Recombinant attenuated salmonella vaccines (RASVS) against infections by avian pathogenic escherichia coli (APEC)

Abstract

The present disclosure provides compositions and methods for making and using Salmonella recombinant bacteria that express LPS O78 antigen and / or virulence factors of avian pathogenic Escherichia coli (APEC) as vaccines to prevent colibacillosis infections.

Description

Attorney Docket No.: 139465-00120 Recombinant Attenuated Salmonella Vaccines (RASVs) Against Infections by Avian Pathogenic Escherichia coli (APEC) REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 568,108, filed on March 21, 2024, the contents of which are incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 17, 2025, is named 139465-00102-SeqList.XML and is 279,169 bytes in size. BACKGROUND

[0003] Avian pathogenic Escherichia coli (APEC) is a type of extraintestinal pathogenic microorganism that causes diverse systemic infections in poultry, including chickens, turkeys, ducks, and many other avian species. The most common diseases caused by APEC in chickens includes airsacculitis, cellulitis, coligranuloma, egg peritonitis, omphalitis, osteomyelitis / arthritis, perihepatitis, pericarditis, and salphingitis (collectively known as colibacillosis infections).

[0004] Colibacillosis caused by APEC is a devastating disease of poultry that results in severe economic losses annually to the poultry industry worldwide. Multiple APEC serotypes have been associated with colibacillosis cases in the field outbreaks; however, three serotypes (O78, O2, and O1) account for the majority (more than 80%) of the cases. The pathogenicity of APEC is still poorly understood. Thus, there is a need for improved vaccines against APEC infection. SUMMARY

[0005] Construction of novel recombinant attenuated Salmonella vaccines (RASVs) protecting avians against systemic infections by avian pathogenic Escherichia coli (APEC) is disclosed herein. In particular, disclosed herein is a method for the inter-genus serotype conversion by replacing the Salmonella O4-antigen gene cluster (rfbO4) in RASV strains with the APEC O78-antigen gene cluster (rfbO78), providing serotype converted RASV strains which successfully produce APEC O78-antigen but no longer produce S. Typhimurium O4- antigen. Additionally, the phosphomannose isomerase (pmi) mutant will have a defect to 1 ME151672980v.1Attorney Docket No.: 139465-00120 produce LPS O-antigen in a mannose limitation environment like in vivo since the phosphomannose is a key building block for the LPS O-antigen production.

[0006] In one aspect, the disclosure provides a recombinant Salmonella bacterium, wherein the bacterium expresses lipopolysaccharide (LPS) O78 antigen of avian pathogenic Escherichia coli (APEC) and does not express Salmonella LPS O4 antigen.

[0007] In some embodiments, the recombinant Salmonella bacterium comprises APEC’s rfbO78gene cluster. In some embodiments, the recombinant Salmonella bacterium further comprises a deletion mutation in the Salmonella rfbO4gene cluster. In some embodiments, the APEC’s rfbO78 gene cluster replaces the entire Salmonella rfbO4 gene cluster in the genome of the Salmonella bacterium.

[0008] In some embodiments, the rfbO4gene cluster comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 4 or wherein the rfbO4gene cluster comprises the nucleic acid sequence of SEQ ID NO: 4.

[0009] In some embodiments, the Salmonella recombinant bacterium further expresses one or more APEC virulence factors. In some embodiments, the Salmonella recombinant bacterium comprises one or more nucleotides encoding one or more APEC virulence factors. In some embodiments, the Salmonella recombinant bacterium comprises one or more APEC plasmids that comprises nucleotides encoding one or more APEC virulence factors.

[0010] In some embodiments, the APEC plasmid is plasmid pAPEC-1, pAPEC-2, pAPEC-3, or a combination thereof, derived from APEC strain χ7122. In some embodiments, the recombinant Salmonella bacterium comprises all three plasmids pAPEC- 1, pAPEC-2 and pAPEC-3.

[0011] In some embodiments, the pAPEC-1 plasmid comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments, the pAPEC-2 plasmid comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments, the pAPEC-3 plasmid comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 3

[0012] In some embodiments, the recombinant Salmonella bacterium further comprises a deletion mutation in the lrp gene (Δlrp).

[0013] In some embodiments, the recombinant Salmonella bacterium comprises an asd gene (asd+). In some embodiments, the asd gene is inserted into the pAPEC-1 plasmid. In some embodiments, the asd gene comprises a nucleic acid sequence having at least 85%, 2 ME151672980v.1Attorney Docket No.: 139465-00120 at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 12, or the asd gene comprises the nucleic acid sequence of SEQ ID NO: 12.

[0014] In some embodiments, the recombinant Salmonella bacterium comprises a pmi gene (pmi+). In some embodiments, the pmi gene comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 11 or the pmi gene comprises the nucleic acid sequence of SEQ ID NO: 11.

[0015] In some embodiments, the recombinant Salmonella bacterium further comprises, or is derived from a parent Salmonella bacterium strain comprising: a araC ParaBAD-regulated murA gene (ΔPmurA::TT araC ParaBAD murA deletion-insertion mutation); an insertion of a c2 gene operably linked to an arabinose regulatable promoter araC ParaBAD(araC ParaBADc2), optionally wherein the araC ParaBADc2 gene cassette is inserted into and thereby inactivate the endogenous asd gene (ΔasdA::TT araC ParaBAD c2 deletion-insertion mutation); a deletion mutation in the gmd and a deletion in the fcl genes (Δ(gmd-fcl));and a deletion- insertion mutation that inactivates the expression of a relA gene and inserts a lacI gene (ΔrelA::araC ParaBADlacI TT deletion-insertion mutation).

[0016] In some embodiments, the recombinant Salmonella bacterium further comprises an araC ParaBAD-regulated murA gene (ΔPmurA::TT araC ParaBADmurA deletion-insertion mutation); an insertion of a c2 gene operably linked to an arabinose regulatable promoter araC ParaBAD(araC ParaBADc2), optionally wherein the araC ParaBADc2 gene cassette is inserted into and thereby inactivate the endogenous asd gene (ΔasdA::TT araC ParaBADc2 deletion-insertion mutation); a deletion mutation of the gene cluster wza-wcaM (Δ(wza- wcaM)); a deletion-insertion mutation that inactivates the expression of a relA gene and inserts a lacI gene (ΔrelA::araC ParaBADlacI TT deletion-insertion mutation) a deletion mutation in the recF gene (ΔrecF); a deletion mutation in the waaL gene (ΔwaaL46); and a deletion in a pagL gene and an insertion of a rhaRS PrhaBAD-regulated waaL gene (ΔpagL::TT rhaRS PrhaBADwaaL).

[0017] In some embodiments, the recombinant Salmonella bacterium is a Salmonella enterica bacterium. In some embodiments, the bacterium is a Salmonella enterica subsp. enterica serovar Paratyphi A bacterium, a Salmonella enterica subsp. enterica serovar Enteritidis bacterium, a Salmonella enterica subsp. enterica serovar Typhi bacterium, a Salmonella enterica subsp. enterica serovar Typhimurium bacterium, Salmonella enterica subsp. enterica serovar Dublin, Salmonella Pullorum, Salmonella Gallinarum, or Salmonella enterica subsp. enterica serovar Choleraesuis.

[0018] In one aspect, the disclosure provides pharmaceutical composition comprising the recombinant Salmonella bacterium described herein, and a pharmaceutically acceptable carrier. 3 ME151672980v.1Attorney Docket No.: 139465-00120

[0019] In a certain aspect, the disclosure provides a vaccine comprising the recombinant Salmonella bacterium or the pharmaceutical composition described herein.

[0020] In another aspect, the disclosure provides a method for eliciting an immune response against APEC in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition or the vaccine described herein.

[0021] In another aspect, the disclosure provides a method for vaccinating a subject against APEC, the method comprising administering to the subject an effective amount of the pharmaceutical composition or the vaccine described herein.

[0022] In some embodiments, the subject is a chicken, turkey, goose, or duck.

[0023] In some embodiments, the pharmaceutical composition or vaccine is administered by spray or oral immunization.

[0024] In another aspect, the disclosure provides a method for producing the recombinant Salmonella bacterium, the method comprising culturing the bacterium in media. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 shows a scheme to develop RASVs against APEC infections.

[0026] Figure 2 shows schematics of APEC virulence plasmids in the wild-type APEC strain χ7122.

[0027] Figure 3 shows schematics of LPS and colonic acid coding areas in S. Typhimurium and APEC.

[0028] Figure 4 shows a schematic of pCHSUI-APEC-O78 (21,542 bp) map.

[0029] Figure 5 shows lineage of the RASV backbone strain χ12341.

[0030] Figure 6 shows construction of the RASV-based APEC vaccines.

[0031] Figure 7 shows siderophore production in the RASV-based APEC vaccines.

[0032] Figure 8 shows LPS O-antigen production in RASV-based APEC vaccines. DETAILED DESCRIPTION

[0033] Before the present disclosure is described, it is to be understood that this disclosure is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 4 ME151672980v.1Attorney Docket No.: 139465-00120 disclosure belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1 %. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0035] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entireties. Definitions

[0036] The articles “a” and “an,” as used herein, should be understood to mean “at least one,” unless clearly indicated to the contrary.

[0037] The phrase “and / or,” when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, “A, B, and / or C” indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase “and / or” may be used interchangeably with “at least one of” or “one or more of” the elements in a list.

[0038] As used herein, the term “derived from” or “derivative of” refers to a composition (e.g., a gene, a plasmid, a bacterium) that results from modifications or manipulation of an original composition. For example, insertion of a selective marker gene into an original pAPEC-1 plasmid results in a plasmid derived from pAPEC-1, or a pAPEC-1 derivative plasmid

[0039] As used herein, the term “gene” refers to a nucleic acid fragment that encodes a protein or a fragment thereof, or a functional or structural RNA molecule, and may optionally include a regulatory sequence preceding (5’ non-coding sequences) and following (3’ non- coding sequences) the coding sequence of the nucleic acid. In some embodiments, a “gene” does not include regulatory sequences preceding and following the coding sequence.

[0040] In one embodiment, the gene is a heterologous gene. In another embodiment, the nucleic acid is a heterologous nucleic acid. As used herein, the terms “heterologous gene” or “heterologous nucleic acid” refer to a gene or a nucleic acid sequence present in a recombinant cell, e.g., bacterium, that is not normally found in the wild-type cell, e.g., bacterium, in nature. In some embodiments, the heterologous gene or heterologous nucleic acid is exogenously introduced into a given cell. In some embodiments, a heterologous gene may include a gene, or fragment thereof, introduced into a non-native host cell. In some embodiments, the term “heterologous gene” includes a second copy of a native gene, or fragment thereof, that has been introduced into the host cell in addition to the 5 ME151672980v.1Attorney Docket No.: 139465-00120 corresponding native gene. A heterologous nucleic acid may also include, in some embodiments, a gene sequence that is naturally-found in a given cell but which has been modified, e.g., by regulation by a different promoter sequence, to expresses an unnatural amount of the nucleic acid and / or the polypeptide which it encodes; and / or two or more nucleic acid sequences that are not found in the same relationship to each other in nature.

[0041] As used herein, the term “endogenous gene” refers to a native gene that is present in its natural location in the genome of an organism (e.g., a bacterial chromosome).

[0042] A “promoter” as used herein, refers to a nucleic acid sequence that is capable of controlling the expression of a coding sequence or gene. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of a nucleic acid. For example, a promoter may include one or more nucleic acids that are specifically recognized by a transcriptional activator protein (e.g., an enhancer element), a transcriptional repressor protein, a polymerase, and the like. The term “operably linked,” as used herein, means that expression of a nucleic acid sequence is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) of the nucleic acid sequence under its control. The distance between the promoter and a nucleic acid sequence to be expressed may be approximately the same as the distance between that promoter and the native nucleic acid sequence it controls. As is known in the art, variation in this distance may be accommodated without loss of promoter function. The nucleic acid sequences of the promoters described herein are known in the art, and methods of operably- linking these promoters to a gene (e.g., a gene encoding a repressor) are known in the art.

[0043] In some embodiments, the promoter for use as described herein may be regulated directly or indirectly by a sugar. For example, in some embodiments, the promoter is responsive to the level of arabinose, otherwise referred to herein as an “arabinose- regulatable promoter”. Generally speaking, arabinose may be present during the in vitro growth of a bacterium, while typically absent from host tissue. In one embodiment, the promoter is derived from an araC-ParaBADsystem from Escherichia coli. The araC ParaBADsystem is a tightly regulated expression system, which has been shown to work as a strong promoter induced by the addition of low levels of arabinose. The araC-araBAD promoter is a bidirectional promoter controlling expression of the araBAD nucleic acid sequences in one direction, and the araC nucleic acid sequence in the other direction.

[0044] For convenience, the portion of the araC-araBAD promoter that mediates expression of the araBAD nucleic acid sequences, and which is controlled by the araC nucleic acid sequence product, is referred to herein as ParaBAD. For use as described herein, a cassette with the araC nucleic acid sequence and the araC-araBAD promoter may be 6 ME151672980v.1Attorney Docket No.: 139465-00120 used. This cassette is referred to herein as araC ParaBAD. The AraC protein is both a positive and negative regulator of ParaBAD. In the presence of arabinose, the AraC protein is a positive regulatory element that allows expression from ParaBAD. In the absence of arabinose, the AraC protein represses expression from ParaBAD. Other enteric bacteria contain arabinose regulatory systems homologous to the araC-araBAD system from E. coli, including, for example, S. Typhimurium. For example, the E. coli AraC protein only activates E. coli ParaBAD(in the presence of arabinose) and not S. Typhimurium ParaBAD. Thus, an arabinose regulated promoter may be used in a recombinant bacterium that possesses a similar arabinose operon, without substantial interference between the two, if the promoter and the operon are derived from two different species of bacteria. Generally speaking, the concentration of arabinose necessary to induce expression is typically less than about 2% (w / w) in a culture media. In some embodiments, the concentration is less than about 1.5%, 1%, 0.5%, 0.2%, 0.1%, or 0.05% (w / w) in a culture media. In other embodiments, the concentration is 0.05% or below, e.g. about 0.04%, 0.03%, 0.02%, or 0.01% (w / w). In an exemplary embodiment, the concentration is about 0.05% (w / w) in a culture media.

[0045] In still other embodiments, the promoter used herein is responsive to the level of rhamnose in the environment, otherwise referred to herein as a “rhamnose-regulatable promoter”. Analogous to the araC-ParaBADsystem described above, the rhaRS-PrhaBADactivator-promoter system is tightly regulated by rhamnose. Expression from the rhamnose promoter (Prha) is induced to high levels in the presence of rhamnose. In some embodiments, the bacteria are cultured in the presence of rhamnose. Rhamnose is commonly found in bacteria but rarely found in human subjects. The rhaBAD operon is controlled by the PrhaBADpromoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding nucleic acid sequences belong to one transcription unit that is located in the opposite direction of the rhaBAD nucleic acid sequences. In the presence of L- rhamnose, RhaR binds to the PrhaRSpromoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose, in turn, bind to the PrhaBADand the PrhaTpromoters and activates the transcription of the structural nucleic acid sequences. Full induction of the arabinose, maltose and rhamonse regulated promoters described herein requires binding of the Crp-cAMP complex, which is a key regulator of catabolite repression.

[0046] Although both L-arabinose and L-rhamnose act directly as inducers of the expression of regulons that mediate their catabolism, important differences exist in regard to the regulatory mechanisms. L-Arabinose acts as an inducer with the activator AraC in the positive control of the arabinose regulon. However, the L-rhamnose regulon is subject to a regulatory cascade, and is therefore subject to even tighter control than the araC-ParaBADsystem. L-Rhamnose acts as an inducer with the activator RhaR for synthesis of RhaS, 7 ME151672980v.1Attorney Docket No.: 139465-00120 which in turn acts as an activator in the positive control of the rhamnose regulon. In the present disclosure, rhamnose may be used to interact with the RhaR protein and then the RhaS protein may activate transcription of a nucleic acid sequence operably-linked to the PrhaBADpromoter.

[0047] As used herein, the term "exogenous" refers to a substance (e.g., a nucleic acid or polypeptide) present in a cell other than its native source. The term exogenous can refer to a nucleic acid or a protein that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found or in which it is found in undetectable amounts. A substance can be considered exogenous if it is introduced into a cell or an ancestor of the cell that inherits the substance. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell.

[0048] As used herein, the term “host cell” refers to a cell in an organism to which the recombinant bacterium is being administered in order to, for example, induce an immune response. In one embodiment, a host is a bird, equine, or human and a host cell refers, respectively, to a bird cell, an equine cell, or a human cell. “Host” and “subject” are used interchangeably in the present disclosure.

[0049] A “nucleic acid” or “nucleic acid sequence” may be any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single- stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA, rRNA, and tRNA.

[0050] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (35); Bauer et al. (36); Craik (37); Smith et al. (38); and U.S. Pat. Nos.4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. Any cysteine residue not involved in maintaining the proper conformation of the 8 ME151672980v.1Attorney Docket No.: 139465-00120 polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0051] A "pharmaceutical composition," as used herein, refers to a composition comprising an active ingredient (e.g., a recombinant bacterium described herein) with other components such as a physiologically suitable carrier and / or excipient.

[0052] As used herein, the term “pharmaceutically acceptable carrier” or a “pharmaceutically acceptable excipient” refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline (e.g., phosphate-buffered saline (PBS)); (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (24) C2-C12alcohols, such as ethanol; and (25) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, disintegrating agents, binders, sweetening agents, flavoring agents, perfuming agents, protease inhibitors, plasticizers, emulsifiers, stabilizing agents, viscosity increasing agents, film forming agents, solubilizing agents, surfactants, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable excipient” or the like are used interchangeably herein. 9 ME151672980v.1Attorney Docket No.: 139465-00120

[0053] A “plasmid” or “vector” includes a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. The nucleic acid incorporated into the plasmid can be operatively linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence.

[0054] As used herein, the terms “protein” and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. The terms "protein" and “polypeptide” as used herein refer to both large polypeptides and small peptides. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0055] As used herein, the term “recombinant bacterium” refers to a bacterial cell that has been genetically modified from its native state. For instance, a recombinant bacterium may comprise one or more nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications. These genetic modifications may be introduced into the chromosome of the bacterium, or alternatively be present on an extrachromosomal nucleic acid (e.g., a plasmid). Recombinant bacteria of the disclosure may comprise a nucleic acid located on a plasmid. Alternatively, the recombinant bacteria may comprise a nucleic acid located in the bacterial chromosome (e.g., stably incorporated therein). In some embodiments, the recombinant bacterium is avirulent. In some embodiments the recombinant bacterium exhibits reduced virulence. In some embodiments, the recombinant bacterium is non-virulent. In some embodiments, the recombinant bacterium is pathogenic. In some embodiments, the recombinant bacterium is attenuated. In another embodiment, the recombinant bacterium is a recombinant derivative of a pathogenic bacterium.

[0056] The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0057] As used herein, the term “virulence factor” refers to a molecule that assist a pathogenic bacterium in colonizing the host at the cellular level. Virulence factors can be either secretory, membrane associated or cytosolic in nature. Typically, the cytosolic factors facilitate the bacterium to undergo adaptation such as metabolic, physiological and 10 ME151672980v.1Attorney Docket No.: 139465-00120 morphological shifts. The membrane associated virulence factors usually aid the bacterium in adhesion and evasion of the host cell. The secretory factors are important components of bacterial armoury which help the bacterium wade through the innate and adaptive immune response mounted within the host. In extracellular pathogens, the secretory virulence factors can act synergistically to kill the host cells.

[0058] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0059] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. Compositions

[0060] The present disclosure provides, in one aspect, a Salmonella recombinant bacterium capable of expressing lipopolysaccharide (LPS) O78 antigen of avian pathogenic Escherichia coli (APEC) without expressing the native Salmonella LPS O4 antigen. The invention is based on the surprising finding that conversion of Salmonella LPS O4 serotype to APEC LPS O78 serotype results in strong O78 antigen production by the recombinant Salmonella bacterium.

[0061] In another aspect, the present disclosure further provides a recombinant Salmonella bacterium capable of expressing APEC’s virulence factors, which is effective in inducing specific antibody response against APEC pathogens.

[0062] In some embodiments, the recombinant bacterium described herein is modified such that the expression of one or more genes, e.g., virulence genes, can be regulated in a sugar-responsive manner. In some embodiments, one or more endogenous genes, e.g., virulence genes, are deleted from the bacterial chromosome. In some embodiments, the deletion is a partial deletion of the endogenous gene. In some embodiments, the deletion is a full-length deletion of the endogenous gene. In some embodiments, the gene, e.g., virulence gene, is genetically-altered to prevent transcription and / or translation of the gene encoding the protein. In some embodiments, the endogenous gene is genetically altered to insert a transcriptional terminator in the open reading frame of the gene. In some embodiments, a regulatory region of the gene, e.g., virulence gene, is genetically-modified to 11 ME151672980v.1Attorney Docket No.: 139465-00120 alter (e.g., decrease) the expression of the gene. In some embodiments, the promoter of a gene, e.g., virulence gene, is altered to include one or more regulatory elements (e.g., a sugar-responsive promoter).

[0063] As used herein, a “deletion mutation” or “deletion” in a gene refers to either partial or full-length deletion of a gene that abolishes activity of said gene. In one embodiment, a deletion is a partial deletion of a gene that abolishes activity of said gene. In another embodiment, a deletion is a full-length deletion of a gene that abolishes activity of said gene.

[0064] In some embodiments, the recombinant bacterium described herein is modified to comprise a nucleic acid comprising a gene. In some embodiments, the recombinant bacterium is modified to comprise a nucleic acid comprising a gene, whereby an endogenous copy of the gene in the bacterial chromosome has been altered and / or deleted. In some embodiments, the nucleic acid comprises a gene that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an endogenous gene in the bacterial chromosome that has been deleted and / or altered. In some embodiments, the nucleic acid comprises a gene that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to an endogenous gene in the bacterial chromosome that has been deleted and / or altered. In some embodiments, the nucleic acid comprises a gene from a bacterial species, subspecies, serovar, or strain that is different than the bacterial species of the recombinant bacterium.

[0065] In some embodiments, the nucleic acid comprises a gene from a bacterial species, subspecies, serovar, or strain that is the same as the bacterial species of the recombinant bacterium. In some embodiments, the nucleic acid comprises a gene that is operably-linked to a regulatable promoter (e.g., a sugar-regulatable promoter). In some embodiments, the nucleic acid comprises a gene that is operably-linked to a rhamnose- regulatable promoter, a xylose-regulatable promoter, a galactose-regulatable promoter, an arabinose-regulatable promoter, a mannose-regulatable promoter, or a maltose-regulatable promoter. In some embodiments, the nucleic acid comprising the gene is located in a plasmid in the bacterium. In some embodiments, the nucleic acid comprising the gene is located in the bacterial chromosome. In some embodiments, the nucleic acid comprising the gene is located at the chromosomal locus corresponding to the locus of an endogenous gene that has been deleted or altered in the bacterial chromosome. In some embodiments, 12 ME151672980v.1Attorney Docket No.: 139465-00120 the nucleic acid is codon-optimized (e.g., to improve expression of the nucleic acid in the recombinant bacterium). 1. Lipopolysaccharide O-antigen conversion

[0066] In the case of interactions between animal host and bacterial pathogen, the bacterial surface is the first line of contact with the host immune system. Among the bacterial surface molecules undergoing pathogenic interactions, lipopolysaccharides (LPS) constitute the major structural component of the outer membrane of Gram-negative bacteria. The LPS fraction comprises about 10-15% of the total molecules in the outer membrane and is estimated to occupy about 75% of the bacterial surface [Lerouge and Vanderleyden, 2001].

[0067] LPS consists of three different components or regions: lipid A, core oligosaccharide, and O-polysaccharide. The structure of lipid A is highly conserved among Gram-negative bacteria; among Enterobacteriaceae, lipid A is virtually constant. Lipid A is the lipid component of LPS that contains the hydrophobic, membrane-anchoring region of LPS. The core oligosaccharide regions consist of a short chain of sugars, which connects the lipid A anchor to O-antigen, and can be divided into inner and outer core regions [Osborn and Wu, 1980].

[0068] LPS O-antigen has been reported to be a potent antigen for inducing specific antibody response which is protective against E. coli pathogens in animal models. The O- antigen or O-side chain is attached to the core polysaccharide and extends from the core out into the environment. It consists of repeating oligosaccharide subunits made up of three to five sugars. The individual chains can vary in length ranging up to 40 repeat units. The O- polysaccharide is much longer than the core oligosaccharide and it contains the hydrophilic domain of the LPS. At least 20 different sugars are known to occur and many of these sugars are characteristically unique dideoxyhexoses such as abequose, colitose, paratose, and tyvelose, which are rarely found elsewhere in nature. The composition of sugars in the O-polysaccharide, especially terminal units, confers immunological specificity of the O- antigen and contributes to the wide variety of antigenic types (serotypes) between species and even strains of Gram-negative bacteria [Lerouge and Vanderleyden, 2001].

[0069] LPS O-antigen has been reported to be a potent antigen for inducing specific antibody response which is protective against E. coli pathogens in animal models [Pluschke and Achtman, 1985; Schief et al, 1993; Cryz Jr et al, 1995]. In addition, specific antibody against LPS O-antigen has been shown to be protective against other gram-negative pathogens [Zhang et al, 2009; Ding et al, 1990; Moustafa et al, 2023]. 13 ME151672980v.1Attorney Docket No.: 139465-00120

[0070] Multiple APEC serotypes have been associated with colibacillosis cases in the field outbreaks; however, three serotypes, O78, O2, and O1, account for the majority (more than 80%) of the cases [Dho-Moulin and Fairbrother, 1999; Ghunaim et al, 2014].

