPA/V 30 7 consistently induced protection against lethal challenge with the highly virulent Y. pestis C092 strain at 35LD 50 as well as at 350LD 50 .
• the protection observed with the MVA/V307 construct was significantly lower ( O.05) than that induced by the MVA/IRES/tPA/V307 vaccine.
• the IRES/tPA processing signals appear to potentiate the protective immune response
• MVA/F1 vaccine failed to elicit a significant antibody response against the Fl antigen.
• 50% of immunized mice were protected against challenge with Y. pestis C092 strain with a median survival time of 11 days.
• Either very low antibody response to Fl or a cellular immune response to the protein may contribute to protect from Y. pestis infection.
• MVA/IRES/tPA/V 30 7 construct may account for the high level of protection observed in these studies. However, the MVA/V construct generated high anti-V antibody titers yet less protection from challenge.
• SCID mice immunocompromised mice. Infection of SCID mice with replication competent poxviruses causes significant weight loss and poxvirus lesions analogous to the disseminated viremia that can occur in vaccinated individuals with underlying immune deficiencies. In these studies, it was demonstrated that the MVA constructs expressing plague antigens are safe in SCID mice and fail to induce systemic disease even in the absence of effective B or T cell immunity.
• MVA constructs expressing the V307 antigen in conjunction with signal sequences was shown to be very immunogenic, safe, conferring protection against intranasal or intraperitoneal challenge with Y. pestis.
• FIG. 1 illustrates construction of rMVA/Z pestis antigen viruses.
• expression cassettes for each of the Y. pestis antigens, Fl , V and V 30 7 were inserted into pdlllGFP.
• Another cassette contained the EMCV-EMCV IRES sequence followed by the tPA secretory signal was fused to the V 30 7 antigen coding sequence.
• the cassettes were generated by PCR to contain the Smal and the BamHI restriction sites.
• Each expression cassette was inserted into a plasmid that contained DNA segments (flank 1 and flank 2) adjacent to deletion III within the HindlU A fragment of MVA.
• the plasmid also contained a strong synthetic early/late vaccinia virus promoter upstream of a multiple cloning site (MCS) and coding sequences for GFP under the control of a divergent synthetic vaccinia virus early/late promoter.
• MCS multiple cloning site
• mice were immunized intramuscularly with MVA/ Y. pestis constructs encoding the Fl , V or V 30 7 antigens.
• Antibody titers after a single immunization (pre-boost) and after two immunizations (pre- challenge) were assessed by ELISA analysis.
• Pre-challenge antibody titers elicited by the MVA/V construct were significantly higher ( ⁇ 0.05) than the MVA/V307 construct, however, there was no significant difference between pre-boost titers induced by these constructs (Fig. 3B).
• the effect of the IRES and tPA sequences on immunogenicity was examined. As shown in Fig. 3B, expression of V307 under the control of IRES and secretory signals (MVA/IRES/tPA/V307) significantly enhanced its immunogenicity.
• FIG. 3 illustrates immune responses to MVA/plague vaccines in mice.
• Groups of eight 4-6 week-old BALB/c mice were vaccinated intramuscularly with MVA-plague vaccines. Following two immunizations separated by 28 days, serum samples collected on days 28 and 42 post-initial vaccinations and analyzed by ELISA to determine humoral immune responses to Fl or V antigens.
• A Antibody responses to Fl antigen.
• B Antibody responses to V antigen from mice immunized with MVA/plague vaccines.
• mice vaccinated with the MVA/IRES/tPA/V 3 o7 survived lethal plague challenge with either the C092 (35 LD 50 ) or Java 9 (100 LD 50 ) strain of Y. pestis (Figs. 4A and 4B).
• passive transfer of pooled immune serum from mice immunized with MVA/IRES/tPA/V307 to naive BALB/c mice conferred significant protection ( O.05) against the Java 9 strain of Y. pestis as compared to the control mice (data not shown).
• mice immunized with MVA/ V307, MVA/V, or MVA/IRES/tPA/V survived challenge with the C092 strains of Y. pestis, respectively (Fig. 4A).
• Mice immunized with MVA/Fl or MVA/IRES/tPA/Fl had 50% or 25% survival rate against challenge with C092 or Java 9 strain of Y. pestis, respectively (Figs. 4A and 4B).