[0071] In some embodiments, the recombinant Salmonella bacterium provided herein expresses the LPS O78 antigen. The structure of O78 antigen of E. coli is known in the art and described in Jansson et al., “Structural studies of the Escherichia coli O78 O-antigen polyscharride”, Carbohydrate Research 165(1): 87-92 (1987), and Liu et al., “Structure and genetics of Escherichia coli O antigens”, FEMS Microbiol Rev.44(6): 655-683 (2020), the entire contents of which are incorporated herein by reference.

[0072] In some embodiments, the O78 antigen comprises the following structure:, wherein the sugar residues in this structure areD-N-Acetylglucosamine (D-GlcNAc) andD- mannose (D-Man) which are linked by β1→4 or α1→3 linkages.

[0073] O78 antigen synthesis is carried out by a number of genes in the O78-gene cluster rfbO78: eco78ABCDEFGHIJK depicted in Figure 3, also referred to herein as the rfbO78gene cluster. The O78-gene cluster rfbO78is about 13 kb long. In some embodiments, the O78-gene cluster rfbO78is located between the galF and gnd genes in the chromosome of APEC strain χ7122.

[0074] In some embodiments, the recombinant Salmonella bacterium provided herein comprises all O78 antigen synthesis genes. In some embodiments, the recombinant bacterium comprises the O78-gene cluster rfbO78.

[0075] In some embodiments, the rfbO78gene cluster comprises a nucleic acid sequence having at least 50%, at least 65%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. In one embodiment, the rfbO78gene cluster comprises a nucleic acid sequence comprising SEQ ID NO: 4. In one embodiment, the rfbO78gene cluster comprises a nucleic acid sequence consisting of SEQ ID NO: 4.

[0076] In some embodiments, the recombinant Salmonella bacterium does not express the Salmonella LPS O4 antigen. The structure of O4 antigen of Salmonella typhirium is known in the art and described in Liu et al., “Structural diversity in Salmonella O antigens and its genetic basis”, FEMS Microbiol Rev 38: 56–89 (2014), the entire content of which is incorporated herein by reference.

[0077] In some embodiments, the O4 antigen comprises the following structure: 14 ME151672980v.1Attorney Docket No.: 139465-00120 →2)-D-Man-(α1→4)-L-Rha-(α1→3)-D-Gal-(α1→ wherein the sugar residues in this structure areD-mannose (D-Man),L-rhamnose (L-Rha), andD-galactose (D-Gal) which are linked by α1→4 or α1→3 linkages.

[0078] O4 antigen synthesis is carried out by a number of genes in the O4-gene cluster rfbO4depicted in Figure 3, also referred to herein as the rfbO4gene cluster. The O4-gene cluster rfbO4is about 18 kb long. In some embodiments, the O4-gene cluster rfbO4is located between the galF and gnd genes in the chromosome of Salmonella bacterium.

[0079] In some embodiments, the recombinant Salmonella bacterium provided herein comprises a deletion mutation in one or more O4 antigen synthesis genes such that O4 antigen synthesis is abolished. In some embodiments, the recombinant bacterium comprises a deletion mutation in the O4-gene cluster rfbO4.

[0080] In some embodiments, the APEC O78-gene cluster rfbO78replaces the entire Salmonella O4-gene cluster rfbO4between the galF and gnd genes in the recombinant Salmonella bacterium.

[0081] In some embodiments, the recombinant bacterium comprises genetic features or modification that enhance expression of O78 antigen and / or enhance immunogenicity of the bacterium.

[0082] In some embodiments, the recombinant bacterium comprises a phosphomannose isomerase, or pmi, gene (pmi+). In some embodiments, the pmi gene comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 11. In one embodiment, the pmi gene has a nucleic acid sequence comprising SEQ ID NO: 11. In another embodiment, the pmi gene has a nucleic acid sequence that consists of SEQ ID NO: 11.In some embodiments, the endogenous pmi gene is not deleted in the bacterium. In some other embodiments, a heterologous wild-type pmi gene is added to the bacterium. The presence of the pmi gene enables the recombinant bacterium to be mannose-independent. The nucleic acid sequence of an exemplary Salmonella pmi gene is provided below:

[0083] atgcaaaaactcattaactcagtgcaaaactatgcctggggaagtaaaactgcgttaacggaactttatggcatcgccaatccgcagcagcagccaatggctgaactctggatgggcgcgcatcccaaaagcagctcgcga atcaccaccgccaacggcgaaaccgtctccctgcgtgacgccatcgaaaagaataaaaccgccatgctgggcgaa gcggtagccaaccgtttcggcgaactgccgtttctgtttaaagtactgtgcgccgcacaaccgctctctattcag gtgcacccgaataaacgcaactccgaaatcggtttcgcgaaagaaaatgcggcgggtatccccatggatgccgca gagcggaactataaagatcctaaccataaaccagagctggtttttgccctgacgcctttcctggcgatgaacgcg 15 ME151672980v.1Attorney Docket No.: 139465-00120 ttccgcgaattttctgacattgtctctttactgcaacctgtcgccggcgcgcattccgctatcgcccactttttg caggtgccgaatgctgaacgtctgagccagcttttcgccagcctgttgaatatgcaaggcgaagaaaaatcccgc gcgttagccgtactcaaagcggcgcttaacagccagcaaggcgaaccgtggcaaacgatccgcgtgatttcagag tattatcctgacgacagcgggcttttctctcctttgttgctgaatgtggtcaaactgaatcccggcgaggcgatg ttcctgtttgctgaaacgcctcatgcttatctgcagggcgttgcgctggaagtcatggcgaactccgataacgtt ctgcgcgctggccttacgccaaaatatatcgacatccctgagctggtcgcgaacgtgaagttcgaacctaagcct gccggcgagttgctgactgccccggtgaaaagcggcgcggagctggacttcccaattccggttgacgattttgct ttttcactgcacgacctggcgcttcaggagacgagcatcggccaacacagcgccgcgattctgttctgcgttgag ggtgaggcggtgttacgtaaagatgaacagcgtctggtactgaagccgggtgaatctgcctttatcggcgcggat gagtctccggttaacgccagcggcacgggccgtttagcgcgtgtttataacaagctgtag (SEQ ID NO: 11).

[0084] In some embodiments, the recombinant bacterium comprises a deletion in the leucine-responsive regulatory (lrp) gene. The Lrp protein appears to be an antivirulence regulator in Salmonella since the deletion mutant (Δlrp) showed enhanced cell invasion, cytotoxicity, and hypervirulence. The lrp gene and methods for creating the deletion mutation Δlrp is described in the art, such as in Baek et al., “Leucine-responsive regulatory protein (Lrp) acts as a virulence repressor in Salmonella enterica serovar Typhimurium.” J Bacteriol 191:1278-1292., incorporated herein by reference in its entirety.

[0085] Any methods known in the art can be used to determine whether a recombinant Salmonella bacterium express O78 or O4 antigen. In a non-limiting embodiments, expression of O78 and O4 can be determined using anti-O78 and anti-O4 antibody, as demonstrated in Examples 2-3 of the present disclosure. 2. APEC virulence factors

[0086] Avian pathogenic E. coli (APEC) is one type of extraintestinal pathogenic E. coli (ExPEC) pathotype [Russo and Johnson, 2000]. APEC causes diverse systemic infections in poultry including chickens, turkeys, ducks, and many other avian species [Barnes et al, 2003; Dho-Moulin and Fairbrother, 1999]. The most common diseases caused by APEC in chickens collectively known as colibacillosis infections including airsacculitis, cellulitis, coligranuloma, egg peritonitis, omphalitis, osteomyelitis / arthritis, perihepatitis, pericarditis, and salphingitis [Dziva and Stevens, 2008]. Colibacillosis caused by APEC is a devastating disease of poultry that results in severe economic losses annually to the poultry industry worldwide [Newman et al., 2021].

[0087] The most prevalent virulence traits associated with APEC are adhesins (type 1 and P fimbriae, Temperature-sensitive hemagglutinin Tsh, and curli), capsules, iron acquisition systems, and serum resistance [Dho-Moulin and Fairbrother, 1999]. Large plasmids are commonly found in APEC strains and are associated with the virulence of 16 ME151672980v.1Attorney Docket No.: 139465-00120 APEC [Dozois et al, 2000; Doetkott et al, 1996; Rodriguez-Siek et al, 2005]. Their importance has been demonstrated by the occurrence of plasmid-encoded genes shared among APEC strains [Dozois et al, 2003] and the presence of virulence genes in plasmids [Johnson et al, 2002; Stehling et al, 2003]. Most virulence genes associated with APEC are often located on IncF plasmids, named ColV plasmids because their ability to code for production of colicin V, a small protein from the microcine family [Johnson et al, 2002; Johnson et al, 2004; Tivendale et al, 2004]. ColV plasmids are associated with E. coli in general and with APEC in particular. Their link with the ability of bacteria to cause disease in production animals has been demonstrated [Skyberg et al, 2006; Smith et al, 1980; Wooley et al, 1998]. In vivo expression of genes encoded by the ColV plasmid, including Tsh, aerobactin, and the Iro system, has been confirmed by using SCOTS [Dozois et al, 2003] and the occurrence of the putative virulence genes of APEC on non-ColV plasmids has also been shown [Dozois et al, 2003; Johnson et al, 2006; Dozois et al, 2000].

[0088] The complete nucleotide sequence analysis of the large ColV / IncF virulence plasmid pAPEC-1 (103,275-bp) from the APEC strain χ7122 (serogroup O78:K80:H9) revealed that a putative virulence region spanning an 80-kb region of pAPEC-1 comprises genes responsible for the potential virulence factors including four iron acquisition systems (iutA, iucABCD, sitABCD, and iroBCDN), temperature-sensitive hemagglutinin (tsh), a colicin V operon, increasing serum sensitivity (iss), ompT, hlyF, and etsABC [Mellata et al, 2009]. In addition, it has been reported that pAPEC-1-cured χ7122 bacteria were attenuated in chickens, caused few lesions of pericarditis and perihepatitis, did not persist in the blood, and poorly colonized the lung, spleen, and liver [Dozois et al, 2000]. This APEC strain χ7122 has two additional large plasmids (Figure 1), pAPEC-2 (82,676-bp) and pAPEC-3 (56,676-bp), that also have virulence attributes [Mellata et al, 2010].

[0089] In some embodiments, the recombinant Salmonella bacterium disclosed herein comprises one or more nucleotide encoding one or more virulence factor of APEC.

[0090] In some embodiments, the recombinant Salmonella bacterium comprises one or more APEC plasmids that comprises at least one gene encoding a virulence factor of APEC.

[0091] In some embodiments, the APEC plasmid is a pAPEC-1, a pAPEC-2, or a pAPEC-3 plasmid of APEC strain χ7122. As used here in, pAPEC-1, pAPEC-2 and pAPEC- 3 plasmids refer to the original pAPEC-1, pAPEC-2, or pAPEC-3 plasmids of APEC strain χ7122, respectively, as well as plasmids derived from the original pAPEC-1, pAPEC-2, or pAPEC-3 plasmids of APEC strain χ7122.

[0092] In some embodiments, the recombinant Salmonella bacterium comprises a pAPEC-1, a pAPEC-2, a pAPEC-3 plasmid, or a combination thereof. In some embodiments, 17 ME151672980v.1Attorney Docket No.: 139465-00120 the recombinant Salmonella bacterium comprises both pAPEC-1, pAPEC-2, and pAPEC-3 plasmids.

[0093] In some embodiments, the pAPEC-1 plasmid comprises a Δtra::asd mutation, which replaces the tra (traM-traX) genes on pAPEC-1 with asd gene. In some embodiments the pAPEC-1 plasmid comprises the nucleic acid sequence of SEQ ID NO: 1. In some embodiments the pAPEC-1 plasmid comprises a nucleic acid sequence having at least 50%, at least 65%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments the pAPEC-1 plasmid comprises a nucleic acid sequence consisting of SEQ ID NO: 1.

[0094] In some embodiments, the pAPEC-2 plasmid has the antimicrobial resistance (AMR) genes removed. In some embodiments, the pAPEC-2 plasmid comprises the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the pAPEC-2 plasmid comprises a nucleic acid sequence consisting of SEQ ID NO: 2. In some embodiments, the pAPEC-2 plasmid comprises a nucleic acid sequence having at least 50%, at least 65%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.

[0095] In some embodiments, the pAPEC-3 plasmid comprises the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the pAPEC-3 plasmid comprises a nucleic acid sequence consisting of SEQ ID NO: 3. In other embodiments, the pAPEC-3 plasmid comprises a nucleic acid sequence having at least 50%, at least 65%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3. 3. Recombinant attenuated Salmonella

[0096] The recombinant Salmonella bacterium provided herein further comprises additional modifications which enable it to effectively express the O78 antigen and virulence factors of APEC. In some embodiments, the recombinant bacterium is particularly effective in eliciting an immune response (e.g., protective immunity) against the pathogen of interest because the bacterium comprise multiple recombinant regulatory systems that permit the 18 ME151672980v.1Attorney Docket No.: 139465-00120 bacterium to replicate upon administration and to colonize lymphoid tissues in a subject in order to elicit potent immune responses. In other embodiments, the recombinant bacterium provided herein may ultimately exhibits an attenuated phenotype after multiple replication cycles in vivo, which allows for safe administration to a subject, for example, as a vaccine composition.

[0097] Recombinant attenuated Salmonella strains described in that art can be used to generate the recombinant Salmonella bacterium of the present disclosure. Examples for recombinant attenuated Salmonella strains may be found at least in US Patent. No. 10,988,729, incorporated herein by reference in its entirety.

[0098] The recombinant regulatory systems of the bacteria described herein depend, in part, on multiple genetic regulatory elements that are responsive to one or more sugars (e.g., arabinose, rhamnose, mannose, maltose, xylose, and galactose) that not available to the bacterium in vivo. Thus, the phenotype of the recombinant bacteria described herein can be altered upon administration to a subject. In some embodiments, the subject is administered one or more sugars before, after or concurrently with the administration of a recombinant bacterium described herein in order to activate and / or repress a sugar- responsive regulatory system of the bacteria. In some embodiments, the recombinant bacterium described herein comprises at least three regulatory systems, each dependent on a different sugar, which facilitates initial invasion of a host cell in the subject, delayed attenuation, and improved immunogenicity.

[0099] In some embodiments, the recombinant bacterium described herein can be regulated for delayed attenuation in vivo. In some embodiments, the recombinant bacterium described herein exhibits regulated production of Generalized Modules for Membrane Antigens (GMMA), or outer membrane vesicles, in vivo, which may lead to enhanced production of conserved outer membrane proteins present in the bacterium, and ultimately improved immunogenicity.

[0100] Some embodiments of the instant disclosure comprise a species or subspecies of the Salmonella genera (e.g., S. enterica or S. bongori). For instance, the recombinant bacterium may be a Salmonella enterica serovar, including, for example, Paratyphi A, Enteritidis, Typhi, and Typhimurium. In some embodiments, the recombinant bacterium is of the serovar S. Typhimurium, S. Typhi, S. Paratyphi, S. Gallinarum, S. Enteritidis, S. Choleraesius, S. Arizonae, S. Newport, S. Heidelberg, S. Infantis, S. Cholerasiuis, or S. Dublin.

[0101] A recombinant bacterium derived from Salmonella may be particularly suited to use as a vaccine. For example, oral infection of a host with a Salmonella strain typically leads to colonization of the gut-associated lymphoid tissue (GALT) or Peyer's patches, which 19 ME151672980v.1Attorney Docket No.: 139465-00120 leads to the induction of a generalized mucosal immune response to the recombinant bacterium. Further penetration of the bacterium into the mesenteric lymph nodes, liver and spleen may augment the induction of systemic and cellular immune responses directed against the bacterium. Thus, the use of recombinant Salmonella for oral immunization stimulates all three branches of the immune system, which is particularly important for immunizing against infectious disease agents that colonize on and / or invade through mucosal surfaces.

[0102] In some embodiments, the recombinant bacterium comprises a deletion in an endogenous O-antigen synthesis gene. In some embodiments, the recombinant bacterium comprises a deletion in an endogenous O-antigen ligase gene. In some embodiments, the deletion is a partial deletion of the endogenous O-antigen ligase gene. In some embodiments, the deletion is a full-length deletion of the endogenous O-antigen ligase gene. In some embodiments, the endogenous O-antigen ligase gene is genetically altered to insert a transcriptional terminator in the open reading frame of the gene. In some embodiments, a regulatory region of the endogenous O-antigen ligase gene is genetically-modified to alter (e.g., decrease) the expression of the gene. In some embodiments, the promoter of a endogenous O-antigen ligase gene is altered to include one or more regulatory elements (e.g., a sugar-responsive promoter). In some embodiments, the promoter of a endogenous O-antigen ligase gene is altered to increase the spacing between the Shine-Delgarno sequence and the start codon of the gene. In some embodiments, the promoter of a endogenous O-antigen ligase gene is altered to decrease the spacing between the Shine- Delgarno sequence and the start codon of the gene. In some embodiments, the Shine- Delgarno (SD) sequence, the start codon, the second codon and / or third codons of the O- antigen ligase gene is altered to increase the frequency of adenine nucleobases in order to enhance the translation efficiency of the gene. In some embodiments, the Shine-Delgarno (SD) sequence, the start codon, the second codon and / or third codons of the O-antigen ligase gene is altered to reduce the frequency of adenine nucleobases in order to decrease the translation efficiency of the gene. In some embodiments, the O-antigen ligase gene is waaL (also known as rfaL). The O-antigen ligase WaaL is necessary to ligate polysaccharide to the lipid A-LPS core moiety. Deletion of waaL results in an intact lipid A- LPS core with no O-antigen or individual sugars attached to it. In some embodiments, the O-antigen ligase gene is selected from the group consisting of waaG (also known as rfaG), waaI (also known as rfaI), rfaH, waaJ (also known as rfaJ), wbaP (also known as rfbP), wzy (also known as rfc), waaP, waaQ, waaF, waaP, waaC, and waaA.

[0103] In some embodiments, the recombinant bacterium described herein is modified to comprise a nucleic acid comprising an O-antigen ligase gene. In some embodiments, the 20 ME151672980v.1Attorney Docket No.: 139465-00120 nucleic acid comprising an O-antigen ligase gene is located on a plasmid in the bacterium. In some embodiments, the nucleic acid comprising an O-antigen ligase gene is located on a chromosome of the bacterium. In some embodiments, the nucleic acid comprising an O- antigen ligase gene is located at the chromosomal locus corresponding to the locus of an endogenous O-antigen ligase gene that has been deleted or altered in the bacterial chromosome. In some embodiments, the recombinant bacterium is modified to comprise a nucleic acid comprising an O-antigen ligase gene, whereby an endogenous copy of the gene in the bacterial chromosome has been altered and / or deleted. In some embodiments, the nucleic acid comprises a Salmonella O-antigen ligase gene.

[0104] The nucleic acid sequence of an exemplary Salmonella waaL gene is provided below: atgctaaccacatcattaacgttaaataaagagaaatggaagccgatctggaataaagcgctggtttttctt tttgttgccacgtattttctggatggtattacgcgttataaacatttgataatcatacttatggttatcacc gcgatttatcaggtctcacgctcaccgaaaagtttcccccctcttttcaaaaatagcgtattttatagcgta gcagtattatcattaatccttgtttattccatactcatatcgccagatatgaaagaaagtttcaaggaattt gaaaatacggtactggagggcttcttattatatactttattaattcccgtactattaaaagatgaaacaaaa gaaacggttgcgaaaatagtacttttctcctttttaacaagtttaggacttcgctgccttgcagagagtatt ctgtatatcgaggactataataaagggattatgccattcataagctatgcgcatcgacatatgtccgattcc atggttttcttatttccagcattattgaatatttggctgtttagaaaaaatgcaattaagttggtttttttg gtgcttagcgccatctaccttttctttatcctgggaaccctatcgcgaggggcatggttggcggtgcttata gtaggtgttctgtgggcaatactgaaccgccaatggaagttaataggagttggtgccattttattagccatt atcggcgctttggttatcactcaacataataacaaaccagacccagaacatttactgtataaattacagcag acagatagctcatatcgttatactaacggaacccagggcaccgcgtggatactgattcaggaaaacccgatc aagggctacggctatggtaatgatgtgtatgatggtgtttataataaacgcgttgtcgattatccaacgtgg acctttaaagaatctatcggtccgcataataccattctgtacatctggtttagtgcaggcatattgggtctg gcgagcctggtctatttatatggcgctatcatcagggaaacagccagctctaccctcaggaaagtagagata agcccctacaatgctcatctcttgctatttttatctttcgtcggtttttatatcgttcgtggcaattttgaa caggtcgatattgctcaaattggtatcattaccggttttctgctggcgctaagaaatagataa (SEQ ID NO: 5).

[0105] The amino acid sequence of the WaaL protein encoded by the nucleic acid of SEQ ID NO: 5 is provided below: MLTTSLTLNKEKWKPIWNKALVFLFVATYFLDGITRYKHLIIILMVITAIYQVSRSPKSFPPL FKNSVFYSVAVLSLILVYSILISPDMKESFKEFENTVLEGFLLYTLLIPVLLKDETKETVAKI VLFSFLTSLGLRCLAESILYIEDYNKGIMPFISYAHRHMSDSMVFLFPALLNIWLFRKNAIK LVFLVLSAIYLFFILGTLSRGAWLAVLIVGVLWAILNRQWKLIGVGAILLAIIGALVITQHNN KPDPEHLLYKLQQTDSSYRYTNGTQGTAWILIQENPIKGYGYGNDVYDGVYNKRVVDY 21 ME151672980v.1Attorney Docket No.: 139465-00120 PTWTFKESIGPHNTILYIWFSAGILGLASLVYLYGAIIRETASSTLRKVEISPYNAHLLLFLS FVGFYIVRGNFEQVDIAQIGIITGFLLALRNR (SEQ ID NO: 6).

[0106] In some embodiments, the nucleic acid comprises a Salmonella waaL gene (provided as SEQ ID NO: 5). In some embodiments, the nucleic acid comprises a waaL gene, wherein the waaL gene comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the nucleic acid comprises a waaL gene, wherein the waaL gene comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the nucleic acid sequence of SEQ ID NO: 5.

[0107] In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding an O-antigen ligase, wherein said O-antigen ligase comprises an amino acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding an O-antigen ligase, wherein said O-antigen ligase comprises an amino acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the nucleic acid sequence of SEQ ID NO: 6.

[0108] In some embodiments, the nucleic acid comprises an O-antigen ligase gene from a bacterial species, subspecies, serovar, or strain that is different than the bacterial species of the recombinant bacterium. In some embodiments, the nucleic acid comprises an O- antigen ligase gene from a bacterial species, subspecies, serovar, or strain that is the same as the bacterial species of the recombinant bacterium.

[0109] In some embodiments, the nucleic acid comprises an O-antigen ligase gene that is operably-linked to a regulatable promoter (e.g., a sugar-regulatable promoter). In some embodiments, the nucleic acid comprises an O-antigen ligase gene (e.g., waaL) that is operably-linked to a sugar-regulatable promoter. Advantageously, recombinant bacterial 22 ME151672980v.1Attorney Docket No.: 139465-00120 strains comprising a nucleic acid comprising an O-antigen ligase gene (e.g., waaL) that is operably linked to a sugar regulatable promoter will synthesize normal LPS in the presence of the sugar (e.g., rhamnose) in vitro, but will form rough LPS in vivo due to the absence of the sugar that activates the promoter and therefore, the expression of the O-antigen ligase. Without wishing to be bound by any particular theory, using this strategy, the bacterium will expose conserved LPS core oligosaccharide and have enhanced production of conserved outer membrane proteins (OMPs; e.g., porins) which may lead to improved immunogenicity and aid in the production of a cross-protective immune response against an antigen of interest synthesized in the bacterium in vivo. In some embodiments, the sugar regulatable promoter exhibits increased activity (e.g., increased transcription) in the presence of a specific sugar and decreased activity in the absence of a sugar. In some embodiments, the nucleic acid comprises an O-antigen ligase gene that is operably-linked to a rhamnose- regulatable promoter (e.g., a sugar-regulatable promoter). In some embodiments, the nucleic acid comprises an O-antigen ligase gene that is operably-linked to an arabinose- regulatable promoter (e.g., a sugar-regulatable promoter). In some embodiments, the use of a rhamnose-regulatable promoter (e.g., rhaSR PrhaBAD) may be preferable to an arabinose- regulatable promoter because a relatively higher concentration is required to activate an arabinose-regulatable promoter as compared to a rhamnose-regulatable promoter (see, e.g., Giacalone et al. (2006) BioTechniques 40(3): 355-366 (39), the entire contents of which are incorporated herein by reference). In some embodiments, the recombinant bacterium comprises the mutation ΔwaaL / ΔpagL::TT rhaSR PrhaBADwaaL.

[0110] In some embodiments, the recombinant bacterium comprises a deletion in an endogenous lipid A deacylase gene. In some embodiments, the deletion is a partial deletion of the endogenous lipid A deacylase gene. In some embodiments, the deletion is a full- length deletion of the endogenous lipid A deacylase gene. In some embodiments, the endogenous lipid A deacylase gene is genetically altered to insert a transcriptional terminator in the open reading frame of the gene. In some embodiments, a regulatory region of the endogenous lipid A deacylase gene is genetically-modified to alter (e.g., decrease) the expression of the gene. In some embodiments, the promoter of an endogenous lipid A deacylase gene is altered to include one or more regulatory elements (e.g., a sugar- responsive promoter). In some embodiments, the lipid A deacylase gene is pagL. Bacterial comprising a deletion of the lipid A deacylase gene pagL have been found to produced increased amounts of outer membrane vesicles (see, e.g., Elhenawy et al. (2016) mBio 7(4): e00940-16 (40)). Deletion of the pagL gene of Salmonella does not impair bacterial virulence (see, e.g., Man et al. Proc. Nat’l. Acad. Sci. USA 111: 7403-8 (41)). Without wishing to be bound by any particular theory, in some embodiments, the recombinant 23 ME151672980v.1Attorney Docket No.: 139465-00120 bacterium described herein comprise one or more genetic modifications which results in increased vesiculation (i.e., increased vesicle production) which may be particularly advantageous in inducing an immune response in the host against an antigen of interest that is expressed by the bacterium.

[0111] In some embodiments, the recombinant bacterium comprises a deletion in an endogenous phosphomannose isomerase gene. Phosphomannose isomerase, also known as mannose-6 phosphate isomerase, catalyzes the reversible interconversion of fructose 6- phosphate to mannose 6-phosphate. Mannose 6-phosphate is then converted to GDP- mannose and used for the synthesis of O-antigen side chains. Bacteria with deletions of the phosphomannose isomerase gene pmi are not mannose sensitive and are partially attenuated (see, e.g., Collins et al. (1991) Infect. Immun.59(3): 1079–85 (42)). These pmi mutants synthesize wild-type levels of LPS O-antigen side chains when grown in media containing mannose, and are both attenuated but highly immunogenic (see, e.g., Curtiss et al. (2007) “Induction of host immune responses using Salmonella-vectored vaccines.” In: Brogden KA, Minion FC, Cornick N, Stanton TB, Zhang Q, Nolan LK, Wannemuehler MJ, ed. Virulence Mechanisms of Bacterial Pathogens.4th ed. Washington DC: ASM Press (43)). In some embodiments, the deletion of the endogenous phosphoisomerase gene is a partial deletion. In some embodiments, the deletion of the endogenous phosphomannose isomerase gene is a full-length deletion. In some embodiments, the endogenous phosphomannose isomerase gene is genetically altered to insert a transcriptional terminator in the open reading frame of the gene. In some embodiments, a regulatory region of the endogenous phosphomannose isomerase gene is genetically-modified to alter (e.g., decrease) the expression of the phosphomannose isomerase gene. In some embodiments, the promoter of an endogenous phosphomannose isomerase gene is altered to include one or more regulatory elements (e.g., a sugar-responsive promoter). In some embodiments, the phosphomannose isomerase gene is pmi.

[0112] In some embodiments, the bacterium comprises a deletion of a pmi gene. In some embodiments, the bacterium comprises a deletion in the pmi gene (Δpmi), e.g., Δpmi- 2426 mutation. A bacterium comprising a Δpmi-2426 mutation, grown in the presence of mannose, is capable of synthesizing a complete LPS O-antigen. Non-phosphorylated mannose, which is the form required for bacterial uptake, is unavailable in vivo. Hence, a bacterium comprising a Δpmi-2426 mutation loses the ability to synthesize LPS O-antigen serotype specific side chains in vivo and the number of O-antigen side chains attached to the LPS core decreases by about half after each cell division in vivo. The LPS that is synthesized comprises a core structure that is substantially similar across all Salmonella enterica serotypes except S. Arizona. This results in a bacterium that is capable of eliciting 24 ME151672980v.1Attorney Docket No.: 139465-00120 an immune response against at least two Salmonella serotypes without substantially inducing an immune response specific to the serotype of the bacterial vector. In some embodiments, the bacterium is capable of eliciting an immune response against all Salmonella serotypes without substantially inducing an immune response specific to the serotype of the bacterial vector.

[0113] A recombinant bacterium described herein that comprises a deletion in a pmi mutation may also comprise other mutations that ensure that mannose available to the bacterium during in vitro growth is used for LPS O-antigen synthesis. For instance, a bacterium may comprise a deleteion in the gmd-fcl genes (Δ(gmd-fcl)), e.g., Δ(gmd-fcl)-26 mutation. This mutation deletes two nucleic acid sequences that encode enzymes for conversion of GDP-mannose to GDP-fucose, ensuring that mannose available to the bacterium during in vitro growth is used for LPS O-antigen synthesis and not colanic acid production. Similarly, a bacterium may comprise a deletion in the wcaM-wza gene cluster (Δ(wcaM-wza)) e.g., the Δ(wcaM-wza)-8 mutation, which deletes all 20 nucleic acid sequences necessary for colanic acid production, and also precludes conversion of GDP- mannose to GDP-fucose.

[0114] In some embodiments, the recombinant bacterium has been genetically-altered such that the bacterium is capable of escaping the endosomal compartment of a host cell. A recombinant bacterium may exhibit a temporal delay in escaping an endosome following invasion of the host cell. Methods of detecting escape from an endosomal compartment of a host cell are well known in the art, and include, for example, microscopic analysis.

[0115] In some embodiments, the recombinant bacterium comprises a deletion in an endogenous sifA gene. In some embodiments, the recombinant bacterium comprises a mutation that alters the function of SifA. SifA is an effector protein necessary for the formation of Salmonella-induced filaments and for the maintenance of the vacuolar membrane enclosing the bacterium. Bacteria comprising a deletion of sifA are capable of escaping the host cell endosome (also called the Salmonella-containing vesicle, or SCV) following cellular invasion. In some embodiments, the deletion of the endogenous sifA gene is a partial deletion. In some embodiments, the deletion of the endogenous sifA gene is a full-length deletion. In some embodiments, the endogenous sifA gene is genetically altered to insert a transcriptional terminator in the open reading frame of the gene. In some embodiments, a regulatory region of the endogenous sifA gene is genetically-modified to alter (e.g., decrease) the expression of the sifA gene. In some embodiments, the promoter of an endogenous sifA gene is altered to include one or more regulatory elements (e.g., a sugar-responsive promoter). 25 ME151672980v.1Attorney Docket No.: 139465-00120

[0116] In some embodiments, the recombinant bacterium described herein is modified to comprise a nucleic acid comprising a sifA gene. In some embodiments, the nucleic acid comprising a sifA gene is located on a plasmid in the bacterium. In some embodiments, the nucleic acid comprising a sifA gene is located on a chromosome of the bacterium. In some embodiments, the nucleic acid comprising a sifA gene is located at the chromosomal locus corresponding to the locus of an endogenous a sifA that has been deleted or altered in the bacterial chromosome. In some embodiments, the recombinant bacterium is modified to comprise a nucleic acid comprising a sifA gene, whereby an endogenous copy of the sifA gene in the bacterial chromosome has been altered and / or deleted.

[0117] The nucleic acid sequence of an exemplary Salmonella sifA gene is provided below: atgccgattactatagggaatggttttttaaaaagtgaaatccttaccaactccccaaggaatacgaa agaagcatggtggaaagttttatgggaaaaaattaaagacttctttttttctactggcaaagcaaaag cggaccgttgtctacatgagatgttgtttgccgaacgcgcccccacacgagagcggcttacagagatt ttttttgagttgaaagagttagcctgcgcatcgcaaagagatagatttcaggttcataatcctcatga aaatgatgccaccattattcttcgcatcatggatcaaaacgaagagaacgaattgttacgtatcactc aaaataccgatacctttagctgtgaagtcatggggaatctttattttttaatgaaagatcgcccggat attttaaaatcgcatccacaaatgacggccatgattaagagaagatatagcgaaatcgtagactaccc cctcccttcgacattatgtctcaatcctgctggcgcgccgatattatcggttccattagacaacatag aggggtatttatatactgaattgagaaaaggacatttagatgggtggaaagcgcaagaaaaggcaacc tacctggcagcgaaaattcagtctgggattgaaaagacaacgcgcattttacaccatgcgaatatatc cgaaagtactcagcaaaacgcatttttagaaacaatggcgatgtgtggattaaaacagcttgaaatac caccaccgcatacccacatacctattgaaaaaatggtaaaagaggttttactagcggataagacgttt caggcgttcctcgtaacggatcccagcaccagccaaagtatgttagctgagatagtcgaagccatctc tgatcaggtttttcacgccatttttagaatagacccccaggctatacaaaaaatggcggaagaacagt taaccacgctacacgttcgctcagaacaacaaagcggctgtttatgttgttttttataa (SEQ ID NO: 7).

[0118] The amino acid sequence of the SifA protein encoded by the nucleic acid of SEQ ID NO: 7 is provided below: MPITIGNGFLKSEILTNSPRNTKEAWWKVLWEKIKDFFFSTGKAKADRCLHEMLFAERA PTRERLTEIFFELKELACASQRDRFQVHNPHENDATIILRIMDQNEENELLRITQNTDTFS CEVMGNLYFLMKDRPDILKSHPQMTAMIKRRYSEIVDYPLPSTLCLNPAGAPILSVPLDN IEGYLYTELRKGHLDGWKAQEKATYLAAKIQSGIEKTTRILHHANISESTQQNAFLETMA MCGLKQLEIPPPHTHIPIEKMVKEVLLADKTFQAFLVTDPSTSQSMLAEIVEAISDQVFHA IFRIDPQAIQKMAEEQLTTLHVRSEQQSGCLCCFL (SEQ ID NO: 8). 26 ME151672980v.1Attorney Docket No.: 139465-00120

[0119] In some embodiments, the nucleic acid comprises a Salmonella sifA gene (provided as SEQ ID NO: 7). In some embodiments, the nucleic acid comprises a sifA gene, wherein the sifA gene comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the nucleic acid comprises a sifA gene, wherein the sifA gene comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the nucleic acid sequence of SEQ ID NO: 7.

[0120] In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding a SifA protein, wherein said SifA protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding a SifA protein, wherein said SifA protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the nucleic acid sequence of SEQ ID NO: 8.

[0121] In some embodiments, the nucleic acid comprises a sifA gene from a bacterial species, subspecies, serovar, or strain that is different than the bacterial species of the recombinant bacterium. In some embodiments, the nucleic acid comprises a sifA gene from a bacterial species, subspecies, serovar, or strain that is the same as the bacterial species of the recombinant bacterium.

[0122] In some embodiments, the nucleic acid comprises a sifA gene that is operably- linked to a regulatable promoter (e.g., a sugar-regulatable promoter). In some embodiments, the nucleic acid comprises a sifA gene that is operably-linked to a sugar- regulatable promoter. In some embodiments, the sugar regulatable promoter exhibits increased activity (e.g., increased transcription) in the presence of a specific sugar and decreased activity in the absence of a sugar. In some embodiments, the nucleic acid comprises a sifA gene that is operably-linked to a rhamnose-regulatable promoter (e.g., a 27 ME151672980v.1Attorney Docket No.: 139465-00120 sugar-regulatable promoter). In some embodiments, the nucleic acid comprises a sifA gene that is operably-linked to an arabinose-regulatable promoter. In some embodiments, the arabinose-regulatable promoter is ParaBAD. In some embodiments, the recombinant bacterium comprises the mutation ΔsifA::TT araC ParaBADsifA. In some embodiments, the recombinant bacterium comprises the mutation ΔPsifA::TT araC ParaBADsifA. When the expression of the nucleic acid comprising a sifA gene is under the control of an arabinose- regulated promoter, the bacterial escape from the host endosome can be delayed. Since arabinose is absent in host cells, arabinose cannot induce the expression of the sifA gene. Thus, if the recombinant bacterium is cultured in the presence of arabinose prior to administration to the subject, the expression of sifA will gradually decrease with each round of bacterial cell division thereby allowing escape of the bacterium from the host cell endosome during the initial cell division cycles. Similar delayed-escape mutations may be constructed using other regulatable promoters, such as from the xylose-regulatable or rhamnose-regulatable promoter systems.

[0123] In some embodiments, the recombinant bacterium comprises a deletion in an endogenous relA gene, which encodes the GTP pyrophosphokinase RelA. The inclusion of a relA deletion in the recombinant bacterium uncouples the occurrence of growth-dependent lysis to the need for continued protein synthesis. In some embodiments, the deletion of the endogenous relA gene is a partial deletion. In some embodiments, the deletion of the endogenous relA gene is a full-length deletion.

[0124] Other methods of attenuation are known in the art. For instance, attenuation may be accomplished by altering (e.g., deleting) native nucleic acid sequences found in the wild-type bacterium. For instance, if the bacterium is Salmonella, non-limiting examples of nucleic acid sequences which may be used for attenuation include: a pab nucleic acid sequence, a pur nucleic acid sequence, an aro nucleic acid sequence, asd, a dap nucleic acid sequence, nadA, pncB, galE, pmi, fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, dam, phoP, phoQ, rfc, poxA, galU, mviA, sodC, recA, ssrA, sirA, inv, hilA, rpoE, flgM, tonB, slyA, and any combination thereof. Exemplary attenuating mutations may be aroA, aroC, aroD, cdt, cya, crp, phoP, phoQ, ompR, galE, and htrA.

[0125] In certain embodiments, the above nucleic acid sequences may be placed under the control of a sugar regulated promoter wherein the sugar is present during in vitro growth of the recombinant bacterium, but substantially absent within an animal or human host. The cessation in transcription of the nucleic acid sequences listed above would then result in attenuation and the inability of the recombinant bacterium to induce disease symptoms.

[0126] In some embodiments, the recombinant bacterium comprises a deletion in an endogenous recF gene, which encodes the DNA replication and repair protein RecF. In 28 ME151672980v.1Attorney Docket No.: 139465-00120 some embodiments, the deletion of the endogenous recF gene is a partial deletion. In some embodiments, the deletion of the endogenous recF gene is a full-length deletion. In some embodiments, the endogenous recF gene is genetically altered to insert a transcriptional terminator in the open reading frame of the gene.

[0127] In some embodiments, the recombinant bacterium comprises a deletion in an endogenous recJ gene, which encodes the exonuclease RecJ. In some embodiments, the deletion of the endogenous recJ gene is a partial deletion. In some embodiments, the deletion of the endogenous recJ gene is a full-length deletion. In some embodiments, the endogenous recJ gene is genetically altered to insert a transcriptional terminator in the open reading frame of the gene.

[0128] In some embodiments, the recombinant bacterium comprises a deletion in an endogenous asd gene. As used herein, the terms asd and asdA refer to the same gene and are used interchangeably. In some embodiments, the deletion of the endogenous asd gene is a partial deletion. In some embodiments, the deletion of the endogenous asd gene is a full-length deletion. In some embodiments, the endogenous asd gene is genetically altered to insert a transcriptional terminator in the open reading frame of the gene. In some embodiments, the promoter of a endogenous asd gene is altered to include one or more regulatory elements (e.g., a sugar-responsive promoter). In one example, the bacterium is modified by using a ΔasdA mutation to eliminate the bacterium’s ability to produce β- aspartate semialdehyde dehydrogenase, an enzyme essential for the synthesis of DAP. Other mutations that result in the abolition of the synthesis of DAP include, but are not limited to, dapA, dapB, dapC, dapD, dapE, dapF, and asd (see, e.g., U.S. Patent No. 6,872,547, incorporated herein by reference). Other modifications that may be employed include modifications to a bacterium’s ability to synthesize D-alanine or to synthesize D- glutamic acid (e.g., ΔmurI mutations), which are both unique constituents of the peptidoglycan layer of the bacterial cell wall.

[0129] In some embodiments, the recombinant bacterium comprises an insertion of a c2 gene operably linked to an arabinose regulatable promoter. In some embodiments, the arabinose regulatable promoter is araC ParaBADpromoter, which is described in US Patent. No.10,988,729, incorporated herein by reference. Various embodiments may comprise the araC ParaBADc2 gene cassette inserted into the asd nucleic acid sequence that encodes aspartate semialdehyde dehydrogenase. Since the araC nucleic acid sequence is transcribed in a direction that could lead to interference in the expression of adjacent nucleic acid sequences and adversely affect vaccine strain performance, a transcription termination (TT) sequence is generally inserted 3’ to the araC nucleic acid sequence. The chromosomal asd nucleic acid sequence is typically inactivated to enable use of plasmid vectors encoding 29 ME151672980v.1Attorney Docket No.: 139465-00120 the wild-type asd nucleic acid sequence in the balanced lethal host-vector system. This allows for stable maintenance of plasmids in vivo in the absence of any drug resistance attributes that are not permissible in live bacterial vaccines. In some of these embodiments, the wild-type asd nucleic acid sequence may be encoded by the vector described herein. The vector enables the regulated expression of an antigen encoding sequence through the repressible promoter.

[0130] In some embodiments, the recombinant bacterium comprises an asd gene (asd+). In some embodiments, the endogenous asd gene is not deleted in the bacterium. In some other embodiments, a heterologous wild-type asd gene is added to the bacterium (e.g., added to pAPEC-1 plasmid that was transferred into the bacterium). In some embodiments, the asd gene comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 12. In one embodiment, the asd gene has a nucleic acid sequence comprising SEQ ID NO: 12. In another embodiment, the asd gene has a nucleic acid sequence that consists of SEQ ID NO: 12. The presence of the asd gene enables the recombinant bacterium to grow without diaminopimelic acid (DAP). The nucleic acid sequence of an exemplary Salmonella asd gene is provided below: atgaaaaatgttggttttatcggctggcgcggagtggtcggctctgttctcatgcaacgcatggtaga ggagcgcgatttcgacgctattcgccctgttttcttttctacctcccagtttggacaggcggcgccca ccttcggcgacacctccggcacgctacaggacgcttttgacctggatgcgctaaaagcgctcgatatc atcgtgacctgccagggcggcgattataccaacgaaatttatccaaagctgcgcgaaagcggatggca gggttactggattgatgcggcttctacgctgcgcatgaaagatgatgccattattattctcgacccgg tcaaccaggacgtgattaccgacggcctgaacaatggcgtgaagacctttgtgggcggtaactgtacc gttagcctgatgttgatgtcgctgggcggtctctttgcccataatctcgttgactgggtatccgtcgc gacctatcaggccgcctccggcggcggcgcgcgccatatgcgcgagctgttaacccagatgggtcagt tgtatggccatgtcgccgatgaactggcgacgccgtcttccgcaattcttgatattgaacgcaaagtt acggcattgacccgcagcggcgagctgccggttgataactttggcgtaccgctggcgggaagcctgat cccctggatcgacaaacagctcgataacggccagagccgcgaagagtggaaaggccaggcggaaacca acaagattctcaatactgcctctgtgattccggttgatggtttgtgtgtgcgcgtcggcgcgctgcgc tgtcacagccaggcgttcaccatcaagctgaaaaaagaggtatccattccgacggtggaagaactgct ggcggcacataatccgtgggcgaaagtggtgccgaacgatcgtgatatcactatgcgcgaattaaccc cggcggcggtgaccggcacgttgactacgccggttggtcgtctgcgtaagctgaacatggggccagag ttcttgtcggcgtttaccgtaggcgaccagttgttatggggcgccgccgagccgctgcgtcgaatgct gcgccagttggcctag (SEQ ID NO: 12) 30 ME151672980v.1Attorney Docket No.: 139465-00120

[0131] In some embodiments, the recombinant bacterium comprises a nucleic acid (e.g., a gene) that is operably linked to a repressor-regulatable promoter to facilitate the regulatable expression of the gene. Thus, in some embodiments, the recombinant bacterium comprises a nucleic acid comprising a gene encoding a repressor. In some embodiments, the gene encoding the repressor is operably-linked to a regulatable promoter. Methods of chromosomally integrating a nucleic acid sequence encoding a repressor operably-linked to a regulatable promoter are known in the art and detailed in the examples. In some embodiments, the nucleic acid sequence encoding a repressor is not integrated into a chromosomal locus such that the ability of the bacterium to colonize a host cell is disrupted. In some embodiments, the recombinant bacterium comprises a nucleic acid encoding a repressor that is integrated into the relA locus of the bacterial chromosome. In some embodiments, the recombinant bacterium comprises a nucleic acid encoding a repressor that is integrated into the endA locus of the bacterial chromosome. In some embodiments, the recombinant bacterium comprises at least one nucleic acid sequence encoding a repressor. In some embodiments, the recombinant bacterium comprises at least two, at least three, at least four, at least five, at least six or more nucleic acids encoding a repressor. In some embodiments, the nucleic acid encoding the repressor is present on a plasmid in the bacterium. In some embodiments, the nucleic acid encoding the repressor is located in the bacterial chromosome. If there is more than one nucleic acid sequence encoding a repressor, each nucleic acid sequence encoding a repressor may be operably linked to a regulatable promoter, such that each promoter is regulated by the same compound or condition. Alternatively, each nucleic acid sequence encoding a repressor may be operably linked to a regulatable promoter, each of which is regulated by a different compound or condition.

[0132] As used herein, a “repressor” refers to a biomolecule that represses the transcriptional activity of a promoter. In some embodiments, the repressor is synthesized by the recombinant bacterium in high enough quantities during in vitro culture, such that the transcription of a nucleic acid that is operably linked to a repressor-regulatable promoter is repressed. This may be particularly advantageous if, for example, expression of the product encoded by said nucleic acid impedes the in vitro growth of the bacterium, and / or the ability of the bacterium to infect and / or colonize a subject. In some embodiments, the nucleic acid that is operably-linked to the repressor-regulatable promoter expresses an antigen of interest. In some embodiments, the concentration of the repressor within the cell gradually decreases with each cell division cycle after transcription of the gene encoding the repressor decreases or ceases (e.g., in vivo). The use of a particular repressor, as described herein, may depend, in part, on the species, subspecies, strain or serovar of the recombinant 31 ME151672980v.1Attorney Docket No.: 139465-00120 bacterium being used. In some embodiments, the repressor is derived from the same species (e.g., the same bacterial species or the same phage) from which the repressor- regulatable promoter is derived. In some embodiments the repressor is not derived from the same bacterial species as the bacterial species in which the repressor is expressed. For example, in some embodiments, the repressor is derived from E. coli if the recombinant bacterium is of the genus Salmonella. Other suitable repressors include repressors derived from a bacteriophage.

[0133] A nucleic acid sequence encoding a repressor and regulatable promoter detailed above may be modified so as to optimize the expression level of the nucleic acid sequence encoding the repressor. The optimal level of expression of the nucleic acid sequence encoding the repressor may be estimated, or may be determined by experimentation. Such a determination should take into consideration whether the repressor acts as a monomer, dimer, trimer, tetramer, or higher multiple, and should also take into consideration the copy number of the vector encoding the antigen of interest. In an exemplary embodiment, the level of expression is optimized so that the repressor is synthesized while in a permissive environment (i.e., in vitro growth) at a level that substantially inhibits the expression of the nucleic acid encoding an antigen of interest, and is substantially not synthesized in a non- permissive environment, thereby allowing expression of the nucleic acid encoding an antigen of interest.

[0134] In some embodiments, the recombinant bacterium described herein is modified to comprise a nucleic acid comprising a lacI gene, which encodes the LacI repressor protein. The expression of the lacI-encoded repressor in the recombinant bacterium described herein may be used to regulate the expression of a gene encoding an antigen of interest expressed by the bacterium. For example, in some embodiments, the expression of the lacI gene is regulated by a sugar-regulatable promoter (e.g., an arabinose-regulatable promoter). When cultured in the presence of arabinose, the recombinant bacterium will synthesize the LacI repressor protein, which in turn will repress the expression of a gene encoding an antigen of interest that is operably-linked to a LacI-responsive promoter (e.g., Ptrc, Plac, PT7lacand Ptac). Upon administration to the subject and in the absence of a source of arabinose, the synthesis of LacI repressor ceases, leading to de-repression of the LacI-responsive promoter and the subsequence causing expression of the antigen of interest. The concentration of LacI in the cell decreases by about half at each cell division in vivo, leading to a gradual decreased level of repression and gradual increased synthesis of the antigen of interest.

[0135] In some embodiments, the nucleic acid comprising a lacI gene is located on a plasmid in the bacterium. In some embodiments, the nucleic acid comprising a lacI gene is 32 ME151672980v.1Attorney Docket No.: 139465-00120 located on a chromosome of the bacterium. In some embodiments, the nucleic acid comprising a lacI gene is located at the chromosomal locus corresponding to the locus of an endogenous relA gene that has been deleted or altered in the bacterial chromosome. In some embodiments, the recombinant bacterium is modified to comprise a nucleic acid comprising a lacI gene, whereby an endogenous copy of the lacI gene in the bacterial chromosome has been altered and / or deleted.

[0136] In some embodiments, the nucleic acid comprises an Escherichia coli lacI gene. The nucleic acid sequence of the E. coli lacI gene is provided below: gtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggcca gccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcac aacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgc ggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgta aagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgc tgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatga agacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttc tgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggc gactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgcca acgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggat acgacgataccgaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaacca gcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaa aaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggttt cccgactggaaagcgggcagtga (SEQ ID NO: 9).

[0137] The amino acid sequence of the E. coli LacI protein encoded by the nucleic acid of SEQ ID NO: 9 is provided below: MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAG KQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQ RVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALG HQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNE GIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRL LGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQV SRLESGQ (SEQ ID NO: 10).