• Fig. 4 illustrates a Kaplan-Meier survival analysis of mice immunized with MVA/plague vaccines. Two weeks following booster immunizations, mice were challenged (A) intranasally with 1 X 10 5 pfu (35LDso) of Y pestis (C092) or (B) intraperitoneally with 100 cfu (100 LD 50 ) of Y. pestis (Java 9) and survival rates were recorded over a period of 2 weeks.
• A intranasally with 1 X 10 5 pfu (35LDso) of Y pestis (C092) or (B) intraperitoneally with 100 cfu (100 LD 50 ) of Y. pestis (Java 9) and survival rates were recorded over a period of 2 weeks.
• mice were immunized (prime and boost) with increasing doses (5 X 10 5 pfu, 5 X 10 6 pfu or 5 X 10 7 pfu) of MVA/IRES/tP A/V307 and then challenged with either 35 or 350 LD 50 s of the C092 Y. pestis strain. Mice immunized with increasing doses of
• MVA/IRES/tPA/V307 elicited corresponding increased immune responses with pre- challenge antibody titers of 3.38, 3.75 or 4.25 (loglO), respectively.
• the highest immunization dose (5 X 10 7 ) elicited significantly higher antibody titer ( O.05) compared to the lower doses and it conferred significant protection (87.5%) against challenge with either 35 or 350 LD 50 s of the C092 Y. pestis strain, respectively (Table 2, below).
• a - Median survival time is the time at which 50% of animals have died. This value is not applicable (N/A) for groups with >50% survival rates.
• mice in the vaccinia- Wyeth inoculated-group developed clinical disease symptoms characterized by pox lesions on their tails and feet and persistent weight loss; they died within 5 - 7 weeks post-infection. None of the animals from the MVAwt or MVA/7. pestis vaccine constructs developed any pox lesions. Weight loss in the vaccinia- Wyeth group was significantly greater ( ⁇ 0.0001) than in groups that were infected with MVAwt or MVA/plague vaccine constructs (Fig. 5).
• Antibody titers (Mean ⁇ SD) following prime and boost immunization with MVA/Plague vaccines were analyzed by Elisa.
• Post-boost titers from mice immunized with MVA/tPA/V 30 7 (IM) or MVA/IRES/tPA/V 30 7 (ID) were significantly higher than
• MVA/C13L/V307 or MVA/IRES/tPA/V307 (IM) groups (P values ** ⁇ 0.01 , * ⁇ 0.05).
• the transfer plasmid pdlllGFP (provided) was used to generate recombinant MVA expressing Y. pestis antigens.
• This plasmid contained: 1) DNA segments (flank 1 and flank 2) adjacent to deletion III within the Hindlll A fragment of MVA, 2) a strong synthetic early/late (SEL) vaccinia virus promoter upstream to a multiple cloning site (MCS), and 3) the green fluorescent protein (GFP) gene under the control of a divergent SEL promoter (Fig. 1).
• a second transfer plasmid, pdlllGFP/IRES/tPA, containing the ECMV IRES sequence followed by the tPA secretory signal was generated by insertion of an IRES/tPA cassette into pdlllGFP.
• Expression cassettes were generated by PCR (Table 1) to contain appropriate restriction sites for insertion into pdlllGFP or pdlllGFP/IRES/tPA.
• PCR products were cloned into the MCS of pdlllGFP or pdlllGFP/IRES/tPA and the resulting plasmids were designated as pdlllGFP/F l , pdlllGFP/IRES/tPA/Fl , pdlllGFP/V, pdlllGFP/IRES/tPA/V, pdIIIGFP/V 3 07 and pdlHGFP/iRES/tPA/Vso?.
• Recombinant MVA-plague viruses were generated as described previously. Briefly, chicken embryo fibroblasts (CEF), were infected with wild type MVA at a multiplicity of 0.05 and one hour (h) later the cells were transfected with each of the transfer vectors using LipofectamineTM (Invitrogen, Carlsbad, CA) . At 48 - 72 h post- transfection, monolayers were harvested, centrifuged at 500 RCF for 5 minutes at 4°C and cells disrupted by freeze-thaw and sonication (2 times for 15 seconds using a Virtis600 at setting 3).