[0138] In some embodiments, the nucleic acid comprises a lacI gene, wherein the lacI gene comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleic 33 ME151672980v.1Attorney Docket No.: 139465-00120 acid sequence of SEQ ID NO: 9. In some embodiments, the nucleic acid comprises a lacI gene, wherein the lacI gene comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the nucleic acid sequence of SEQ ID NO: 9.

[0139] In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding a LacI protein, wherein said LacI protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding a LacI protein, wherein said LacI protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the nucleic acid sequence of SEQ ID NO: 10.

[0140] In some embodiments, the nucleic acid comprises a lacI gene that is operably- linked to a regulatable promoter (e.g., a sugar-regulatable promoter). In some embodiments, the nucleic acid comprises a lacI gene that is operably-linked to a sugar- regulatable promoter. In some embodiments, the sugar regulatable promoter exhibits increased activity (e.g., increased transcription) in the presence of a specific sugar and decreased activity in the absence of a sugar. In some embodiments, the nucleic acid comprises a lacI gene that is operably-linked to a rhamnose-regulatable promoter (e.g., a sugar-regulatable promoter). In some embodiments, the nucleic acid comprises a lacI gene that is operably-linked to an arabinose-regulatable promoter. In some embodiments, the arabinose-regulatable promoter is ParaBAD. In some embodiments, the recombinant bacterium comprises the mutation ΔrelA::araC ParaBADlacI TT.

[0141] The recombinant Salmonella bacterium of the present disclosure may be derived from any recombinant attenuated Salmonella bacterium, such as any of the bacterium disclosed in US Patent. No.10,988,729, incorporated herein by reference in its entirety. The recombinant Salmonella bacterium of the present disclosure will contains all of the genetic features or modifications of the bacterium from which it is derived that have not been altered.

[0142] In some embodiments, the recombinant Salmonella bacterium of the present disclosure comprises, or is derived from a Salmonella bacterium comprising: a araC ParaBAD- 34 ME151672980v.1Attorney Docket No.: 139465-00120 regulated murA gene (ΔPmurA::TT araC ParaBADmurA deletion-insertion mutation); a deletion- insertion mutation that inactivates the expression of asdA gene and inserts a c2 gene (ΔasdA::TT araC ParaBADc2 deletion-insertion mutation); a deletion mutation in the gmd and a deletion in the fcl genes (Δ(gmd-fcl)); and a deletion-insertion mutation that inactivates the expression of a relA gene and inserts a lacI gene (ΔrelA::araC ParaBADlacI TT deletion- insertion mutation).

[0143] In some other embodiments, the recombinant Salmonella bacterium of the present disclosure comprises, or is derived from a Salmonella bacterium comprising: anaraC ParaBAD-regulated murA gene (ΔPmurA::TT araC ParaBAD murA deletion-insertionmutation); a deletion-insertion mutation that inactivates the expression of asdA gene and inserts a c2 gene (ΔasdA::TT araC ParaBADc2 deletion-insertion mutation); a deletion mutation of the gene cluster wza-wcaM (Δ(wza-wcaM)); a deletion-insertion mutation that inactivates the expression of a relA gene and inserts a lacI gene (ΔrelA::araC ParaBADlacI TT deletion-insertion mutation); a deletion mutation in the recF gene (ΔrecF); a deletion mutation in the sifA gene (ΔsifA); a deletion mutation in the waaL gene (ΔwaaL46); and a deletion in a pagL gene and an insertion of a rhaRS PrhaBAD-regulated waaL gene (ΔpagL::TT rhaRS PrhaBADwaaL).

[0144] In some embodiments, the recombinant Salmonella bacterium comprises the genetic features of any of the strains listed in Table A below: Table A: Non-limiting examples of recombinant Salmonella bacterium35 ME151672980v.1Attorney Docket No.: 139465-00120Pharmaceutical Compositions and Methods of Use

[0145] A recombinant bacterium provided in the present disclosure may be administered to a subject as a pharmaceutical composition. In some embodiments, the pharmaceutical composition may be used as a vaccine to elicit an immune response to an APEC. In an exemplary embodiment, the immune response is protective. 36 ME151672980v.1Attorney Docket No.: 139465-00120

[0146] Pharmaceutical compositions may be administered to any host capable of mounting an immune response. Such hosts may include all vertebrates, for example, mammals, including domestic animals, agricultural animals, laboratory animals, and humans, and various species of birds, including domestic birds and birds of agricultural importance. Preferably, the host is a warm-blooded animal. In one embodiment, the host is a cow. In some embodiments, the host is an equine. In another embodiment, the host is an avian. In another embodiment, the host is a human. The pharmaceutical composition can be administered to the subject as a prophylactic or for treatment purposes.

[0147] In some embodiments, the recombinant bacterium is alive when administered to a host in a pharmaceutical composition described herein. Suitable vaccine composition formulations and methods of administration are detailed below.

[0148] A pharmaceutical composition comprising a recombinant bacterium may optionally comprise one or more possible additives, such as carriers, preservatives, stabilizers, adjuvants, and other substances.

[0149] In one embodiment, the pharmaceutical composition comprises an adjuvant. Adjuvants are optionally added to increase the ability of the vaccine to trigger, enhance, or prolong an immune response. In exemplary embodiments, the use of a live attenuated recombinant bacterium may act as a natural adjuvant. In some embodiments, the recombinant bacterium synthesizes and secretes an immune modulator. Additional materials, such as cytokines, chemokines, and bacterial nucleic acid sequences naturally found in bacteria, like CpG, are also potential vaccine adjuvants.

[0150] In some embodiments, the pharmaceutical composition comprises buffered saline (e.g., phosphate-buffered saline (PBS)).

[0151] In some embodiments, the pharmaceutical composition comprises a food product.

[0152] In another embodiment, the pharmaceutical may comprise a pharmaceutical carrier (or excipient). Such a carrier may be any solvent or solid material for encapsulation that is non-toxic to the inoculated host and compatible with the recombinant bacterium. A carrier may give form or consistency, or act as a diluent. Suitable pharmaceutical carriers may include liquid carriers, such as normal saline and other non-toxic salts at or near physiological concentrations, and solid carriers not used for humans, such as talc or sucrose, or animal feed. Carriers may also include stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Carriers and excipients as well as formulations for parenteral and nonparenteral drug delivery are set forth in Remington’s Pharmaceutical Sciences 19th Ed. 37 ME151672980v.1Attorney Docket No.: 139465-00120 Mack Publishing (1995). When used for administering via the bronchial tubes, the pharmaceutical composition is preferably presented in the form of an aerosol.

[0153] In some embodiments, the pharmaceutical composition is delivered to a farm animal (e.g., poultry). In some embodiments, the pharmaceutical composition is delivered as a course spray (e.g., for use in hatcheries for delivery to poultry). In some embodiments, the pharmaceutical composition is delivered through oral immunization, such as in drinking water or mixed with food.

[0154] Care should be taken when using additives so that the live recombinant bacterium is not killed, or have its ability to effectively colonize lymphoid tissues such as the gut-associated lymphoid tissue (GALT), nasal-associated lymphoid tissue (NALT) and bronchus-associated lymphoid tissue (BALT) compromised by the use of additives. Stabilizers, such as lactose or monosodium glutamate (MSG), may be added to stabilize the pharmaceutical composition against a variety of conditions, such as temperature variations or a freeze-drying process. The recombinant bacterium may also be co-administered with glutamate and / or arginine as described herein.

[0155] The dosages of a pharmaceutical composition can and will vary depending on the recombinant bacterium, the regulated antigen, and the intended host, as will be appreciated by one of skill in the art. Generally speaking, the dosage need only be sufficient to elicit a protective immune response in a majority of hosts. Routine experimentation may readily establish the required dosage. Typical initial dosages of vaccine for oral administration could be about 1 x107to 1 x 1010CFU depending upon the age of the host to be immunized. Administering multiple dosages may also be used as needed to provide the desired level of protective immunity.

[0156] In order to stimulate a preferred response of the GALT, NALT or BALT cells, administration of the pharmaceutical composition directly into the gut, nasopharynx, or bronchus is preferred, such as by oral administration, intranasal administration, gastric intubation or in the form of aerosols, although other methods of administering the recombinant bacterium, such as intravenous, intramuscular, subcutaneous injection or intramammary, intrapenial, intrarectal, vaginal administration, or other parenteral routes, are possible, e.g., for anti-cancer applications.

[0157] In some embodiments, these compositions are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intradermally, intramuscularly, etc.).

[0158] In an aspect, the disclosure provides a method for eliciting an immune response against an avian pathogenic E. coli in a subject. In another aspect, the disclosure provides a method for vaccinating a subject against an avian pathogenic E. coli. The method comprises 38 ME151672980v.1Attorney Docket No.: 139465-00120 administering to the subject an effective amount of a pharmaceutical composition comprising a recombinant bacterium described herein.

[0159] In a further embodiment, a recombinant bacterium described herein may be used in a method for ameliorating one or more symptoms of an infectious disease in a host in need thereof. The method comprises administering an effective amount of a pharmaceutical composition comprising a recombinant bacterium as described herein.

[0160] In some embodiments, the recombinant bacterium described herein is used to induce an immune response in poultry (e.g., as a vaccine). When used in poultry (e.g., chicken, turkey, goose, or duck), the recombinant bacterium may be administered by course spray and thereby inoculate the conjunctiva-associated lymphoid tissue (CALT) via eye exposure, the nasal-associated lymphoid tissue (NALT) and bronchus-associated lymphoid tissue (BALT) via respiratory exposure and the gut-associated lymphoid tissue (GALT) via oral exposure. In some embodiments, the recombinant bacterium described herein is administered to newly-hatched chicks. EXAMPLES

[0161] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Example 1: Background

[0162] Pathogenic Escherichia coli isolates have been classified as either intestinal pathogenic E. coli (IPEC) or extraintestinal pathogenic E. coli (ExPEC) based on the anamnestic clinical reports and virulence features [Russo and Johnson, 2000; Russo and Johnson, 2003]. Extraintestinal pathogenic E. coli (ExPEC) pathotype includes newborn avian pathogenic E. coli (APEC), meningitis E. coli (NMEC), uropathogenic E. coli (UPEC), and septicemic E. coli (SEPEC). [Russo and Johnson, 2000]. APEC causes diverse systemic infections in poultry including chickens, turkeys, ducks, and many other avian species [Barnes et al, 2003; Dho-Moulin and Fairbrother, 1999]. The most common diseases caused by APEC in chickens collectively known as colibacillosis infections including airsacculitis, cellulitis, coligranuloma, egg peritonitis, omphalitis, osteomyelitis / arthritis, perihepatitis, pericarditis, and salphingitis [Dziva and Stevens, 2008]. Colibacillosis caused by APEC is a devastating disease of poultry that results in severe economic losses annually to the poultry industry worldwide [Newman et al., 2021]. Multiple APEC serotypes have been associated with colibacillosis cases in the field outbreaks; however, three serotypes (O78, 39 ME151672980v.1Attorney Docket No.: 139465-00120 O2, and O1) account for the majority (more than 80%) of the cases [Dho-Moulin and Fairbrother, 1999; Ghunaim et al, 2014]. The pathogenicity of APEC is still poorly understood. The most prevalent virulence traits associated with APEC are adhesins (type 1 and P fimbriae, Temperature-sensitive hemagglutinin Tsh, and curli), capsules, iron acquisition systems, and serum resistance [Dho-Moulin and Fairbrother, 1999]. Large plasmids are commonly found in APEC strains and are associated with the virulence of APEC [Dozois et al, 2000; Doetkott et al, 1996; Rodriguez-Siek et al, 2005]. Their importance has been demonstrated by the occurrence of plasmid-encoded genes shared among APEC strains [Dozois et al, 2003] and the presence of virulence genes in plasmids [Johnson et al, 2002; Stehling et al, 2003]. Most virulence genes associated with APEC are often located on IncF plasmids, named ColV plasmids because their ability to code for production of colicin V, a small protein from the microcine family [Johnson et al, 2002; Johnson et al, 2004; Tivendale et al, 2004]. ColV plasmids are associated with E. coli in general and with APEC in particular. Their link with the ability of bacteria to cause disease in production animals has been demonstrated [Skyberg et al, 2006; Smith et al, 1980; Wooley et al, 1998]. In vivo expression of genes encoded by the ColV plasmid, including Tsh, aerobactin, and the Iro system, has been confirmed by using SCOTS [Dozois et al, 2003] and the occurrence of the putative virulence genes of APEC on non-ColV plasmids has also been shown [Dozois et al, 2003; Johnson et al, 2006; Dozois et al, 2000]. The complete nucleotide sequence analysis of the large ColV / IncF virulence plasmid pAPEC-1 (103,275- bp) from the APEC strain χ7122 (serogroup O78:K80:H9) revealed that a putative virulence region spanning an 80-kb region of pAPEC-1 comprises genes responsible for the potential virulence factors including four iron acquisition systems (iutA, iucABCD, sitABCD, and iroBCDN), temperature-sensitive hemagglutinin (tsh), a colicin V operon, increasing serum sensitivity (iss), ompT, hlyF, and etsABC [Mellata et al, 2009]. In addition, it has been reported that pAPEC-1-cured χ7122 bacteria were attenuated in chickens, caused few lesions of pericarditis and perihepatitis, did not persist in the blood, and poorly colonized the lung, spleen, and liver [Dozois et al, 2000]. This APEC strain χ7122 has two additional large plasmids (Figure 1), pAPEC-2 (82,676-bp) and pAPEC-3 (56,676-bp), that also have virulence attributes [Mellata et al, 2010].

[0163] In the case of interactions between animal host and bacterial pathogen, the bacterial surface is the first line of contact with the host immune system. Among the bacterial surface molecules undergoing pathogenic interactions, LPS constitute the major structural component of the outer membrane of Gram-negative bacteria. The LPS fraction comprises about 10-15% of the total molecules in the outer membrane and is estimated to occupy about 75% of the bacterial surface [Lerouge and Vanderleyden, 2001]. LPS consists of three 40 ME151672980v.1Attorney Docket No.: 139465-00120 different components or regions: lipid A, core oligosaccharide, and O-polysaccharide. The structure of lipid A is highly conserved among Gram-negative bacteria. Among Enterobacteriaceae, lipid A is virtually constant. Lipid A is the lipid component of LPS that contains the hydrophobic, membrane-anchoring region of LPS. The core oligosaccharide regions consist of a short chain of sugars, which connects the lipid A anchor to O-antigen, and can be divided into inner and outer core regions [Osborn and Wu, 1980]. The O-antigen or O-side chain is attached to the core polysaccharide and extends from the core out into the environment. It consists of repeating oligosaccharide subunits made up of three to five sugars. The individual chains can vary in length ranging up to 40 repeat units. The O- polysaccharide is much longer than the core oligosaccharide and it contains the hydrophilic domain of the LPS. At least 20 different sugars are known to occur and many of these sugars are characteristically unique dideoxyhexoses such as abequose, colitose, paratose, and tyvelose, which are rarely found elsewhere in nature. The composition of sugars in the O-polysaccharide, especially terminal units, confers immunological specificity of the O- antigen and contributes to the wide variety of antigenic types (serotypes) between species and even strains of Gram-negative bacteria [Lerouge and Vanderleyden, 2001]. LPS O- antigen has been reported to be a potent antigen for inducing specific antibody response which is protective against E. coli pathogens in animal models [Pluschke and Achtman, 1985; Schief et al, 1993; Cryz Jr et al, 1995]. In addition, specific antibody against LPS O- antigen has been shown to be protective against other gram-negative pathogens [Zhang et al, 2009; Ding et al, 1990; Moustafa et al, 2023].

[0164] Live attenuated bacterial vaccines have been created by recombinant DNA technologies for nearly 4 decades [Lewis, 2007]. Over this time, a number of animal studies have shown that these live attenuated recombinant bacterial vaccines can induce protective immunity by delivering antigens expressed by the bacterium itself and DNA vaccines to be expressed in target eukaryotic cells. Recombinant attenuated Salmonella vaccines (RASVs) have been significantly improved to exhibit the same or better attributes than wild-type parental strains to colonize internal lymphoid tissues and persist there to serve as factories to continuously synthesize and deliver recombinant antigens which are selected to induce protective immunity to infection by that pathogen [Clark-Curtiss and Curtiss, 2018]. These RASVs are designed to attributes maximize their abilities to elicit mucosal and systemic antibody responses and cell-mediated immune responses. Additionally, RASVs can be administered by a convenient oral vaccination route which is a significant advantage in the vaccine industry. 41 ME151672980v.1Attorney Docket No.: 139465-00120

[0165] In the present study, the RASV-based APEC vaccines are designed (Figure 1) to produce the APEC virulence plasmid-encoded virulence factors and APEC O78-antigen to induce protective immunity against APEC infections. Example 2: Materials and Methods Bacterial strains, plasmids, culture conditions, and reagents

[0166] The bacterial strains and plasmids used in the present invention are listed in Table 1. The starting strains, χ11534 and χ12341, originated from the wild-type Salmonella enterica serovar Typhimurium strain χ3761 (UK-1). The construction history of χ12341- lineage strains including χ11534 and χ12341 is described in Figure 2. The wild-type strain χ3761(UK-1) was recovered from the spleen of a chicken orally infected with a highly virulent serovar Typhimurium strain originally isolated from a horse [Curtiss 3rd et al, 1991]. This wild-type strain UK-1 is highly virulent in chicks and mice [Curtiss 3rd et al, 1991; Zhang et al, 1997]. Bacterial strains were grown in SBM broth or LB medium [Bertani, 1951] at 37oC. SBM broth is a modified LB medium containing 0.5% yeast extract, 2% tryptone, and 5% NaCl. Diaminopimelic acid (50 µg / ml) was added to the culture media for growing strains with Δasd mutations. Antibiotics were used as needed at the following concentrations: streptomycin (Sm), 20 or 100 µg / ml; chloramphenicol (Cm), 20 µg / ml, and tetracycline (Tc), 10 µg / ml. All antibiotics and chemicals were purchased from MilliporeSigma or Thermo Fisher Scientific. Anti-O4 rabbit antibody was purchased from Abcam. Goat anti-rabbit IgG- alkaline phosphatase antibody was purchased from Thermo Fisher Scientific. Table 1: Bacterial strains and plasmids42 ME151672980v.1Attorney Docket No.: 139465-0012043 ME151672980v.1Attorney Docket No.: 139465-00120SmR, streptomycin resistance; CmR, chloramphenicol resistance; TcR, tetracycline resistance DNA manipulations

[0167] Plasmid DNA and genomic DNA were isolated by using QIAprep Spin Miniprep Kit (Qiagen) and Genomic DNA Purification Kit (Thermo Fisher Scientific), respectively. Restriction enzymes and DNA-modifying enzymes were used as recommended by the manufacturers (New England Biolabs or Thermo Fisher Scientific). The primers used in the present study are listed in Table 2. Table 2. Primers used in this study44 ME151672980v.1Attorney Docket No.: 139465-00120

[0168] The lrp-deletion (Δlrp) mutation was introduced into the RASV strains by the gene replacement method [Kaniga et al, 1991] using a suicide vector pCHSUI-Δlrp. Two DNA fragments, Δlrp-L (1,286 bp, upstream region of lrp gene) and Δlrp-R (1,015 bp, downstream region of lrp gene) were amplified by PCR from the wild-type χ3761 using the primer set 1 (CHB455 and CHB456) and set 2 (CHB453 and CHB454), respectively. These two DNA fragments were cloned into the SpeI site of the suicide vector pCHSUI-1 using GeneArt™ Seamless Cloning and Assembly Enzyme Mix (Thermo Fisher Scientific) to generate pCHSUI-Δlrp.

[0169] The pmi mutations in the RASV strains were replaced with the wild-type pmi gene using a suicide vector pCHSUI-pmi. A 3,224 bp DNA fragment containing the wild-type pmi gene was amplified by PCR from the wild-type χ3761 using the primers CHB433 and CHB435. This DNA fragment was cloned into the SpeI site of the suicide vector pCHSUI-1 using GeneArt™ Seamless Cloning and Assembly Enzyme Mix (Thermo Fisher Scientific) to generate pCHSUI-pmi.

[0170] A deletion-insertion mutation Δ(rfbB-rfbP)::eco78ABCDEFGHIJK for the serotype conversion was introduced into the RASV strains by the gene replacement method [Kaniga et al, 1991] using a suicide vector pCHSUI-APEC-O78. Two DNA fragments, ST-galF arm (831 bp) and ST-gndA arm (931 bp) were amplified by PCR from the wild-type χ3761 using the primer set 3 (CHB250 and CHB560) and set 4 (CHB251 and CHB559), respectively. A 12,900 bp DNA fragment containing APEC serotype specific O78-antigen gene cluster (rfbO78: eco78ABCDEFGHIJK) (Figure 3) was amplified by PCR from the wild-type APEC strain χ7122 using the primer set 5 (CHB561 and CHB562). These three DNA fragments, ST-galF (831 bp), APEC-O78 (12,900 bp), and ST-gndA (931 bp), were assembled with the SpeI-digested pCHSUI-1 using GeneArt™ Seamless Cloning and Assembly Enzyme Mix (Thermo Fisher Scientific) to generate pCHSUI-APEC-O78 (Figure 4).

[0171] To replace tra (traM-traX) genes on pAPEC-1 with asd gene, the three DNA fragments, Δtra_L-arm (811 bp), Δtra_R-arm (766 bp), and asd (1584 bp), were amplified by 45 ME151672980v.1Attorney Docket No.: 139465-00120 PCR using the primer set 6 (CHB541 and CHB542), set 7 (CHB543 and CHB544), and set 8 (CHB550 and CHB551), respectively. These three DNA fragments were assembled with the SpeI-digested pCHSUI-2 using GeneArt™ Seamless Cloning and Assembly Enzyme Mix (Thermo Fisher Scientific) to generate pCHSUI-APEC-1-Δtra::asd.

[0172] To delete the antimicrobial resistance (AMR) genes on pAPEC-2, the two DNA fragments, ΔAMR_L-arm (830 bp) and ΔAMR_R-arm (811 bp) were amplified by PCR using the primer set 9 (CHB535 and CHB536), and set 10 (CHB537 and CHB538), respectively. These two DNA fragments were assembled with the SpeI-digested pCHSUI-2 using GeneArt™ Seamless Cloning and Assembly Enzyme Mix (Thermo Fisher Scientific) to generate pCHSUI-APEC-2-ΔAMR.

[0173] A 1,052 bp DNA fragment was amplified by PCR from pAPEC-3 using the primer set 11 (CHB546 and CHB547) and assembled with the SpeI-digested pCHSUI-1 using GeneArt™ Seamless Cloning and Assembly Enzyme Mix (Thermo Fisher Scientific) to generate pCHSUI-APEC-3. Chrome azurol S (CAS) assay

[0174] To assess the production of siderophores, the CAS agar medium was prepared as previously described [Schwyn and Neilands, 1987]. The ternary complex chrome azurol S / iron(lII) / hexadecyltrimethylammonium bromide is a blue compound and serves as an indicator. When siderophore (a strong chelator) removes the iron from the dye, its color turns from blue to orange. The RASV strains were inoculated on the blue CAS agar medium and incubated at 37oC for 24h and then further incubated at room temperature for 3 more days. The orange halo formation by reaction with siderophores was recorded daily with visual inspection. Preparation of antiserum to O78-antigen

[0175] To raise chicken antibodies to APEC O78-antigen, ten 3-week-old SPF chickens were injected with an inactivated APEC strain χ7122 with aluminum hydroxide as the adjuvant. Three weeks after the first injection, the birds were boosted. The serum was collected at 8 weeks and tested the ability to interact with O78-antigen using LPS immunoblot analysis. LPS O-antigen detection

[0176] Bacterial cells were grown overnight in SBM broth. The culture samples were adjusted to the same cell density (OD600 = 2.0) and 1 ml of each culture was pelleted by centrifugation. LPS samples were prepared as previously described [Hitchcock and Brown, 1983]. LPS samples were run on 12% SDS-PAGE gels, transferred to nitrocellulose membranes, and subjected to western blot analysis. Membranes were first incubated with a specific anti-O-antigen antibody followed by a secondary antibody (goat anti-rabbit IgG 46 ME151672980v.1Attorney Docket No.: 139465-00120 conjugated with alkaline phosphatase, Thermo Fisher Scientific). LPS bands were visualized using a chromogenic substrate, 1-Step NBT / BCIP (Thermo Fisher Scientific). Example 3: In vitro Results and Discussion

[0177] The APEC strain χ7122 (O78:K80:H9) harbors three large virulence plasmids (Figure 2), pAPEC-1 (103 kb), pAPEC-2 (83 kb), and pAPEC-3 (57 kb), and all three are known to have significant virulence attributes [Mellata et al, 2010]. Therefore, the virulence factors encoded by these large virulence plasmids would be potential target antigens to induce protective immunity against APEC. To develop RASV vaccines against APEC infections, all three APEC virulence plasmids are introduced into the RASV strains by conjugation (biparental mating) to display APEC antigens (Figure 1). Two RASV backbone strains χ11534 and χ12341 were used as starting strains. These χ12341-lineage strains (Figure 5) already have multiple mutations for the delayed-lysis system to ensure antigen presentation and biocontainment in vivo [Curtiss 3rdand Wang, 2018]. The construction of the RASV-based APEC vaccines is summarized in Figure 6. The virulence plasmids pAPEC- 1 and pAPEC-2 are self-conjugative with genes responsible for the conjugation machinery on the plasmids. However, pAPEC-1 doesn’t have a selection marker on the plasmid while the plasmid pAPEC-2 has the streptomycin-resistant (SmR) gene as a selection marker in the AMR area of the plasmid (Figures 1 and 2). For the conjugation selection of pAPEC-1, a suicide vector (pCHSUI2-Δtra::asd) with a chloramphenicol-resistant (CmR) marker gene was constructed and inserted into the pAPEC-1 plasmid before conjugation. This suicide vector pCHSUI2-Δtra::asd was also designed to introduce a deletion / insertion mutation Δ(traM-traX)::asd at the suicide vector removal step after conjugation. The resulting plasmid pAPEC1-Δtra::asd will be no longer conjugative and allow the Asd- RASV strains can grow without diaminopimelic acid (DAP). The virulence plasmid pAPEC-3 doesn’t have genes responsible for the conjugation and selection marker on the plasmid, so a conjugative suicide vector pCHSUI-APEC-3 with the selection marker (TcR) gene was constructed and inserted into pAPEC-3 for the conjugal transfer and selection. All three virulence plasmids, pAPEC-1, pAPEC-2, and pAPEC-3 with the selection markers were transferred from the APEC strain χ7122 to the RASV strains by conjugation. Then, the suicide vector part including the selection marker was removed from pAPEC-1 and pAPEC-3. The AMR genes on pAPEC-2 were deleted by the gene replacement method using a suicide vector pCHSUI- APEC-2-ΔAMR. The RASV-based APEC vaccines, Curtiss 272, Curtiss 273, Curtiss 274, Curtiss 275, Curtiss 276, and Curtiss 277, harboring all three APEC virulence plasmids have been verified by PCR and whole genome sequencing. This is a unique feature of the APEC vaccine formula which has never been done before. 47 ME151672980v.1Attorney Docket No.: 139465-00120

[0178] The bacterial iron uptake system is an important virulence factor for pathogenic bacteria [Braun, 2005; Fischbach et al, 2006; Griffiths et al, 1988]. The plasmid pAPEC-1 possesses four iron acquisition systems (iutA iucABCD, sitABCD, and iroBCDN) [Mellata et al, 2009; Mellata et al, 2010]. To test the expression of pAPEC-1 iron acquisition systems, siderophore production was assessed on the CAS assay agar medium [Schwyn and Neilands, 1987]. As shown in Figure 7, the RASV strains Curtiss 276 and Curtiss 277 harboring pAPEC-1,2,3 showed a much bigger orange halo (representing the level of siderophore production) around the colony than Curtiss 280 and Curtiss 281 harboring the control empty plasmid pCHC116.

[0179] It has been reported that the O-specific polysaccharide (O-antigen) biosynthesis (rfb) gene cluster is located between galF and gnd genes in E. coli [Amor and Whitfield, 1997; Bao et al, 2018]. Attempts have been made to construct RASV strains that will produce APEC O-antigens (O1, O2, and O78) which are predominantly associated with APEC infection cases [Han et al, 2017; Han et al, 2018; Han et al, 2021; Roland et al, 1999; Roland, 1997]. Han et al (2021) introduced a plasmid harboring the APEC O78-antigen gene cluster (rfbO78) into the RASV strain with a Δrfp mutation. However, the resulting RASV strains showed poor O78-antigen production. In a different approach, the cya gene in the RASV strain with a Δrfc mutation was replaced with the rfbO78gene cluster from the APEC strain χ7122 [Roland et al, 1999; Roland, 1997]. This resulting RASV strain also showed a weaker production of O78-antigen in comparison with APEC strain χ7122.

[0180] The present invention is directed to a method for the inter-genus serotype conversion by replacing the Salmonella O4-antigen gene cluster (rfbO4) in RASV strains with the APEC O78-antigen gene cluster (rfbO78). To design the conversion of the RASV serotype (O4) to the APEC serotype (O78), the nucleotide sequences of the genetic loci for the colonic acid and LPS synthesis in the S. Typhimurium (UK-1) and APEC strain χ7122 are compared (Figure 3). The serotype-specific O-antigen gene cluster between galF and gnd genes does not share any significant sequence homology while the colonic acid coding area and part of the LPS coding area have significant sequence homologies at both nucleotide and amino acid sequence levels (>90%) (Figure 3). With this notion, a positive-selection suicide vector pCHSUI-APEC-O78 was constructed (Figure 4) to replace the entire serotype- specific gene cluster (18 kb DNA fragment between galF and gnd genes) in S. Typhimurium with the entire serotype-specific gene cluster (13 kb DNA fragment between galF and gnd genes) in APEC strain χ7122. The serotype converted RASV strains using this suicide vector are Curtiss 274, Curtiss 275, Curtiss 276, Curtiss 277, Curtiss 280, and Curtiss 281 (Figure 6). All of these RASV-based APEC vaccines successfully produced APEC O78- antigen but no longer produced S. Typhimurium O4-antigen (Figure 8). This serotype 48 ME151672980v.1Attorney Docket No.: 139465-00120 conversion approach by replacing the entire Salmonella O4-antigen gene cluster (rfbO4) with the APEC O78-antigen gene cluster (rfbO78) has not been done before. Additionally, the phosphomannose isomerase (pmi) mutant will have a defect to produce LPS O-antigen in a mannose limitation environment like in vivo since the phosphomannose is a key building block for the LPS O-antigen production. The pmi deletion (Δpmi) mutation in Curtiss 274 and Curtiss 275 was replaced with the wild-type pmi (pmi+) gene using the suicide vector pCHSUI-pmi to generate Curtiss 276 and Curtiss 277, respectively. The pmi+ resulting strains Curtiss 276 and Curtiss 277 showed mannose-independent LPS O78-antigen production while the parental pmi mutants Curtiss 274 and Curtiss 275 showed a significant reduction of LPS O-antigen production in the absence of mannose (Figure 8C and 8D). Similarly, the pmi+ strains Curtiss 280, and Curtiss 281 also showed mannose-independent LPS O78-antigen production (Figure 8F and 8H).

[0181] Leucine-responsive regulatory protein (Lrp) appears to be an antivirulence regulator in Salmonella since the deletion mutant (Δlrp) showed enhanced cell invasion, cytotoxicity, and hypervirulence in BALB / c mice [Baek et al, 2009]. To improve the invasion activity in vivo, the Δlrp mutation was introduced into the RASV strains by gene replacement method using the suicide vector pCHSUI-Δlrp (Figure 6) so that the resulting RASV strains can exert enhanced immunogenicity. Example 4: In vivo efficacy of RASV-based APEC vaccines

[0182] To assess the efficacy of the RASV-based APEC vaccines in this study, SPF chickens will be spray vaccinated on Day 0 after hatching, boosted via drinking water on Day 14, and challenged with a virulent APEC isolate on Day 21. Then the clinical signs and survivability of birds will be monitored to evaluate the protective immunity of the vaccines in comparison with unvaccinated birds.

[0183] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Sequences

[0184] Nucleic acid sequence of rfbO78gene cluster (eco78ABCDEFGHIJK) (SEQ ID NO: 4): ATTTtgaaagcttgatcggatatgacggctgcgaaaaattggaataattgcaggttattttgaatatgggggatttctaaaacaccccattaaca atgtgttgacaattgattttgaccaagttgttaatgtttttcttgcgttttcaattggttgattagataatattatatagatctataatctattctagttgggttgg 49 ME151672980v.1Attorney Docket No.: 139465-00120 gttgggttgggttgggttgggttgggttggggaaattgttcgatcggaatatatagtttctggtgttctgatagccataagtcaggggcggtagcgtg gatttttattgcgtaaaaacgtcagtaaacatagaggtttaatgaaaaaaatactattaatatccatgagtttgggaaagggcgattatggtggtg gtatagtatcaaatacaaacttttttgctttaaaggaattagagcattatgagttattttcagtaggtattgtaaaaaacaccaacgatgcaccaaac tttattaacatggttttgcctggcaacgcgagtaaattctcaactgcaattaataatatattagggtttgccgggcagttaaataataaaactacgaa aaacataaaatacataattgacgagtttgagcctgatattgtataccttgattcaagccttctggggtgtatagcttcttattgcaagaaaaaacata aaactattcaaataataacgttctttcataatattgagtttgattttgaaattgcacgtattatgtcagggttattgcatttttttccctcgttaatatccaca actctcgcagaatatgcggctgtaaggtatagtgacaagattatcgcattacataaaaaagattcttttagactagaggaaaaatatggaagga aggctgattatatcgtacccgtttgcattaaagatacacaaagagaaaaaagttttaaactcgtcaacgaaaagaaaaaagaaggcaaaaa gttaaaagttggctttataggtacagctttttttgcaaatgtagagtctgctaagattatatctcaatacattgcgccaaaggtggaggggattgcta atttttatatatgtggcaatggatttgaaaaatataaagcattgaattcaactaatgtaaatgtttcaggctatattgattctttggatgatttctataatg aaatggatgtgatgatatttcctatttttagcggcgcaggaatgaaggtgaaaatagcagagtcattaatgtataataaaccaattctagcttctgc atttgcattagttggttacgaaaaaataatagatggaactaatgtgattagctgtgagtctcacgaatcttttgtttatcatataaaacaatttagaag ggataataacacattatataataggaagtcttattataaatatttctctgataaagcatgtctgcattattttagaaatatattacgagaaattgttaata acgaatagatagatggactaataacgtcatgaatataatacaattatcaaaattctaccctccgatatttggtggaatagagcaagttgcacagg acataaccgaagggatgcaaaatttgtgcaatgttgatgttttaagcgtaaataacagttctaaaacaatatattgtaagaatataattcgagcttc attactatttacactattatctaccccagtatcgattagctatatactaatctggagtagaatcagaaataattatgatataattcatgttcatttgccaa atcctcttgctattattgcactgttgctgttcccgcccaaagctcccgtcgtagtgcattggcatagcgatatagtaaaacagaaaatagcgttaaa gttttttcggcctttacagaatttatttttgaataaagtagaaaaaattattgtgacatctgaaatttatgggagttcttctcctcaattacaaagatttca agataaaataatttgtataccgatcggtataaaatcagagcgattacctaagaatgaaacgctattaaagcatctaaaagaaaaatataaaaa taaaaagatagttttctctcttgggaggctggtttattacaaagggtttgaaaatctagttaatgcagctaactttcttcctgaagatacaataatcctt attggtgggtgcggtgagttatatgacgagttggctgatagcattttatccaataaactagaagggaaagtggttttgctgggggagataaaatat gaacagctttctgcttattatcaggtttgtgacgtattttgtcttccttccattcatgaatcagaagcttttggcgtggttcaaattgaggcaatgagctat ggaaaacctgttgtatccacaaatattaaaaatagtggggtcccatgggtaaatgaaaatggtatttcaggtgttgtagttgaacctaatgagcct catgaactagccaaagctatcttgacaattttaaataatccagctggtttttccttaggggcattagagcgatatcgcaaattattcactagagata aaatgatctcaagtttgattggactttatcagaatattaaaataggaaatgaaaaagaatgatcttacctgttattctggccggtggtgcaggtagt cgcctttggccactttcccgcttactttaccccaaacagtttttatgtctgaaaggcgagctttctatgctgcaaacaactatctgccgcctgaatggtt tggggtgcgaaaatccggtggtgatttgtaatgagcagcaccgctttattgttgcggaacagttgtatcaactgaacaaactcaccgagaacatt attcttgaaccggcaggacgtaatactgcacctgctattgcgctggcggcgctagcaacacaatggcataaaccagatagtgatccgttactgtt ggtgctggcagcggatcatgtgattgctgatgaagacgcattccgcgccgctgtgagtaatgctatgccgtatgccgaagcgggcaagttggtg acttttggcattgtgccaaggcatccagaaacaggttatgggtatattcgtcgtggtgcagtgtctgcgggcgagcaggagggagtagcctttga agtggcacagtttgtcgaaaaaccgaatctggaaaccgcgcaggcctatgtggcaagcggcgaatactactggaacagcggtatgttcctgtt ccgcgccggacgctatctcgaagaactgaaaaagtatcgtccggatattctcgacgcctgtaaaaaagcgatgagcgccgttgagccagatc ttaattttattcgtgtagatgaagaagcgtttctcgcctgtccggaagagtcggttgattacgcggtaatggaacgaacagcggatgctgttgtagt accgatggatgcgggctggagtgatgtgggctcttgggcttcgttatgggagagctgcactcacacagctgaaggcaacgtttgccgcggtga cgttattaattataaatccgaaaacagttatgtgtatgccgaatcgggcctggtcaccaccgtcggggtgaaagatttggtggtagtgcagacca aagatgcggtgttaatagccgatcgcagctcggttcaagatgtgaaacatgtggttgagcagctcaaagccgacggtcgacacgagtatcgg atgcatcgcgaagtataccgtccgtgggggcaatatgactctatcgatgcaggcgctcgctaccaggtgaaacgcataactgtgaaaccggg cgagggcttgtcggtacagatgcatcatcaccgcgcagaacactgggtggtggttgcgggaacggcaaaagtcactattaatggtgacatca aactgcttggtgaaaatgaatccatctatattccactgggggcgacacattgtttggaaaatccaggaaaaatacctttagaaataattgaggttc 50 ME151672980v.1Attorney Docket No.: 139465-00120 gctcaggttcttatcttgaagaagatgatatagttaagttttatgatcgttatgggcgagattaatttcattactgaagttgctaattgtttaacaaaata cttctgtgtattttttcatataagtttgtaaatatgactagattaagctgctttaaagcctatgatattcgcgggaaattaggcgaagaactgaatgaa gatatcgcctggcgcattggtcgcgcctatggcgaatttcttaaaccgaaaaccattgttttaggcggtgatgtccgcctcaccagcgaaacctta aaacgggcgctggcgaaaggtttacaggatgcgggcgtcgacgtgctggatatcggtatgtccggcacagaagagatctatttcgccacgttc catctcggcgtggatggcggcattgaagttaccgccagccataacccgatggattataacggcatgaagctggtgcgtgagggggcccgccc gatcagcggcgataccggactgcgcgatgtccagcgtctggcagaagccaacgactttcctcccgtcgatgaaaccaaacgcggtcgctatc agcaaatcaacctgcgtgatgcatacgttgatcacctgttcggttatatcaatgtcaaaaaccttactccgctgaaacttgtgatcaactccggaa acggcgcagcaggtccggtggtggacgccatcgaagcccgctttaaagccctcggcgcaccggtggaattaatcaaagtgcacaacacgc cggacggcaatttccccaacggtattcctaacccgctgctgccggaatgccgcgacgacacccgcaatgcggtcatcaaacacggcgcgg atatgggcattgcctttgatggcgattttgaccgctgttttctgtttgacgaaaaagggcagtttatcgagggctactacattgtcggcctgctggcag aagcgttcctcgaaaaaaatcccggcgcgaagatcatccacgatccacgtctctcctggaacaccgttgatgtggtgactgccgcaggcggc accccggtaatgtcgaaaaccggacacgcctttattaaagaacgtatgcgcaaggaagacgccatctacggtggcgaaatgagcgcccac cattacttccgtgatttcgcttactgcgacagcggcatgatcccgtggctgctggtcgccgaactggtgtgcctgaaaggaaaaacgctgggcg aactggtgcgcgaccggatggcggtgttcccggcaagcggtgagatcaacagcaaactggcgcaacccgttgaggcgattaaccgtgtgg aacagcattttagccgtgaggcgctggcggtggatcgcaccgatggcatcagcatgacctttgccaactggcgctttaacctgcgctcctccaa caccgaaccggtggtgcggttgaatgtagaatctcgtggtgatttaggacttgttgaaaagcagacaacatatatcattaacttattaacaacccc ccccaatgatgaaaagtgaaaatattacattcgtagtgcaaggtccagtcagaaatgaaacaaaagatactttagaaagtattaggctaaattt taaagaagctaaaataattttgtccacatgggaggggagtaacgttaaaggtctttcatttgacgatattgtttttagtaacgatcctggccctttaaa tataaaaagaggtactaaaatagttgctcaggaaaatacaaatcgtcaaataatttcaacataccaaggtttaattaaggttcgaacaccatatg cggttaagttgagaacagatacgccattgcgaaatgataatattataaaaaaatatataaaagctcaagcatatgggagagattataatttttcat acttagaggaaaggattcttgtatcatctataaatactatagatccaaaaagctatattcaattcccataccatatttctgattggatctactttggtaa aactaacgatttattaaaaatttgggatggggaattaatagatgataatgattattttactgagcaggatataaaagaagagattagttgcaaaag aggatttgagtttggaagatacacagctgaacagcttgttctatactcatttttaaaaaaaaataaaattagcgaatataaacattattgtgataca aataatgacagggttgaaaaggtattaaaagtaatcctttctaatttctattgcgtaagtcctaaacaaatgggactctgttttgataaatactcaga cttaataacccctagaatgaattacaggagtctccgtagtttcttagggttttattttgttacaattaatgagagagtttggcgaaattattacagccaa ttctataaacttgacactacaatatcagaaaaaatagaaatatgtgcacgcaggatatttataaagatcatgaagaagaggaaaaaatgagta aaagcatgttatatatctacacacataatggttacaatgatgcatttttaaaaaaagatgtagggatggtaccatctgttatgttaaatggtgggaa agttgattcgctaatttattgtaaagaaaagaaaggaaaggctgtttttaataatttagaaatatattttactggatgtggatggtttattagtaacttca aggcgatactgtttctttgtcgaaatgcaatatttaatagacatttagatttattgatatttcatctcagccttagaaatatcgtgcctgtgttaacatata aacttttatttaaaggggaattaatatgtaagatggatttaaatactgaaagtgccatcgaatattctgataaaagccataaaataaatgggatca aaaaaaaactaataagaatgattatttcccgcacagatcaattttatgttgagactataagaaattataacataataaataaaggcatttttgatgt gccgatttcaggaaagttaaaattaatgcccaacggagttgatgctaaattagttagggaggttattaaaactgaaggtcctgatgtatttaaaga aaatacaataacaataatctctcgccatgcttgtgaagcaaaggctccaaacagaatattcgatgtaattgacgtaatgtctgaattgaaactta ataactggacattgcaaatattgggctatttcccaacaatatttacggatgctctaagaataagagctcttgaatgtggagtgaatatcatatgtga cggttataatttagagctctatgatgtgtataaaagattatttaaaagtaagatgtttatatgtctttcgctgcaagagtcatattgtatatcactagttga agctgctgtatttaataatcatataatatctacaaatgttggagtagcagaagatctttcattaaaatacggtaatattaaaattatcgaaaattattc gaaaaatttactatccaaaactattgaaaagacaattaaaaatgatattaaaataaaaaaagcaagcatttctgatagaatgttagactcatatt cttgggataatatcataacaggggtcatgagcgaataatatgggagtatatttcattttaatgacagtggcactattgatctctattatcgtattattcc acaatgatgttaaatttacgaatgtatatatatatacttatattttactgctctcattacttgctggtttaagatttcataccggctgggattgggaagctta 51 ME151672980v.1Attorney Docket No.: 139465-00120 tgactattttttttatgagctgacaagttctaatttatttgaaataagcaaagcaaatgtttttaattatgaaccagggttcgttttactatcttatatatctgt tttattgcacataccaccatttctctttttctctttaataaccgttacattaattataaagtcggcttataagtatttagggaactttgtttacatttttttattaat atatttatattacggttattttcataatttttctattgtacgacaaggtgttgctgctgcgttattcgcttattccattcgttatttgatagcgaaaagctataa atattatatattaatatttgtcgctgcattattccatatgtctgctattatgttattaattgtcccatttctttataaaatcgctaatcgtataccattttccgccc tattgctattttcattgttaatggtttatacctctgtaagtgagctcataggtatgaaatcattattgtcaggaataaatgcactaaatatatatatcaata atagttctttaagctataaagttggtttttcgtttaaatatttggagttattaatcattctagttctattttataataagagattttcagaactgattgctagca aatttagtaaaaagcaatattatgtatttagatgtttgattgtaattgaagtgatgatatattcgcttttcaatgattttagcattatttatgaaaggctaac tgtttacttcgaattttctcatgctatatgtatagcaatgattatatcagcctttaagtataaaagagttcagttgtttttactgttaattttattgtctcttattttt gtaagatattatcaattattcaattctcctgttcgtgtcgatggagaaatgacacattacgaacgatttgaaaactattgttctgtttttaacacaaaag attgtcaaaggtaacaatgaagcgaattattatagacttggataatactatcacaatccataacgatgaaccatacggtaaaaaaccagtaaat caagatgttgtaaataaaatgaagatgtataaagagctgggttatacaatcacaatatttaccgcccgtaatatgaatactttttcaggtaacattg ggaagataaatgttcatacaatgcctgttataattgactggttaaatcgtaataatattccatatgatgaaattattgtgggtaaaccatggtgtggg acaaatggattttatgttgatgataaggcgattaggccttcagagtttataagcttgtcagaaacggagattgctgaactactggataaggaggta aaaaaatgattctaataaactctgctgcttacgttaatgacgacctaagagctgaattcggtttgctaccaccgtgctttttaccccttggtaataga agactatatcatcatcagattgagtcgttaaggaaagcttttcctgaagaaaaaatatatttatctctgccggaagagtatcacattggtaggtatg atcaacaatactttaatgaaaatgatattacaatactaaaaacttcttatactttatcacaaggacatagtctgtatcggttgttaaatgattttaactta atcgaatgtgataaactaattattaattatggtgatactttttatcatacctttgaagctacgagtgatgattatttttatgtatctaataacatcggctact ataaccgagcatctgttgtaattaatggtgataagcttattataaataaagataatccagctgttgataatggtgagttggttatttctggtttcttttgttt cagtaatattgggaggttatgcgaatgcttgaaaaagatgaactatgattttctgtcgtcttctgaattgtattactcgtcggttgcaacaaaagtttta aaaaatgatcattggtatgattttggtcatttaaacagcttttttacatcaagaacgagtatcactactgagcgtgagtttaatagtcttcaaataaat aatttttgtgtgataaaaaaaagtaagaagaaagcaaaaatgctaggcgaagcgaattggtttttgagtttgccgcctgcattgagtatcttcact ccgagggtttttgatgttagagaagataatgattatgcagagtatacaatagaatatctttataatcttcctttgtctgatatggcagtgttttgtgagtta cctaagtcatgttgggtgaatgtgtttaactcgtgtcgagatttcttaaatagatcaaaagaatactctaatagtaaaaaaacgctagatatgtctg aactggcatcatatgataaattgtactatgataagaccctaaaaaggttacatgattttgaattgacaacaaacatacaattgactaaagatgtta tatttaatggtaaaagatttccatcgataaaaaaatagcagaaatatcatcacgctttattaacccaactaagagtgatgatgttataattacacat ggcgatttttgctttagtaatatattatacgattttagaagtcagagaattaaattaatcgatcccagaggtattaactataacaatgagttaagcata tggggagatataaggtatgacttggctaagttatgtcactcggtaattggatgttatgatcttattatcgctcagaaatttttgctggaatacgattatgt agataacagaattgaatttgaactatatgatgataactacagcgacatagtgaattcatttttaggcgtgtttaattcttttgaagaatttgggtatact attgaagagatacaatgtataaccatacatttatttttatcaatgttgccgcttcattctgattctaaacttagacaagaagcatttatagcaaatgctt atagactgttcagtttgttaaatttggagtatttatgacaacgattgtaattcctatggcaggacaatcatcacgcttttataaagcaggttatgatgttc ctaagtataagttaaaaattaataacaaaagcgtattttttcattcactacgaagttttctaaaatttaaggaccatttttttctaattataggtattaaag gcatactagatgaaaagtttgttcaatatgaaatggccgaattaggtataaatgattttgaaattgttttgctcgaacagcaaacaaatggtcaag cagagacggttgtgaaaggagtaaaaaattgcaagagagatattagtgacattttaatttttaatatagatacttttcgcagagagattaatttgcct gataattttagtttgaaagattgctctgggtatttggaaacatttttagggtcaggtgagaattggagtaatattatgcctgaatcagatgtccctaatc aagtaaaaatgactgcggaaaaacaaaatatatctgaatattgttgcactggtttatattattttggtgattataagttgttgaatatagcttataataa ctggctagaaaatgaagatactactaaagaggtgtatatcgctcctctatataatcatttgattacatgtgggcacaaaattttttataccgttataaa cagatgtgatgtggtgttctgtggtgtgcctaatgaatatgaaaaaataaagaatgggtttgactggaaatgagacagcacagaaaattagtca gttatgctgtgattattacttttttgtttatacctatacaagttattctgtcgttaatttttaactattatttagctaaaaacgttgctctggatgttttaaatgctt ggtatatgttttttgcactcatttcccttttttctttgttggaattttcctatcccatattagcaattaatttttttaacagctatcagggaaattgtaaaggagc 52 ME151672980v.1Attorney Docket No.: 139465-00120 tctgtctttcttcataaagaaatcattaggggtagtaataccccttcagatcatatttgttgtaatttatagtttatttgttaacccgctttttatttattttggtat gggtttggtggtacgtagtatatcgaatattattaatgcatgtgtatatgcaaaacaaaatattattatcgataaagtttatcgatttctgtacgccata attatgccaactgctttccttttcttgttttatcattctgataaaaacattgatttgtatgatttaactcttgtatggtttttgagctcactattctgcttaatatatt ctttgtcatttttactccttaagcataagaagtctgtcaacaggagcgaaaaatcattccatctaccattcaagtataaccttaagcttttttttactgtttt tcctgcaattttcatctatacattgtctatatattatttgaaaatatttggttcagaaagcagttctagttctactattatatatggtttctttttgcaaatattta atgtttataatttgataattattttggttgcaagttatttgatgcctacgctctcaaagcgtttccatgatggtgttgatgttatccctctcacacttaaacta ttagatatcagtgtacttatatctgttatttctacttcttttgtggcattgttaggtattcctgttgcggagtatattcttaaggggaaaatagatgtaatatc ctactattatatagtagcaataattattatattctatattgaaacatgtcaggttattctaacctcaattggcactgccattggtatttacgattttcatagg caaagtattatatcagcagttatggttttaattctgtcatgttttttaatacctcattacaatgctgagggtttgatgtatagtatcattatatctcaattcttta catgctttatttataaccctaaacgtgttataaataaaattggattagataaagttaattatattaaaagattaatggtgcactttttgtttatgttgactctt atttttatggggttatattttcaaaatgatgatatattgccgagagttgtgattctttcttttatcacattgattggtgtttgttttctctacccaaatgtgaaaa aaataattgttgatttcttgtagttttttaattttatttgcattttaaatggaaaatcttgttctatcttagaaaggagttaacaatgctgaagcaacagatc ggcAAAT