• the disrupted cell extracts containing possible recombinant viruses expressing GFP were plated onto fresh CEF cells and overlaid with 0.8% agarose. After 48 - 72 h, recombinant virus -generated plaques were detected by fluorescence and picked into media with a glass pipette. The cell/virus samples were sonicated and plated as described above. After three consecutive rounds of plaque isolation, high titer virus stocks were prepared in CEF cells for subsequent in vitro and in vivo characterization. In vitro expression of Y. pestis antigens
• the infected cells were harvested in the presence of a protease inhibitor cocktail (Mini Protease tabs, Roche Diagnostics, Indianapolis, ⁇ ), washed, resuspended in IX loading buffer and heated to 95°C for 5 min.
• the supernatants from the infected cells were centrifuged and concentrated by ultrafiltration with a 3 kDa cutoff membrane (Nanosep 3K Omega, Pall, Inc., East Hills, NY). The supernatants were then combined with an equal volume of 2X loading buffer and heated to 95°C for 5 min.
• Supernatant and cell samples were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane for immuno-blot analysis using polyclonal rabbit anti-F l or anti-V serum produced in-house.
• the polyclonal antibodies were generated by inoculating specific pathogen free (SPF) rabbits with purified F 1 (from cafl operon expression system) or V (ATCC, BEI Resources, Manassas, VA, cat# NR- 3832) proteins. These antibodies showed minor background to MVA wild type expressed from CEF or Vero when used in immunoblot analyses.
• SPPF pathogen free
• the Fl glycosylation state was analyzed using a protein deglycosylation enzyme mix (New England Biolabs (NEB), Ipswich, MA, cat# P6039S). Briefly, Vero and CEF cells were infected with MVA/F 1 or MVA/ IRES/tPA/F 1 and harvested as previously described. The cell pellets, suspended in 30 ul H 2 0, and concentrated supernatants were processed using the NEB kit protocol. 18 ⁇ of the resuspended cell pellet and concentrated supernatant samples, were denatured for 10 minutes at 100°C. G7 reaction buffer containing 10% NP40 was added to bring the reaction volume to 50 ⁇ .
• NEB protein deglycosylation enzyme mix
• reaction was split and half was treated with 2.5 ul NEB deglycosylation enzyme cocktail and incubated at 37°C for 4 hr. lOul, of each reaction, was subjected to electrophoresis and analyzed by Western using the polyclonal rabbit anti-Fl serum.
• mice intraperitoneally with 100 ⁇ of post-boost pooled serum with a titer of 100,000 from mice immunized with two doses of MVA/IRES/tPA/V307.
• the passively immunized mice were challenged intraperitoneally with a 100 ⁇ inoculum containing 100 cfu (IOOLD 50 ) of the Java 9 strain of Y. pestis. Challenged animals were monitored for two weeks.
• the stability and virulent phenotype of the Y. pestis C092 or Java 9 frozen stock cultures were validated by testing aliquots for bacterial counts and lethal doses.
• LD 50 Lethal dose
• Serum samples were collected on day 28 post-primary vaccination and day 14 post-boost (pre-challenge) to assess antibody titers against Y. pestis Fl or V antigens.
• Serum total IgG as well as IgGl and IgG2a subclass titers were measured by enzyme-linked immunosorbent assay (ELISA) as described previously. Briefly, 96-well ELISA plates were coated with purified recombinant F 1 or V antigen (O. ⁇ g in 100 carbonate buffer, pH 9.6 per well) at 4°C overnight.
• HRP horseradish peroxidase
• Abeam Inc Cambridge, MA
• TMB tetra-methyl- benzidine
• mice Groups of six, five-week old BALB/c SCID mice (Harlan Sprague Dawley, Indianapolis, IN) were inoculated intraperitoneally with 1 X 10 8 pfu of MVA/F1, MVA/V, MVA/V307, MVA/IRES/tPA/V307 or wild type MVA (MVAwt).
• An additional group received 1 X 10 6 pfu of the vaccinia Wyeth strain via the same route. Mice were monitored daily for 3 months and their weight was recorded weekly. Mice died naturally or were euthanized when showing body-conditioning score less than two (BCS ⁇ 2) as previously described.

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