[0185] Nucleic acid sequence of pAPEC1-Δtra::asd plasmid (SEQ ID NO: 1): ttggctatcctaagcagccgattatgcgttgaggctttcagtgatggctgatatagcaacaatgagaatgaaggctctgcacttctgggataaac acggtatttctgcagcttctgaagcctttggcgtatcctgccgcacgctttactggtggcgtcagttactgaacaagggaggacctgaggggcta atcccgcacagtaaggcacctctggtgcgacgaaaaaagcactggcatcccgatgtgctgaaagagattcgacgcctcaggacagagctg ccgaacctcggtaaagagcagatttttgttcgcctgaagccctggtgcgcataacgccatcagacctgcccgagtgtttccaccatcggcagaa tgatcgccaccgcacacgataaaatgcggatgataccagtacgtctgagctcgcggggtaaggctctgcttgtcaaaaagcgaaccaccaa acccccgcaggccaaagcactaccgtccggtaaagacaggagagctcattgggatggacgccattgagctcagaatgggtgaaatgcgtc gctatatcattaccatgatcgacgaacacagcgattacgtactggcgctggctgtgccgtcgctcaacagtgatatcgtcaatcactttttcagcc gtgcagcccggctgttccctgtcggtatcagccagatcatcacagataacgggaaagagttcctgggaagctttgacaaaacgctgcaggaa gccgccatcaaacacctctggacctatccctacacgccaaaaatgaacgctatctgtgaacgttttaaccgggcattaagagagcaattcattg aatttaatgaaattttactctttgaggatctggcgttgtttaacctgaaactggcggaatatctggcgctgtataacagcaaaagactccacaagg cgctcgcactaacgacgcccgtggaatatattttaaaagagaacaagaattgcaatatgtggtggacccatacacagcaaacgcacaggcc gccccggttgggtagatatgtgggatggctggttcagacttcaggatatggttgaggctggtgatgaagtctctttatcaggagatctgatcaaga gacagccgcgcagggggattcactatgctggattcttttaacaaattctccgctaaccaaaaaatagactttttgcattcgtaaatgcgttaagag ataatttttaaagttccatcaatctcccggccagttcgtctcgcggcattactcactatccgcacaacttcagtaagatcatgactttttcaatcgttat ctgttgaccaagctttgaaaccactagcgtcccgtcaacctgacctggaatcgtcgagaaccgcccatagttgtgaattggctagagtgtgatgg aaagcaacgctaaacgacctagtcacacattctcttttccctaacaatgaccatttagtgaaataacgtatccaaaatgatgacgtgaaagcct gacactggcagcgaaaagccgaaaaaactcacctggtcaggagcaaaaatcggattattcgccaacagcgtaatatcagcagtagttctcg acacgcttgcgctatagacagaggatgaaacgctgctggtcggagttctctatacatttttggcgacggcagaaccgctcaccaatgccttaac gtctatcagccttccggtgagttggaacagcatgctcagcgtttgcaagatattctccacggtctgtagcagcgtaggtttcaatttaaatatcccca gatagccctctgggagcttccctttttattaagttttaattaccgttgcaatagttaaatgatattggtaattattatcattagcatttgtgtctgtgttgtttct ggtaaggctgaaagggaacaaacatcgcctgtacaacggcgtttttacttacagaccagcgaaggcgagactatcaggatgatgcatcgtgt gtctaagaaatcacggttctttccagattcgttgttcggtctaacactgcacaaatgcgtcagaacatcccggcgtagctgtccgacgccatttgg gctgttcaacaggacaatggcgcgtaagcgttaatggaagtggggatacggagttccctgttcacctgtgtatgtcacggaaaagtctctgactg agctgtccataacgctggtgaagcacaaatggcgcagcatctggtaaagcaggtttaacgaaaagtcgtgtcctgctgctggcatatgagtgc gtcgactgcctgatttagatcgtcaagcggagagggattttctcatgcgtattttgtttgtaggcccgcctctctatgggctgttgtatcctgtgctgtct 53 ME151672980v.1Attorney Docket No.: 139465-00120 ctggcgcaggcatttcgcgttaacggtcatgaagtgctgatcgccagtggaggacaattcgcacaaaaagcggcagaggccgggttggtggt gtttgacgccgcacccggtctggattccgaagctggttaccgccaccatgaggcacagcggaaaaaaagtaatatcggaacccaaatgggc aacttctcattcttcagcgaagaaatggccgaccatctggtggagtttgccggacactggcggcctgacctcatcatctaccctccgctcggagt catcgggccgctgattgccgccaaatatgatataccggtcgtgatgcaaaccgtaggcttcggccataccccctggcatatcaagggtgtaac gcgttcgctcacggacgcctatcgtcgccacaatgtgggggccacaccacgtgatatggcctggatcgacgtaacaccgccgagtatgagca ttctggaaaacgacggtgaacccattattccgatgcagtacgtcccgtataacggtggcgcggtgtgggaaccatggtgggagagaaggccc gagcgtaaacgtctgctggtgagtctcggcactgtcaaaccgatggttgacggccttgatctaatcgcctgggttatggattctgccagtgaggtc gatgccgagatcatcctgcatatttcagccaatgcgcggtccgatttgcgctcgctgccgtcaaatgttcgcctggttgactggatacccatgggt gtcttcctcaacggcgcagatggtttcattcatcatggcggagcaggcaataccctgacagcgctacacgctggtattccacaaatcgtcttcgg ccagggagccgatcgcccggtaaatgcccgcgttgttgccgaacgcggctgcgggattatccctggcgacgtcggcctgtcgagtaacatga tcaatgctttcctcaacaatcgctcacttcgcaaagcctctgaagaggtagcagcggaaatggcggcgcaaccctgcccaggtgaagtggcg aaaagcctgatcacaatggtacagaaagggtaaccactacaccttcatgataatcatatgccagcacccatccaggagtcatgcatccataa caatgcgggaccggagcgtgctggcttttttgcggaacgatccggccagtggatcgtattttacagacggaggaggcttaatgcctgcgaatca cactcccacaccggctcagtcatggatagttcgcctagcgcgcgtgtgctgggaacgtaagaaacttagtgtcatcgtggtggtagcgtcagtat cgactattttgctggctgcgctgacgccactgctgacaagacaggctgtgaatgacgcactggcgggcaatccggcccgcctgccgtggctgg cctgcgggttactgttgatcgctttttttgatttcatcggtaactatgtgcgccgtggttatgccgggatgctctcactctgggtgcagcataccctcag aggacgggtattcgacagtattcagaaacttgacggcgcaggccaggatgcgctgcgcaccgggcaggtgatttcacggaccaacagcga tctgcagcaggtgcataccctgctgcagatgtgcccggtgccgctggcagtgttcacttattacattgccggcattgccgtgatgctgtggatgtcc cctgccatgacgcttatcgtcgtgtgcgtactggtatgcctggcgatcaccgcgcttcgtgcgcgtcgtagggtcttcgcgcaaaccgggctggc ctcggaccaactggcgaatctcaccgaacatatacgcgaggtgctggcacagatctcagtggtaaaatcctgtgtggcagagatgcgtgaaa cgcactggctcgataggcagtcgcggcagattgtgcgtgtacgcatcggtgcggttatctcgcaggcgatgcctggggccaccatgctggcgc taccggtgctcgggcaaatcgtcctgctgtgctacggcgggtggtcggtcatgcacgggcggatcgatctcggtaccttcgttgcattcgccagc ttcctcgcgatgctgaccgggccaacccgcgtactggcatcgtttctggttatcgcacagcgcactcaggcgtccgtggagcgggtgtttgcact gatcgacacccgttcacagatggaggacgggacggagtcgattaacagtcaggttgtcggactggaactggagaatatgagctttgactacc accatggcgacagacatatcctcagcgatatctccttttccctgcgcgccggtgaaaccgtggcggtggtgggcgcatcgggttcaggaaaat cgaccctgttgatgctgctggcgcgtttttatgatccctgctccggaaagatatggctcaacaccagcgaaggccgacaaaatcttcgcgatatc agactggaggcgcttcgtcgccgggtaggcatcgtatttgaagatgcttttctgtttgccggtacggtggcggaaaatatcgcctatggccaccct caggcaacggcggacgacattcgccgtgcggcagctgctgcaggagccagcgattttatcaacgccctgccgaaaggcttcgatagcctgtt aaccgaacggggtacgaatctttccggcgggcagaggcagcgaatagcgctggcgcgggcgctcattactgcaccggacgtgttaatcctg gatgataccacctcagcggttgatgctgttacggaagcggagattaataccgcgctgggtcgctatgctgacgaagggcatatgctgctggtga ttgcccgacggcgttcaacacttcagctagccagccgggttgtggtgctggataagggccgtatggtggataccggaaccccggcagaacttg aagcgcgctgtccggcgttccgcgcactgatgaccggcgacagcgattttctggccacgtcccacaatagccacaacgaattgtggccggct gaaccagcgacacaagacgatgtaacggatacgggggataaaggttttgtcgcccgtatgacccgcgtaccggaaaatgcagtacagcag gcgctggccggtaaaggccgcaaagtcacgtcactactgaagcctgtggcgtggatgttcgtcatcgccgctctgctgatcgcactcgattctgc ggcaggcgtaggggtgctgatactgttgcagcacggcattgactccggtgtcgccgcaggcgatatgtcgaccatcggcctctgtgccctgctc gccctgagcctggtcattgtgggctggtgcagttattctctgcagacggtcttcgccgccagagcggcggaatcagttcagcattcggtgcgcttg cgcagcttcggccatatgctgcgtcttggactcccctggcatgaaaagcatgccgattcgcgtcttacccgcatgaccgttgatgtggactctctc gcccgctttctgcaaaacggccttgccggtgcggccaccagtctggtgacgatgttcgcaatcgccgccaccatgttctggctcgacccgttcct ggcgctgacggcattaagcgcagtgccagtggccgcactggcaaccatgatttatcgccgcctcagtacccctgcttatgcacaggcacggct 54 ME151672980v.1Attorney Docket No.: 139465-00120 ggaaataggcaaagtcaacagcaccctgcaggaaaaagtctctggcatgcgtgtcgtgcaatcgcatggtcagcaggaactggagggcgc ccggctgcgcgcgttatcggagcgtttccgcgcaacccgtgtgcgagcacaaaaataccttgcagtctattttccgttcctgacattctgcaccga ggcctcctatgccgctgttctgttagtgggagcttcgcaggtcgccgctggagaaatgactgccggggtactggcggctttcttcctgttgctgggg caattctatgggccagtgcagcagttatcagggattgtcgacgcctggcagcaggcgacagccagcggcaaacatattgatgaactactggc gacagaaggcactgagaacctcggctcctcttcggtcctccctgtcaccggtgcactgcatcttgatgaggtcacgttcagttatcccgacagtc acgagccagctctgaacaaacttaccctgacgatcccagagggaatggttgttgcggtcgtcggtcgcagcggtgcgggtaagtcgacgctg attaagctgattgccgggttgtatttccccacgcacggcaacatcagaatcggtgtgcaaatgctcgatgatgcctcgctcgctgagtatcgtcgc cagattgggcttgtcgatcaggatgtagcactgtttagtagtgatattgcagaaaacattcgttattcacggccatccgccaccaatgaagacgtt gaaattgcctcacagcgggcagggctgtatgagatggtgtgcaatctgccgcagggattccggacaccagtgaataacggcggagccgatc tgtccgcaggtcagcgccagttgattgcgctgtcccgcgcgcaactggctaatgctcacatcctgctgctcgacgaagccacgtcatgtctggat cgcacatccgaagaacgactgatgtcatcgttaacagatgtcgtgcatgccgggaagcactcggcgctgattgttgcacatcgtctgaccacc gcgcaacgctgcgatctgattgccgttattgataaggggttacttgcggaatacggaacccacgaacagctgttatctgcgggcggcctctatac ccgcttatggcatgacagcgtcagcagtactgcgctccatcgccagcacaacatgaaggaggaaaccccgggatagttactggacacgtaa tgtattaaaaacacagtcagaagcggcggtaccgtgaatagccgctttaattatttatactgacatccttaatttttaaagagtatgaatgctgaac atgcaacaacatccctctgctatcgccagcctgcgcaaccaactggcagcgggccacattgctaaccttactgacttctggcgcgaagctgag tcgctgaatgttcctcttgtgacgccagtcgaaggagcggaagatgagcgagaagttacctttctgtggcgcgcccgacatcctctgcagggcg tttatctgcgtctgaaccgggtgacggataaagagcacgtagaaaaaggaatgatgagcgcccttcccgaaacggatatctggacactgaca ctgcgtttacccgcaagttactgcggctcctattcgctgctggaaatcccccccggcactacggctgagacgattgcactgtccggaggccgtttt tccaccctcgccggaaaagccgatccgctaaacaaaatgccggagatcaacgttcgggggaacgcaaaggaatcagtgctgacacttgat aaagctcccgccctgtcggaatggaacggcggcttccacaccggacaactgcttacctccatgcgcattatcgccgggaaatctcgccagatt cggctctatattccggatgttgatatttctcagcccctcgggctggtcgtgctgcccgatggtgaaacctggtttgatcaccttggcgtatgcgcggc aattgacgccgccataaataacgggcgcatcgtgcccgtggctgtactgggcattgacaacattaatgaacatgaacgcactgagatactcg gcgggcgcagcaaactgataaaggatatcgccggacatctgctgccgatgattcgcgctgaacaaccgcagcgtcagtgggcagaccgttc gcgcacagtgctggccgggcagagcctcggcgggatcagtgcgctaatgggggctcgttacgcaccggaaacgttcggtctggtgctcagc cactctccttcaatgtggtggacgccagaaagaaccagtcgaccaggcttgttcagcgaaaccgatacctcatgggtgagtgagcatctgcttt ctgccccaccgcagggcgtacgtatcagcctgtgcgtgggatcgctggaaggttcgacagtgcctcacgttcagcagcttcaccagcggctga ttaccgctggcgtcgaaagccattgcgcaatttacaccggtggtcacgattacgcatggtggcgcggtgcactgattgacgggattggtttacta cagggttgagttgacccacaaacactttcaggaaacggtacagacttcctgaataaatcaaattgtcacctgcggaaaaggaataatcatcag atgtatgcccgcgagtatcgctcaacacgcccgcataaagcgattttctttcatctttcttgcctcacccttatctgtagtgcgcaagtttatgcggag ccggatatgcggccactggggccgaatatagccgataaaggctccgtgttttaccatttcagcaccacctctttcgactctgtcgatggcacacg ccattatcgggtatggacggccgtgccgaatacaaccgcaccggcatcgggttacccgattttatatatgcttgacggtaacgcagttatggacc gcctggatgacgaactgctcaaacaattgtcagaaaaaacaccgccagtgatcgtggctgtcgggtatcagaccaacctccctttcgatctca acagcagggcttacgactatacgccagcagcagaaagcagaaaaacagatctccactcagggcgttttagccgtaagagtggtggcagca acaacttccgccagttactggaaacgcgtattgccccaaaagtggaacagggactgaatatcgatcggcaacgccgcggcttatgggggca ctcctacggcggcctcttcgtgctggattcctggctgtcctcctcttacttccggtcgtactacagtgccagcccgtcgttgggcagaggttatgatg ctttgctaagccgcgttacggcggttgagcctctgcaattctgcgccaaacacctggcgataatggaaggctcggcgacacagggtgataacc gggaaacgcatgctgtcggggtgctgtcgaaaattcataccaccctcactatactgaaagataaaggcgtcaatgtcgtattttgggatttcccc aacttgggacacgggccgatgttcaatgcctcctttcgccaggcactgttagatatcagtggtgaaaacgcaaattacacagcaggttgtcatg agttaagccactaaacactgcccgcttttacgcgggcagtacgcctgaaacactacgatcagaatgatgcggtaactccggcatagtaagcc 55 ME151672980v.1Attorney Docket No.: 139465-00120 cggcctggctcgttataggtattcgccccttcagaagatcggaagatctgtttattgaggatattactgacgccgacattaagacgcagatttttatt aatatcgtaattgaagttcgtccccaccagtgaataagcgcccagctctttacctgacagaccgccagtatcttcactgcgggtttccgcatgagt acgcggtttttgtctgccatataacgtccagttgacgctggcagaaaacgcctgggtgatggtccagttaagcgagttattgatagtatatttcggg atgaccgacagaggattaccggtgtctttttgctccgaagtgatcatccatgtggcattggtattccagttcagacgatctttcaccagtgggaaag acatactggcttcgataccgtccaccagagctttcccgccattctgccacttgaggatatatgcgcctgaagcggtttgcccgataacgttatccc cggccacgatcttattctggtaatcattgcggaagtaggtcacacttgcgtggtaatcttcccaggtgaactccagcccaatttctttattgacgctg atttccggatcgagatctttattaccgatcaggtagcacccgcctgatgtaatatcttttggacagccattgcctttcgagtagagcagatagccttc actggattgatacaggtttggggctttaaaggttcgggcaacccctgctttgactttgaaataatcgcccaattcctgcgaaagattcagactggg gctgaagttcccgccggagtcgctgagataatcaaagcgcaggccgggaatgatattcgtgccaggaaccggctcaatgttatcttcaatata cagcgcactgatttgagaatgatttttactgctgcgatccgcagcagagccagaaataccgctgatatcactgtcattcaccgtcaggctggtag aggaaggatcatcgagcttatcgcggttccactctgcaccaacggtcagcgtttgatcaaccatcacattcaaaggaatattaagctcgccgct ggttcgccaggaactcaggcgattggtcgtaaacttttcacccgctaaaatacgcccttcaccaccgccggataatccttcattcatgcgggtatt attggttttctcgtaataaacaccaaagcgactttgtccccagtcccagataccattatgcgtaatgccataattctgtcggtacaggcggttcgtct ctttgccggattttgccaggctttcggtaactgcactggaagaactgttttgcgtatcgcccgcatagatattcccctggcggctatatccggcttcg aaatcgagaatctgctgcggatttaatttccacgagacaacgccgttaatatctttgttacgtaccccttcatgcccggctgcgtttttcgtaccgac cggagaattaatatcccaactgtcagcatccgttttattcaggttaccatacaaacgcgtggtaagagcattaccagccagaggcccactaagg ctgaaattggcgcgacgcgtagcgccctcatcgctactttccggctgattggtgtataacgacagcgaaccgtgccagtcgttggtgggacgttt ggtaatgatgttcaccacccccccggctgcccccgaaccgtagcgcgccgccgcagggccgcggatcacttcaatacgctcaacctgttccg gtggcacccagttggtgtcaccgcgggtatcacgctctccacgccagctataacgcacggagttacgtgacgtcaccggtacaccatcaatta aaattaaggtgttttccggccccataccacgaatatcgatctggcggttgttaccgcgtgtgcccgaggcgctattgccggtaagattgacgcca ggcattttacgaataatatctgaaaggtcgtttaccggaggggtctttttaatatcctcgctggtaataaccgacacgcccggctgctgttttaatac ctgctcagcggtggcttccaccaccagagtctcgtcattatcatcgtcggaggatttggctactgatacctggctattcaacccaaccaggagca cagttagcgaccagaggattttgttaattctcatacctattccctaataaatgcctaacttaaaatgtttgatcgttaagctcacatccttgccagatat ttcttactgccattattgtttttatataagaatgataattaatatcatttagtaaaagaaaaagcaatccctcacaagataaatatattgatttttcataa atatcaaattgatatataacatatgttttttatttcattgcacttgtcgatcatcgggaattttgtcacaatttctcacggatagtgttcacattgtttctgac ctgcatttcaggcgcgggcgctgcttatgtatataagggcctgtggtcgacgaatcagcataatcagatcttcctgcgaacaatttctttcagcgta tcaggcagcactatcggattatctacgagcgtttttgattcacattgtgtgtgaaacttcatacatcatgattcgctttccctactcagttctgatgattgt tttttattctattaacacggagttaagattctaccctcggtgtcctcattattaatgaggaatgctacctatctaaaaactgaccgaaactttggggcat ccctggaggtttccaaggagaaaaaggtgacagtgatagtgccacattgtgaggaaattcctgagaaactcctgagaaactcctggttcacac acataattcaggagcttgccatagatagtaaccaattgttttatattttaagaccattaaatacgcggctacgatagcgctttatataatcataaact gtagagataaaatatctgcttctctgataacactaattgagtgtcttaaactaagtaatttcatttttccccctttaaaagcaatatatgtgcaatagttg aagttaaagctgtgctgccagagttgataataattgttctgtctgttgccatgatatgcattcatcagtaacagactgaccatactctagcggaaaa ctatcggattgttttccatcttgtaaaaagctttccaccataatgccagctacatatcctggtatttttttccgattatctataacttgacgtgcaacgga aatttgccgtttagccactttaccgctattaccatggctacaatcaattatcagacgatggttaatgccctcatcatgcattaatttcactgccttagtta tatcagacaaaccatagttaggttctctacctccacgtaatattagatgtccatgtggattgccatcggttaatagtgtagatatactatttgttaaag atgtcatatatacaatatgttgttcacgggctgcaataatagcgtcaatagctaagttaatatttccatcagtactgtttttaaatccaactggacaat gtaaaccagacgcaagttgacgatgcgtttgtgattcagtggttctggcaccaatagcaccccaacatattaaatcagcaatatagggagtaag aaaaggatcgaggaattctgtagcggttgcgacacgcatcgttgtgattgaagacaaacactggcgagcatatcgtatccctttctcaacatcat aacttccatttagatcaggatcgtgcattattcccttccagcctttacgggttcgaggtttctcaaaataagtgcgcattacaatgtacattttcgataa 56 ME151672980v.1Attorney Docket No.: 139465-00120 gtacttattctgtaatacgaacaatcgcttcgcataatcaactgcggcctgaacgtcatggattgagcatggaccaacaatcacaagtaagcga ggatcttttcccagcaaaatattcgctataatatctcgttgcagagagatccatgttacggtttcttccgatactgcgatttctttatgaatctcgcccac agtgggtaaactgccaagcaacttcccgcggtagattaatttactagctgactgcatcttgtctcctaaaagcgctatttcagaccatcagttgcac acagtagatgataatgataatcatttactgtataattggcaatatatggggattcatagtcatttctgagataatatacgttaggccttctctcccgtac ggaaatcacttgttttccgtaaattacctgaatcttaatccctgagctgcggctagctgtggaactcacaaaagctgaccgggccgtggcacagc ctctctgctggcagaacagcgtactgcagaagtcgtcgtactgccgctggtcccggcatcaggtctgcccgcagatggcacttctgccactgat ctgccggtggagctgaacggcatgacatatcaggcctgccgtggggattttgtggtgcgcctcgacggcagcacctgtctgcagttatagaata aagaaggcggggaggtacgccgggagggcgatccgctggaagtggcgtagtggctgtaggcctgtaatgatgcaggaatagaagttcgtgt acaggttaatgaaagcatcaccccgtaaaaacatgccggcacggtttacacatcattccatggtggcagccggttaactttcctgcgccacca gtggaaagaggccatgagcagtccgaacaaaaaaccactcgtgatacttataataatggcctcacctgctaccattcctgacggcccccagt acataaaccacattgcacatccccaggaaataccccataagccccccatcagcagcgtaacctgccagaacggcataaagggcaatggc ggaagccggatacccagccgccagagaatacgcagcagaggaggagcataattactccgccacatctttttgctgtccatcagggcaattgc ccgggctttttttgctcaaaagtcacaggagcacctccgttccatgatgtaaacgtgacacaacttattgttattgattattattatcataccgtgattat gttgtcatgcacctgtacactgataaaagaaagggagaacaggaggctggctgaacaaccaatccgttcgtccagttcaatggcggctatca ggttctgctccgcaatgtactcacgaatgcgcttcctgtcctggctggccagcatggtccagaatatctccatcatttaccggcgactttcctgctca tttcatcccgccatgcagcagcggaagattcgaccatgtcatggggaagcacatcgccgcgctcgagttgctgacgtcctgcctcaacctggtc acggaaccaggtattatgctgccgttccacggtctggcgcataaaatcccggattaactgagagccattacggtccatgctttttgcagggccat aaaagcatccttcagttcagcgtcaattcttaaactcatattaacctgtgccatttatcaccccgtcattaccacgtatatacacagtatataacgatt cagatatcgtcactaggatatgccgcgccagcggcatggaaggcggcactccgctgtttcatatgataccgccgaagcccgatgtaagccgc tacagtcgtccgaaagtcaccagcctcctcccccctgccgtcatccgtgcatcagctatgcactgagtatgcctgcccttccctagagaatcctg ccaggcttgccacactgatatatcttgactttatgtaaacgatatgacactttaacatgataatgattaccattctcttttaatatacagagaaactag gaaatagatgaatgagttatgttactttaatattctctgacaataacctaaatcagttagattattgtcatttaataaataatgacattctttcatcataa ataaaaagactattgtttataatattgttctcagcattatatgattatttatcctgataactctcctatgttgtatgtttatatgattttccttgaaacatataat gcaaattttcgatttattttccatcattaatccagataaacaacaaactaatagtatgcaaggagacattatttgtttcgccaggatgctttagaaaa cagaaaaatgaagtggcagggacgggcaatattacttcccggaataccactgtggttaatcatgctgggaagcattgtgtttattacggcatttct gatgttcattattgttggtacctatagccgccgtgttaatgtcagtggtgaggtcacaacctggccaagagctgtcaatatatattcaggtgtacag ggatttgttgtcaggcagtttgttcatgaagggcagttgataaaaaaaggggatcctgtttatctgattgacatcagtaaaagtacacgcaatggt attgtcactgataatcatcgccgggatatagaaaaccagctggttcgtgtgaacaacattatttcccgtctggaagaaagtaaaaaaataacgc tagataccctggaaaaacaacgtctgcaatacacagatgcgttccgtcgctcatcagacattatacagcgtgcagaggaagggataaaaata atgaaaaataatatggagaattacagatactatcagtcaaaaggactaattaataaagatcaattaactaaccaagttgcattatattatcaaca acaaaacaaccttctcagtctgagcggacaaaatgaacaaaatgccctgcagataaccactctggagagtcagattcagactcaggcagca gattttgataatcgtatctatcagatggaactgcaacgactcgaattgcagaaagaactggttaacactgatgtggaaggcgaaatcattatccg ggcgttgtctgacgggaaagttgactccctgagtgtcactgtagggcaaatggtcaataccggagacagccttctgcaggttattcctgagaac attgaaaactattatcttattctctgggtcccgaatgatgctgttccttatatttcggctggtgacaaagtgaatattcgttatgaagccttcccctcaga aaaatttgggcagttctctgctacggttaaaactatatccaggactcctgcgtcaacacaggaaatgttgacctataagggcgcacctcaaaat acgccgggtgcctctgttccctggtataaagtcattgcgacgcctgaaaagcagataatcaggtatgacgaaaaatacctccctctggaaaat ggaatgaaagccgaaagtacactatttctggaaaaaaggcgtatttaccagtggatgctttctcctttctatgacatgaaacacagtgcaacag gaccgatcaatgactaacaggaatttcagacaaattataaatctgcttgatttgcgctggcaacgtcgtgttccggttattcatcagacggagacc gctgaatgtggactggcctgcctagcaatgatatgcggtcattttggtaagaatattgacctgatatatcttcgccggaagtttaatctctctgcccgt 57 ME151672980v.1Attorney Docket No.: 139465-00120 ggagcaacccttgcaggaatcaatggaatagcggagcaactggggatggccacccgggctctttcactggagttggatgaacttcgagtcct caaaacgccgtgtattctccactgggatttcagtcacttcgtcgttctggtcagcgtaaagcgtaaccgttatgtactgcatgatccggccagggg cataagatatatcagccgggaggaaatgagccgatattttacaggcgttgcacttgaggtctggcccggaagtgaattccagtcggaaaccct gcagacccgcataagtcttcgttcactgattaacagtatttacggtattaaaagaacgctggcgaaaattttctgtctgtcagttgtaattgaagca atcaatctgctaatgccggtggggacacagctggttatggatcatgctattcctgcgggggacagagggctactgacgctaatttctgctgctctt atgttttttatattactcaaagctgcaacgagtacgctgcgcgcatggtcttcactggttatgagcacgctcatcaatgtacagtggcagtcggggc tgttcgatcatcttctcagactaccgctggcgttttttgaacgccgaaaattaggtgatatccagtcacgttttgactcccttgacacattgagggcca catttaccaccagtgtgatcgggttcataatggacagcattatggttgtcggtgtttgtgtgatgatgctgttatacggaggatatctcacctggatag ttctctgctttaccacaatttacatttttattcgactggtgacatacggcaattaccgacagatatcagaagaatgtcttgtcagggaggcccgtgcc gcctcctattttatggaaacattatatggtattgccacggtaaaaatccaggggatggtcggaattcggggagcacactggcttaatatgaaaat agatgcgataaattcgggtattaagctaaccaggatggatttgctcttcggaggaataaatacctttgttaccgcctgtgatcagattgtaattttatg gctgggagcaggccttgtgatcgataatcagatgacaataggaatgtttgtagcgtttagttcttttcgtgggcagttttcggaaagagttgcctctct gaccagttttcttcttcagctaagaataatgagtctgcacaatgagcgcattgcagatattgcattacatgaaaaggaggaaaagaaacctgaa attgaaatcgttgctgatatggggccaatatccctggaaaccaatggtttaagctatcgttatgacagtcagtcagcaccgatattcagtgctctga gtttatctgtagctccgggggaaagtgtggctataactggtgcttccggtgcgggaaaaaccacattaatgaaagtactatgtggactatttgaac ctgatagcgggagggtactgataaatggtatagatatacgccaaattggaataaataattatcaccggatgatagcctgtgttatgcaggatga ccggctattttcaggctcaattcgtgaaaatatctgtggttttgcagaggaaatggatgaagagtggatggtagaatgtgccagagcaagtcata ttcatgatgttataatgaatatgccaatgggatatgaaacattaataggtgaacttggggaaggtctttctggcggtcaaaaacagcgtatatttatt gcacgagccttataccggaaaccaggaatattatttatggatgaggcaaccagtgctcttgattcagagagtgaacatttcgtgaatgttgccat aaaaaacatgaatatcaccagggtaattattgcacacagagaaacaacgttgagaactgttgatagagttatttctatttaaaccatagaggaa ttacaagcgtatgaggaatatttcttcctgttataattcctcgttatgctcagatatctgttggaggtggaatggaagatagacaatccaccaagaa gaaatatcattctgtgtggattgtccaataactgttctttcttatattaaataatactatttataaacaaacatcactaagattatttggactccaattaca caatcttcccgcagcatagttccatgcttctgaaggtatcccttcgggtttttgcttaattgttccccctaaaccggatggagacattgcaggattagg tttgtgagtggatgcatagtcatatattgcacctccagccacacccccagcagctgctccaattcctcctgcaacaaattgcccggatagtgttcct atagccatcgcaatatcacgccctgaagcaccaccagaaacagaatctaattcatttagagtcagagttctcatatgatctcctttttatcttatcgg atattgaataataattatcaccaacaaagtaacatattgcagacattaatgcagagaagcaaaatgtatgcatggataaaaagtcctttcctcta aaaacacaatcatatatagctaatgcaatatatattgcggtggcatttattataaatgcaaataacaactctaattttgttctttttctatccattacttttt atcccattactttctatcccattaccacacaaacactaacgataatgattatcgttaacatagtcaagagtgaagggtaggaggccctcaacccc ctataaggggtccgcttggaaaacggatttccccacgtcaagagaattgatttgaacgagtggcttcgctacaaacagctcccgtttgtgtgtga aaaacccctctcaacagatctcaactctgcacgatatctacaggagattgctccaaaaggcagtatcgtgtcttttaactaccgatataaccccg tcatcgacatgataatgcgtctgagaaatgagaagaaattaggtgaactgcatttcttctcagcagaatttaataaaaattctgcactgacccgcc atcacctgacctggcgagactccgcacaacagagcaaaagcagcggtgctctgggcgatctatcctgtcatctgcttgatttattttgttttatagg ggaaagcccggtagtggttcatgggatcaaaacggtaaaggggacacgtgggtacaaaatcagatggtcaggttgaagtggatgataacg gctatgtaatgggcagttcagaaaagggagcttatttcagagttcatgccagtaaatcggaaacagatcataatttgggactgcatatacagctt gtatttgaaaatggtgaaatcagatattcaacccaccatgaaaatcgcctgctcctgattctatttaatgatacgaatacggaaacaattggcttcg acgctctgaaacgcttgcctgatccaccacgggagcttccattctggtctgattcgtttattcatcttcatgacgactggtgtgctcttattaaatatggt cactcgtctcctaagctagcggacctgtcatccggccttcatatacaggaaataattgaggcattctgatatggataccattcttttaaccgggctgt ttgctgcatttttcacaacgtttgcgttcgctccccagagcataaaaactatcaggacacgcaataccgaaggtatctccgttgttatgtatatcatgt tcctgacaggtgtaatctcatggatagcgtatggcattatgcgatctgattttgccgtactgatcgccaatattgtcacactgtttctggctgcaccag 58 ME151672980v.1Attorney Docket No.: 139465-00120 tgcttgtaataacacttatcaaccgcaggaaaaaacacgtatgaatattgcaatcatcggcgcgggtcctgccggcatcatttctgcccgtaatg cgattaaagcaggtcactccgtggttctgtttgagaagaatacccgaattggggagatctggaacccctggagtggtggcgcttaccgtaatgc ctgtatgcagaactctcgttatacatttcattatactggctttccccctggcgatatcgatgaatttctaggagtggaacaggtattcagatatctttcg gccgtggccggagaggatgccctccgtgagtcgatccgactgaatactgaagttgtttcactcagaaaagacgccggacaccgggtgatccg ctgcgcttctgaaggaaaagacacggaagacatttttgaccgggtcatcattgcaacaggtgaactctggcaaccccgccggccccccctgc caggtgaggaaaacttctccggaacgttgatcacgtcgagagattatcaggagccagaggcatttaaaggaaaaaatatcctcatcattggc ggcggtgtcagcggtgcggatattgcctcagaccttgttccctttgccagaagcgtaagtctgtccgtcaaaaagatgggactttatctgccgag acaattcccgactggcccgaatgacatgatgcactcctatctgggcaggtgtctgctgagccaaatgaattatgaagattttatcggttatctcga caccatgatgcctgactacatgcaggcctaccgagcatccagtctgttgcccgacatggcgaacaataacgcggtgcatgttaacgaaaaaa tcataccgaatgtggccgccggtctgataaaagttaaaccccaggcagagcgtttcaccggagaaggcgctattaagttcgtcgacgcatctc aggaaaaatatgatgtaataataacctgtacaggctatgaaatgcctgattactcgttcattcgtaagcgtacagcctgaaccgtctggtcagaa tctgacgaattagacaaagtggtgtccaccaaataagtagtgggaaccaaagtgtcagatatgcagaaaaatgtgactcccggcaggcgaa aaggctgccctaattatcctcccgaatttaaacagctgctcgttgctgcctcctgtgaacccgggatatccatctcaaaacttgctcttgaaaatgg cattaacgccaatctgttgttcaaatggcgacaacaatggcgcgagggaaagctgctattaccttcttcagagagcccccagctacttcctgtga ctctcgatgcagctgccgaacagccagaatcgcttgcagaggacccggaaaccctcagtatcagctgtgaggtaacgttccggcacgggac gctccgcttcaatggcaatgtcagcgaaaagctcctgactctgctgatacaggaactgaagcgatgatcccgttaccttccgggaccaaaattt ggctggttgccggtatcaccgatatgagaaatggcttcaacggcctggctgcgaaagtacagacggcgctgaaagacgatcccatgtccggc catgttttcattttccggggccgcagcggcagtcaggttaaactgctgtggtccaccggtgacgggctgtgcctcctgaccaaacggctggagc gtgggcgcttcgcctggccgtcagcccgtgatggcaaagtgttccttacgcaggcgcagctggcgatgctgctggaaggtatcgactggcgac agcccaagcggctgctgacctccctgaccatgctgtaaatctctttatcctggttgtcacagaataagcccggtaaaatacgggcttatgaacga catctcttctgacgacatcttcctgctgaaacagcgcctggccgaacaggaagcgctgatccacgccctgcaggaaaagctgagcaaccgg gagcgcgaaatagaccatctgcaggcgcagctggataaactccgccggatgaacttcggcagtcgttccgaaaaagtctcccgccgtatcg cacaaatggaagccgatctgaaccggcttcagaaagagagcgatacgctgactggtagggtgtatgacccggcagtacagcgtccgttgcg tcagacccgcacccgtaagccgttccctgaatcactaccccgtgacgaaaagcgactgttgcctgcggcgccgtgctgcccgaactgcggcg gttcactgagctatctgggcgaggataccgccgaacagctggagttgatgcgtagcgccttccgggttatccggacggtacgggaaaaacat gcctgtactcagtgcgatgccatcgtgcaggcacctgcaccttcgcggcccatcgagcggggtatcgccggaccggggctgctggcccgcgt gctgacctcgaagtatgcagagcacaccccgctgtatcgccagtcagaaatatacggccggcaaggtgtggagctgagccgttcactgctgt cgggctgggtggatgcatgctgctggctgctgtctccgctggaagaggcgcttcatggctatgtcatgactgacggcaaactccatgccgatgat accccggtccaggtactgctgccgggtaataagaagacgaagaccgggcggttgtgggcgtatgttcgtgatgaccgcaatgccgggtcag cgttggcacctgcagtgtggttcgcttacagcccggacaggaaaggcatccatccgcagactcatcttgcttgcttcagcggtgtgctgcaagc ggatgcgtacgccgggttcaacgagctgtatcgcaatggtcggataacggaagctgcctgctgggctcatgcccgccgaaagatccacgatg tgcacgtccgcatcccgtcagcactgacggaagaagccctggagcagatcggtcagttgtacgccatagaggcggatataaggggaatgcc ggcagagcagcggcttgctgaacgtcagcgaaaaacgaaaccgctgttgaaatccctggaaagctggttgcgtgaaaagatgaagaccct gtcgcgacactcagagttggcgaaggcgttcgcgtacgcacttaaccagtggccggcactgacgtactatgcgaacgatggctgggtggaa atcgataacaacatcgctgaaaatgccctgcgggcggtcagtctgggtcgtaaaaacttcctgttcttcggctctgaccatggtggtggcgggg agcgctactgtacagcctgatcgggacgtgcaaactgaatgacgtggatccagaaagctaccttcgccatgtgcttggcgtcatagcagactg gccggtcaaccgggtcagcgaactgcttccgtggcgcatagcactgccagctgaataacacatccccgtcaatacggccctcgctgtacgctt accaataaataatatgcttatcgacctgatggctgcaatgtcgcataaagactggctgagtcgtcgtcaacgacaaaaacagggaattgaacg tgcccatactttaggtaagtatcggggcaaacaggcagatcgggaacgacatcagaaagtaatgtactaccggcagatcaagaaactgagc 59 ME151672980v.1Attorney Docket No.: 139465-00120 atccgtgaaacggcagaggtgacaggctatagtacttcccaagtttgtcgcattcaggcgctctataaagaggttaaacctgattaaaaaatag ccttaacgtacaatattttccgtttcccaaacggagcccaatacagatggctaacagaccgtctgcttccagttcaaatggtttaccgtcgcggtcc agggcgcgaattttaactttctctgcgcgtggcactaaagctcgttgttcagggattgacaggcaaccttcttcaatgcctgtttcgccgcttttttcta aaagctccggattgattaacaccagccgttcgtcacggttttccgaaacatcaattacaatgatacgttgatggatatcaacctgggttgccgcca ggccaatacctttttctgcgtacatcgtctcgaacatatcatcgacgatacgctgaatttctgcattcacttcttctaccggtttagcaactttgcgaag ccgctcgtccggaatatgtaacacttgcaaaactgacataaatctccagagatgtgttcaggagttagaaagattatttcttctactgtaccgtaat cagataatcgcagcaaagggacaaagcaatagtccctttgctgcacagcatcagaatgaataacgaatattagcgtttatcgcatcatctgtgtt gtatttaccaaatgcagagcgttcaacttccagccccaaacgcgtattgtcgccaaaccgggcatttaaccccacaccgtaaagcatacgact gtcttttctgccattaatctgatgttctcccgctgcatccttcagatgaacgtcagcactgtccgtcagatcgaactcataatgcaggccggcacgg gctgtcagactccagtccttaccactgaaggttttaccggaaacaacgccggttctgcctaccagaggattaacgctgttacgacgcattgaga catccattccactgtcgttccagttaaatgtttggccctgcagtcttccccagaccagttccgcctgaggttcaacaaacgtcgtatctgtcagatga taacggtatccgacttctgcacctgcatacagtgaatggctgcggaagttctgtttaccagctccggcaaagttcaggtcatatttgttttcattgtga atatatttggcaatcacatcaaagtaagcgccggaccggaacagaccactggcatagaaaccaccaccccatgattttgttttaccgctgtaca ggtctgctgacgcatctgtgtcagtgtaggtggccatcacgccggtaaacaggtccatacttcccagttcgtgcttacggtcagcccccatctgca gcagggtatagtggtcagtgaaaccgccatcagcagagccggaaccgttcagcagacgcacccacgtaccggcttcgccattaatatccctc aaatcgcccatgcgtttgttcaggttgttaacttcagtgatgaagttgttatagctgatgtgcatgaatgtggcggcagccttaccctggccgtcgtta cgtgcaacctggtaaccatcgaggacccactcttttttcccgtcctcttttctgacactaaggatgggggtgacatcactgaatcccacaacccgt gttgatgccctgaacagattatcagctgtcgcttcaggtgcgctgaccagtggaatatcaagcgtgtccttgttagaaggttttttcaggaagttaac ccagatgctgttgtcatgacctgttgccgacttgtttatcaccagtttgtctgccttgttaaggtctgtacgcatgacaaatgctgactgaaccgcgtc cagattatctgttgtcagtgtcgtgaacggtgatgttcccccgttaaaaccgactattgtccggttaagtttcatatttcctgccgtggagtttccgttca tcgaccactgggtgtctgtcatgctgacggtggcatccggtgcattcaggctcccgctccagatattgcggtacccgttaaacaggctgtacaac atctgattctgaagagtcaggtcaggactcagttccccttcccctccgagggtgacagtccctttatcctgaacattgatattacctgacaacatac tgtacggccccacgttcaggcgggcatcgtctcccttcaggttccatgaaccggcataatcgtatacaggtaaaagtgtgtgagatacctcatca ggacggctgttcagactcagggcggcatcagaaatattcactggaccgtcagaaacaaaactctgactggccagagcatttgctcccttgttca gattcagggcggtactggtcagtgttgagttccccagaacggcactgtctgaggagatattcacggtactgttgttcgcctgtattccgccattgaa tatatcattgatattcagcactgactgattatccaggttgacggtgccgttgaagacacttttatctgcatctttagttgcaacagatgtgccttcttcaa gggtaaaggctgttccctggccatcgtttttgtcgataaatacccggctgtcgcccagcgtgacgctggagttatctgcctggatggttgtgttcagt gtggcattgcgaccaagaccaaaatcagtgttcttcagtgacagcctgtcaaaggtaaaactgcggttctcccagtcctcctgactgaatgacgt aggctgagtcagaaccgaatggtctccactggcagccagtttgtcagccacagactgagtattgtatgcatggataaccggatgcccctgcag cgtcagacgcccgttttcctgggtgaatgtaccggagatatccgcagctccgtccatcaccagagcaccggtaacgccttcaggcaggcgatt gtccacattcagattgcctttcagttgtccgtgaaacagataccctgcagtattgaaacggtcagctaccagtttctgtgcatccccctgactgtgcc ccacaaactcccaggtggcgttgctgctctggtccgtggggaaataaccataggtgctctgcttcaggatgaagtaatccgtcgtatttgtgtacg ggttattgtatttatataaatttccggcagttcctttacctgattccgaccagccattcagtgctactttgtcagcccgcagggcatagtccagcgtgat agtggcccgtttatcaacgttattcgccagcacggcaccataatctgccgccttcaactgatgaaacgtcagactgttaccattaacatccagtgt gcccccacgatatccccatgagacggtatccggatttacctgccgctcatcagtcagtaccacggtcggccggccactggcaatattaacact gctgaacgcctgcacctgtcctttattgtccgcctgctggttcagtacaacctttccgtccccgaccttcaggccaccttcattaatacctgtaccctg taccgtcagcgtaccttcaccaattttatgcaggttatcgcccttaacaccattaacctgccagtttacggacaccccgttgtccacaacaatacc ggcaccggtccaggtacttccgttagaggttgttactgtgtaattatcacggaacgtcagggaacccgccccctgagaaaccgaatccttaagg ttaatctgaccattctgcccctgaaatatcaggttcttaccagcattcaggtcatttccctgctgcccgtgcatggcatatgtggtggttccctgtgtca 60 ME151672980v.1Attorney Docket No.: 139465-00120 gcgcaccagctccggtactgctgttaaagctccactccagtgcaccaccttccgatgttctgaacgtgaccggggcatcattgtcttcattaaattt ctgcccgataaaatccagtggaataacagcccagttatttcccctgccccccgcgccattccccgcagtaagaacaccgaccagcacccattt attctgaacagtatcaaaagcaaataaaggcgaaccgctgtcaccggcctcgccataaatgggcattgcaccattaagtttgtaatcaaaaac cagacctgaactggtgctaatcatttctccattctgataggatgacaggctaccgacagtcccgccggtcagccaggaatatgcacctcccattt gtgtcagctgtccgttactgtccttaatatactgagtaccagaccccagacgataaaaaacaggatagcgctccttatccagatatgcgccagc cactgccccctgcgccgtcaccgcagtaggggcaacctctgtcaccagtttatccagccggggggcatgaaaatccagtgacggcgcattatt ccggtccacgatattgtaacggttttcaccatcaccaaagctaacgtttgtatatcccccgttatgtttcacgctggcgatatactgcgggtttatcag tgtcgccacaccgatttccgaatccacagcactgaaatcaggcatagctgccttatccagcgtaccgacaaattctccctgcttattataaatagc gatattcgttgcccccgggcggaacatccccttattttcagcaaaatcacgaaataactgatacccgagttcattattgaccgttcccgcaagact tcctgcagagaatagtaccgggatcagtaataaaaccgggaaacacagacgtctgacagacttatgaacacatttcctggcaaactcagata cggcaataaagccccgggccacagcgctgtagcgaagagaataaattctgttcatttttaattactcctgagaaaaataaaatcagtactactc aggcataacggataatcaccatcatcctgaggaatccccagaaaaaggagacaggcatagcgtgatgtgaactactgattctttcacaaattc agggagagcaccgttatgcctgcacataaagtatgccagaaccggtggcgaatgtgtgctatgcacaaatggtgaaacagttcctgagtcgtg acccgctcgaatgcgtgctgtgtggcggccgaatggtataccggcgtgccatagcggggctgaatgtggacgggctgaagaaaaacgcgc gggatatcagtctgataaggattactttccagccggctgatggctgacgggcgtaatcccattcgttcggccagcgcagactttgttaaccccgct ttttctctcatttcgtataacatttctaccatctccagttctttgtcagcatcttcataccctttaacagcttctggagtattaagcagtttttgctttacctggc tgaacggaacactttttactttcgtcatttcatctccttcaggcgcatccttgaagtggtttactgaatttggccacctgaacagaggtgatatgctcac ctcagaacaacacaggtgctccaatgaaaaaaagaaatttcagcgcagagtttaaacgcgaatccgctcaactggttgttgaccagaactac acggtggcagatgccgccaaagctatggatgttggcctttccacaatgacaagatgggtcaaacaactgcgtgatgagcgtcagggcaaaa caccaaaagcctctccgataacaccagaacaaatcgaaatacgtgagctgaggaaaaagctacaacgcattgaaatggagaatgaaatat taaaaaaggctactgtagattcaattggtcaacgcaacagttatgtgaaaacatggggttgcggaggttttttgaatgagacgaacatttacagc agaggaaaaagcctctgtttttgaactatggaagaacggaacaggcttcagtgaaatagcgaatatcctgggttcaaaacccggaacgatctt cactatgttaagggatactggcggcataaaaccccatgagcgtaagcgggctgtagctcacctgacactgtctgagcgcgaggagatacga gctggtttgtcagccaaaatgagcattcgtgcgatagctactgcgctgaatcgcagtccttcgacgatctcacgtgaagttcagcgtaatcgggg cagacgctattacaaagctgttgatgctaataaccgagccaacagaatggcgaaaaggccaaaaccgtgcttactggatcaaaatttaccatt gcgaaagcttgttctggaaaagctggagatgaaatggtctccagagcaaatatcaggatggttaaggcgaacaaaaccacgtcaaaaaac gctgcgaatatcacctgagacaatttataaaacgctgtactttcgtagccgtgaagcgctacaccacctgaatatacagcatctgcgacggtcg catagccttcgccatggcaggcgtcatacccgcaaaggcgaaagaggtacgattaacatagtgaacggaacaccaattcacgaacgttccc gaaatatcgataacagacgctctctggggcattgggagggcgatttagtctcaggtacaaaaaactctcatatagccacacttgtagaccgaa aatcacgttatacgatcatcctcagactcaggggcaaagattctgtctcagtaaatcaggctcttaccgacaaattcctgagtttaccgtcagaac tcagaaaatcactgacatgggacagaggaatggaactgaccagacatctagaatttactgtcagcaccggcgttaaagtttacttctgcgatcc tcagagtccttggcagcggggaacaaatgagaacacaaatgggctaattcggcagtactttcctaaaaagacatgtcttgcccaatatactca acatgaactagatctggttgctgctcagctaaacaacagaccgagaaagacactgaagttcaaaatgtcaacgacggatgaaaagtgatcc acttatatctccaccaacggcccaatattgatccaccgttttactcaggattagcttctgctataaccccggcctttcgtttctgtctgagtcgatagctt tctcctttgatttgaacgacatgtgagtggtgtaagatacggtccagcatcgctgaggtcagtgctgcatcaccggcgaacgtttgatcccactgc ccgaacggcagattggatgtcaggatcattgcgctcttttcgtaacgtttagcgatgacctggaagaacagctttgcttcttcctgactgaacggc agatagcctatttcatcaatgatgagcaggcggggggccattactccacgctgaagcgtcgttttataacggccctgacgttgtgccgtagataa ctgaagtaacagatctgctgctgttgtgaagcgaactttgatacctgcacggactgcttcatagcccatcgctattgccagatgggttttccccaca cctgatggccccagtaatacgatattttcattacgttctatgaagctgagtgagcgtaacgactggagttgcttctgcggtgctccggtggcgaatg 61 ME151672980v.1Attorney Docket No.: 139465-00120 tgaagtcatactcttcgaacgttttcaccgccgggaaggctgccattcgggtatacatcgcctgtttacgttgatgacgtgccagtttttcttcatgaa gcagatgctccaggaagtccatataactccattcctggtctactgcctgttgtgacagcgcaggcgctgcgcttataaggctttccagttgcaact gcccggcgagcaccatcagtcgttgatgttgcagttccatcatcacgccactcctctgcagaatgagtcgtagatggagagtggatgatgcagg gggtgtttgtcgaagttcaccagattttcatcaagatgcacgtcatactcttttttctccggaggcagtgccagcatggactgctgctcttcgagcca gcgatcgcagggacgtgcctggattgtttcatgctttcgttggttagcgacatcgtgcagccagcgcagaccgtggcggttggctgtttcaacatc gacagtgatccccatcgggcgcaggcgagtcattagtgggatgtaaaaactgttacgggtgtactgcaccatccgttccaccttacctttagtctg tgccctgaaggggcgacacagtcggggagagaagcccatctccttgccgaactgccacagcgaaggatggaaccggtgctgaccggtctg atatgcgtcacgttgcagaaccacagttttcatattgtcatacaacacttcgcgcggcacaccaccaaagaagcggaacgcattacgatggca ggtctccagcgtgtcataacgcatattgtcagtgaattcgatgtacagcattcggctgtatccgagaacagcaacgaacacgtgaagcggtga gcgaccattacgcatagtgccccagtcaacctgcatctgtcgtccgggttcagtttcgaaccgaacggcaggctcctgctcctgaggaaccga gagagaacgaatgaatgccctgagaatggtcattccgccacgatatccctggtctctgatctcgcgagcgattaccgttgccgggattttgtaag gatgagcatcggcgatgcgttgacgaatataatcccggtattcatccaggagtgaagcaacagcaggtcgcggcgtatattttggcggctcag attttgcctgcaaataacgtttaacggtattgcgggagatccccagttctctggcaatcgcccggctactcattccctgcttgtgcaggattttaatttc cataactgtctcaaaagtgaccataagctctcctgaatcaggagagcagattaccccctggatctgatttcaggcgttgggtgtggatcactattg caccgttcgtgacacaaaacaccgaaagagataattgaaaggggtgttgcattgacagattgaatctacaaccgcgctcttgatgtcagactc cctcgagtaggcagcctggcggctgcggcttgtcatggcctggaattaccgttataaaaaaagataatgtcattgtctttcaggtagttatatggcc cgttcagctaaaccccgtaaacgcaaacccgcaccacaaagaagcaaacttccccgctatgttgtgaaacttcatccggatgatttttttgacg aagaagacgctgaagttctgcgctttgataattttgacgatgccgttgagtgctgcgctgacctgggtattccgttctttctggatgcaggaaacaa aaagctggtcttctggtttgttcgtgtcgatgacgaagggtatccggaaatagcccgctgtacggagcgggagtttgcaaccattcttgccggtat cagtgccggtggtatgtactgcccggaatgcggcacagttcactggccggatggcgttaccccacccgtctgatgcttccccgttttgccgatattt ttcagcagggtaaccgctggcttaactggctggagaaacagccggaaggttcagtgcgtccggtggtgactgagtcagtgacaaaaatcatg gcatgcgggaccacgctgatgggctacacgcaatggtgctgttcgtcaccggactgttgccacaccaaaaagatctgcttccggtgtaaagcc gctcctgtccgaactgcggggtgaaggctggcgcacagtggatacagtatctgctgagcctggtccccgactgcccgtggcagcatattgtgtt cacacttccctgccagtactggtccctggtgttccacaaccggtggttactggcagagatgaaccgcattgcagcggatgtgatactggaaatct gccatcaggcagatgtggagccggggatattcacggtgatccacacatgggggagtgaccagcagtggcatccgcatatccatttatcgaca actgccggtggtgtgacgtcgggccacacctggaaaaatcttcatttatacgcccgtaaggtgatgagcatgtggcgctaccggataacgcgg ctactgtcccggaaatacccggagctggtaataccggatgaactggcagtggaaggaaacagcaaacgggactggaatcgcttcctggac acgcattaccgccgcggctggaatgtcaacatatccagggtgatggataacgccacacatgtggcggtgtacttcggctcttacctgaaaaag ccaccggtgccgatgagtcggctggagcattatgccggtcaggatgaaatcggtctgcgttacaacagtcaccgtacaaaacgggaagaat acctgttgatgagtggagatgagttcatggaaaggttctcctggcatgtagcagataaggggttccgtatggtgaggtactacggtttcctgagtc cggtgaagcgccgcttactggaagaagttgtgtacgtcataacggagacggtgagaaaaacggcgatgcaaatcaggtggagagggatgt atcagaggttaccgactggcagagctggtcctgatgcatgagcgactggcacgacagcaggtgtgcggctgagagtcgcagaggggaagtt gcgtccattttaccggaaacggagcaaaaaaccgccattcataccctgtatcaatcagtgtcatcctgtttaatagtcgtttccgctcatatggtgc acaagaggtgttgaagaaatatctgttttgtggtgcttttttagtcttttggggatttaaattcctatcgatcaaggtcacgcatcagctttttatatgagc aaaatccaggctgcttttttatccattcagtatctatttttaaccttattaactttttattatgtgttccatttatagacgtagactcagatatattaccgactct tcgcataaatgatttttctgattcacttaatttttcagtaccgttacataatccgtgaccatatggtaatggagatgtcgttaaccatactttattatacaa aattgttccatcatacaacgttagatatccttctctcaggccttcagatgcaagtatggaatgcaacataggaattgctgtaaagtgatataaatcc ataaataaccctattctgtctatttatattcattttatatcatgttcaggccagactggcgagtcgtgcatagcctgtttactgaatgccgcttcagcgg catgtaaggcggcacgctgtggttacatgtgataccggagtaaaaccgccgaagcccggcgtaagccggtactgattgatagatttcaccttat 62 ME151672980v.1Attorney Docket No.: 139465-00120 ccatccccagccctgtcagaccatacccgctttcagccatgacagagtttctgtgcgcggtcggagtggtcccgacgagggtttacccgaagtc ggggcgtatctccgcgttagcgggccgtcagggccgcttacgagcgtgtattcaatacttccagccagaagactgacagcgacggcaatgctt agttacaacattcataattaaaagcgactctgttccggcccgaagggccggggcggggccgcttttcagttatgagggaggggctttgtggtttc agttctgcgctggttcggggtttttctggaggttggttttgtgtgttgtaactaaagtggctccggttggggcccgccgtttacggtgggaggtgcatat ctgtctgtccacaggacaggcagtgaataggttttctttttaaatgaatgtaattaagtagtttaaaggagatataaacaggtgtttaaaagatacat tgcaccctgtaaggctggcgcctggcgctttatgacatgaacggttgtaaccttatggggaagtccattgcagttaaatgtggataagcaaaattc cccgtctgtgaggcgtgttttgtatcaaaaacaggggggggccagatgcaccagaaggtggatgatgaggttgttttttgtatgaggtgctgcttttt tgttcgctggcaggcttcaggcgtgcgaatgcctccggtgcgggccggattattcagaagaggtcactttcagggggaagctgtggccagccg gctgtaattacggttacgtgacagaatcatgcgctctttcacacgacgctccacttcgcgttttaccgcctcacgattggcagtgaagcgcccttcc gagatttcacgcgtcagctgccgtttcaccagggtgacgatatcctgacgttccctgttcgcatcacgacgcgcacgggcacgctttattccacg ggacttaagctctgtctggtaactgcggaaacgctcacgcacaaaacgccaggctttcgctatcagttcatccatacccagggtatccagcccc tgctttttgcgctgtctgttttcccattcaacacgactgcggcgcgcagcaaccactgcatcctcagacacatcaagggcggcaaataacgccg gtgtgaacgtgatatcggtcggaatgtagcacccgataagcggatcatattccgtctggtaggtaatcagtcccagctctgcaaggaacgtcaa ggctcgggaggcccgggtgatggagagttttccggcagcagactccgtcgccagtccgcactcaatggccagcgtagtgatggagcactgg acgcggttggccagcgggtcatagtgaaaacacagcccctgcagcagcgcatcaatagcccggcgacgcagtaccggtggcatgcgccg acgcagtcccttcgaacgggcatgtgccacatgaatggcgaaatcaaaacgggaggtgaagcccaccgccttttccatcagtttttcgcagaa cttcagcgttccggcaccttcacggggtgtgaacaccggattcggatttttaacctgtgaatagtatttttgaaggagatcagccatgtccccctcc aacactattccccctcctacgggcggtaactagctcgtctgggccgacacgtaaagtttcgaacatatctggtctgaaccagatggttcctccatc aaaagcgatgagatgaagatcattatcaagagttttgagggctctccaaacacgagtgtatgaacctccactacaaactgaaagctctttaaca gataagttaactttgttcgtatcagggatgtagtgtgcgcacattacttgtacgagggaaacataaaaactaaccatcgacgcgctgattttattttt acgtttgccatgcgctattgcaagcaaacctctgatgaagatcagatgcttgaaggagtcaaaatgagcccttttctggagaaggctgcaaaac gggtgaggatacttgctggaatcaatgtgaagatctgattcaatcccacccgctattactgaatgtgtttcatgattagccacaccatcctgcactt acaatgcacagaaggagtgagcacagaaagaagtcttgaacttttccgggcatataactatactccccgcatagctgaattgttggctatgcgg ttttatggggccccggtaatctttctagtcgccaaacttgaagaagattatcggggttttcgcttttctggctcctgtaaatccacatcagaaccagttc cctgccaccttacggcgtggccagccacaaaattccttaaacgatcagcaatctaccactcactcctgagataagcaagaatgtgaatatttac gaagtcgctctgcgtttcagctctgattcaatcagtttttcaagcatctgcgcctgagtaataccttcctcatccgccagttcactcagcctgtcttttaa ccgggcctgaataaccgcatgaaaagccttatgagtgttgcttctgcgagccagagaagctctttgcctctctctggccggaacagggttaccttt ccggtatgtacgcttggtgcctgatgaggaagtcactgcattttctgtctgcgacatctcgcctcctcaattctcaaacagggatcgtttcgcagag aatactacagttttttgaaatcagcgacttgaaagttgtgacgatgatctgtgttggcgcacaaatcatgtctgattacgaccgtttaatgtgattttga taatgaaatgactgaaaagctggaaatgccttagccagggtgccggccccgggcgggaccggctgtttctggtatatagggtgtctgcccctg aaatacctcacacttccggaaagcggggacttccgtccccgcccggcagcttacttccgtgcttcgtaggccagagttacctgcaccactgtatt tcctctgtgaatattcagctcacataaccgttccctgaatatcagtgaaaaacacaacaccgtggcgcacacggcgagcaacccgataaggg catatttcgtcataaactttgacctctcagcaaagaagaggctacaatcagtagtgttaaggtacttcatgcagacctcacgagttaatggattaa cgagtggggtcttcgcatttctggtacctgcgtcttgcctgaaccagaaaggctcaaacacccgccgcgataataccacattccctgttgctgact attacccccatttgcctgtttttgttacaaaaacgccgggagcgggggaaaacttactttttcccgtttccgggggggacaactgagcattgcgaa cgcgtcagctacgatgttccggtgattgctgatcagcccctgttgtattcataaactaattaacattcattctgtctggtgcataaatctgcccgtgaa catctgcagacaataccggccatgaaaagataaataataccagtggaatatatttcctcatgttattctccatatcagataaatggcacctgtattt cagcccccctgaatatcgcagcacacattgaataagaccttattttgtgaaattaatcacaaaatataaaatcgcgacttaatacggaaaaaac tctgtgctactcagaactggccggtcatactgcccgaccggatattaaaaaaagcaaatctcataaactcctttcagccttctgactcctggagta 63 ME151672980v.1Attorney Docket No.: 139465-00120 atgaaggctgcttttttcgttacacccttccccggcagcgccggtggcagtgttcagcccttttccgggcaaactgttgccagtctgtccggggagc tatgcaggcagcccgtcgccatgcttctgccgcttccatccagcgtccactgacctccagaataacggcccgcccgccggcacgctggtaac gccagacctgaccgttccgtgaactgtttctgaatccattcatttttctgctcctcccttttagtcgtcagtattattgtggtaaataattcagtggactgg cttcctttcgggtacggaagttatttccttttaagccttattgtttcagcctccccggtttccggggaggctttttttatttcccatcaagcacggcctgaa gttctgcctttgtccggttctggcggcgaattttctccaaacgccctgccgcatacgcttcctcctcctgagaaatatgctccgtcacatacccttccg tgtcataccggcaggcaccggctttcatggcacacagatagctttctgaacgggtgatggccttcagcgcccggcgcagttttttatgcgagagc gggatattccgctgcgccacgtcttccagtaacacgtcccggataccgcaggccagaagccggggcgtgttaccgtcaaacaatcccggcc accaaggtttcagagtgttcaccgcctcatccagcgagggcagcgtcagataaatggacagcgcctgtctggcctgtgctttcttcgccgccag ctctgcttcccgggcagccttctccgccagtttctgctttttcaccttccattttggtggcgtggtgacattgatgatggttttccggctccgggtcggtgtt tccccttccgttttccgtttcagtgtcagtaccggtcgtttctgctctgtcatgattgccctgacctttgtgaaaatatgtcctccaggtcgttgaaactact cactatgatattaagctgacctcaacgacctctgacatattaataccgacaattaaagatgttttttaaaccattgcgtctgtactgccgccacacgt tcaaaaaacgcgcctttataaatatcataatggcatgcgtcagcttcctcgtataattccttagtaccagaagcaacagcatcatataaggctcttc cttgttctggtggattaacactatcctggccggcaataacaaccaaaactgggcactgtactttggctgcgctctctgccggtttatattgcaacgtc tccataaccgtaaggaaaggtatttttatatccatttcaggatactgaccttttaccttctcgaagaatacctttgattcattatcactcagtacccggg taacccccacaaacatctctttcccggtatttttcttcttttctgccattttattcagcgtggacagaaaagaagctttctccagttcattcatttctccggt aaccagaacctcaccgtcagcaaaagccagctggctgacaatacttttcactcttttatcctgcgcagctgcattgaatacatgacacccaccc agcgatgttccccataatcctatacgctgattatcaatacatacctgcttttctgcccaattgataactgaaataatatcttcggtctgcattgccgga accagacgacctcgttccccttcactctcaccgaatccacgataatcaaaagtaatcgttgcaaacccggcttctgtaaacgcattcgcaaagg aaggcagcagaacattccggataccgcaaaatccatggcataagagaatcaaaggatgttttatatttccttccgggacacgaaatgtcaggg caataccttcagggagtttgtgatccgtaattttcataaatatcctgtgttatgttgtttattaataagcagaatcagcgccagcatcataccagATT Tgccatcgtccacatatccacgggctggcaagggagcgcagcgaccgcgcagggcgaagcccggagagcaagcccgtagggcgccgc agccgccgtaggcggtcacgactttgcgaagcaaagtctagtgagtacgatataagttgttggaagatcttccctaaatttaaatataaacaac gaattatctccttaacgtacgttttcgttccattggccctcaaacccctaattaggatcaataaaacagcgacggaaatgattcccttcctaacgca aattccctgataatcgccactggactttctgcttgcgcggtaaggcaggataagtcgcattactgatggcttcgctatcattgattaatttcacttgcg actttggctgctttttgtatggtgaaggatgcgccacaggatactggcgcgcatacacagcacatctctttgcaggaaaaaaacgctatgaaaa atgttggttttatcggctggcgcggagtggtcggctctgttctcatgcaacgcatggtagaggagcgcgatttcgacgctattcgccctgttttcttttc tacctcccagtttggacaggcggcgcccaccttcggcgacacctccggcacgctacaggacgcttttgacctggatgcgctaaaagcgctcg atatcatcgtgacctgccagggcggcgattataccaacgaaatttatccaaagctgcgcgaaagcggatggcagggttactggattgatgcgg cttctacgctgcgcatgaaagatgatgccattattattctcgacccggtcaaccaggacgtgattaccgacggcctgaacaatggcgtgaagac ctttgtgggcggtaactgtaccgttagcctgatgttgatgtcgctgggcggtctctttgcccataatctcgttgactgggtatccgtcgcgacctatca ggccgcctccggcggcggcgcgcgccatatgcgcgagctgttaacccagatgggtcagttgtatggccatgtcgccgatgaactggcgacgc cgtcttccgcaattcttgatattgaacgcaaagttacggcattgacccgcagcggcgagctgccggttgataactttggcgtaccgctggcggga agcctgatcccctggatcgacaaacagctcgataacggccagagccgcgaagagtggaaaggccaggcggaaaccaacaagattctca atactgcctctgtgattccggttgatggtttgtgtgtgcgcgtcggcgcgctgcgctgtcacagccaggcgttcaccatcaagctgaaaaaagag gtatccattccgacggtggaagaactgctggcggcacataatccgtgggcgaaagtggtgccgaacgatcgtgatatcactatgcgcgaatta accccggcggcggtgaccggcacgttgactacgccggttggtcgtctgcgtaagctgaacatggggccagagttcttgtcggcgtttaccgtag gcgaccagttgttatggggcgccgccgagccgctgcgtcgaatgctgcgccagttggcctagtactcaagcAAATcaaagcggtgtcggcg cgttgttgtagccgcgccgacaccgcttttttaaatatcataaagagagtaagagaaactaatttttcataactctctatttataaagaaaaatcag caaaaacttgtttttgcgtggggtgtggtgcttttggtggtgagaaccaccaacctgttgagcctttttgtggagtgggttaaattatttacggataaa 64 ME151672980v.1Attorney Docket No.: 139465-00120 gtcaccagaggtggaaaaatgaaaaaatggatgttagccatctgcctgatgtttataaatgagatctgccaggccactgattgctttgatcttgca ggccgggattacaaaatagatcctgatttactgagagcaatatcatggcaagaatcccgttaccgggttaatgccatcggtattaatccggtaac gggatatggcagcggactgatgcaggtagattcccagcattttaacgaactggcccgctacggaattaagccagaacatctgacaacagatc cctgcatgaacatttataccggtgcttattatctggcaatagcatttaaaaaatggggcgtcacctgggaggccgttggtgcatacaatgccggat tcaggaaaaccgaacgccagaaccagagacgtcttgcctacgcatcagatgtttaccggatttatactgggataaaaagcagtaaaggtatc cggcttccggccacgaagaaatcacttccggaaattaacagcgtgcagaacaattaatcttacagacattgtcagataccggttatgccgcaa aagcggcatggaaggccacacacaaaaatatcatatgagtgacaatactgactggccgaagccccgcgaagcgggaacccatcttatata tacccggtaactgtgcagcctcctgccagaccatttttcgcgggcacaatatttacagacaaccggaaatccggttgatgaaattgaggtatatc agacgacacagcaatgccgaaaaatgataatataagtaaagccccgaaaattttttcggggctttacttatattatcatgacttccactttctgaac tgttcggcaatcacccataacgccttgttgattcttatatcaccatcaatacctgtaatggctcttgtcctggtatttttcccggaggcacttcgccccg ataatccaccttttatcatattctcctgaacccgctgccaggttgtccagagatcgttctgtttgtcttcccgacgacggggagtaattatctgttcagg tgtcactggcgttttattttcatcttcatacctgaccatcagtgcagctctgccaaataaatgttgctcgtcactgttaagatgaatttctttcatcgcctc catattatcagtgaccttatcaaagacaccgagcacttcatacgctccctcgataacctgcccgacaatatcacctttatgtggaacgcggatttc gccaaaattatttccgcacaccaggccatttgtgcagacaaaacgaaaaattccggggatcatctgataactggatgaaccatcatgtgaatta agcaggataatttcaggaacttcctgtccgttaatatgcccttccctgcgaagacgtaacatatgtttactgtattcgcgacgtcccaaatcacgaa tccgactctgacaggcaaagaatggctggaaaccttcatcacgtaacttattgatgatattgattgttggaatatacgtataacgttcactccggga ctcatgtttatcaccggaaaataccgaaggcacgaactgcattaattcatcatccgttaaaggacgttcacggcggatggaattataccgacca aaacgggaagctaatcgcataacagctactcctttatgaaagaataaatttaataaaaaatttttccgcttaatatcatccccccgatctccattaa caagattgcaacaatcaggagggatattcatcacatccggtcagcagatcgcccggatatcgaggccggcacgcttaatctgacgcaggaa ctgtttgaagtgatcggcttccgcaatgttgatatggcggaactgcttatccacaccctgactgagcgcgtagctataactgaaggccatcttttcc gggtggcttttgcgatattttttcagctcagtcaggctgctgaattccaggacgtcccactcttccccggtgttttttttcttcccggcaacaagaagcg tgtacgacgcggtgttaacgtggatatttcgggtactcatcctgctttctcctctctctggttttcagcccccatcatctgttgagtttcgggagccgcg gttttgacgttttgcggggacctgccttttgttccctccggtattcaccatcacctgtttgagaaccttcgccgcatggggcatcactgccacggccg gaaacgactgtccgggggcagaccgcaaaagtttttgcctgcttctgggcaaaaagttttgcgggatgtgcatttcacccctggactggcggag acggttgtggctgtctatgcccccggcgacaggggcttaaactggtgaatggcaccggacacaacaacgggcagggacccgccgcaacg acgtgaagcgcggtttgcgaaacggcgttgcgccttacatcccggcgccttttgccgggatgccgggagcgcaggtcacggcgcgacagag gcggcgcgaccggaacggtcgctgcaagccaggggttttcacgcatcggaccgtagcgacagcccgaaaccgcgccagcgggttcggcg gagactggcaggtgcgccagcagctgcctgtcggagtcgggcctgacgaccggatgccgccaggcagccgggagccgaaggtacgccc ggcagacaggacaacactgcagtagtcagaatcagagcaggaagaaaaagagaataatcacagggaaggtgatgtgatttctgacaggc cataaccagagaaggcgggggaaaagaacgggcgccgcaggcgcgcgttcatccccgttcctgaagggaaacgaccttcctgaagtcag gttgttcatgcccgtacaacatcagcaaggagaaaggggctaccggcgaaccagcagcccctttataaaggcgcttcagtagtcagaccag catcagtcctgaaaaggcgggcctgcgcccgcctccaggttgctacttaccggattcgtaagccatgaaagccgccacctccctgtgtccgtct ctgtaacgaatctcgcacagcgattttcgtgtcagataagtgaatatcaacagtgtgagacacacgatcaacacacaccagacaagggaact tcgtggtagtttcatggccttcttctccttgcgcaaagcgcggtaagaggctatcctgatgtggactagacatagggatgcctcgtggtggttaatg aaaattaacttactacggggctatcttctttctgccacacaacacggcaacaaaccaccttcacgtcatgaggcagaaagcctcaagcgccgg gcacatcatagcccatatacctgcacgctgaccacactcactttccctgaaaataatccgctcattcagaccgttcacgggaaatccgtgtgatt gttgccgcatcacgctgcctcccggagtttgttcggcaccatccaccgctccatctcacgaacagcatgaccgaccacccttgccgccatacca gcctctgcgatgacctgccagaatggcatatcctgccactgccagcgttccattccggccagtgacggaaaacgctcgccccactgccagaa cagggtttccaggtcatgctctgccacccggtcactccagcgtgcataccagcttccgactgtttccggcgaatggaatgccagttcgatttcagc 65 ME151672980v.1Attorney Docket No.: 139465-00120 ctgagcgaccagcgcctggtacgcacgggctttctgctctgcctccctcgcctgctggcggctgtacttctgcatgaccagcgcggatttatggc gactgcgttccctgacacgaaaccgcaccgacgggaacaagcgttctgctgcatcgcctggaaggggaaggaggcaaacctcgccctgtg tttcaatcagcacatcgatggcatacagccactgaagacggttgccgtgcagttcttctgcggtcagggacggcatgaaaaatcgcaggtcag acagcgatatcctgcggcttccgttcagacagcggaaaaacgcccccgcatacggatattcagccagcgtgttaccctgatggcggcgtttttc ggtctccgccaccgcccgccatgcggcctgttgctctgtgctgagaggaaccagtgcctgtgaacgtgcgctcatgcctgacttccttccgccat caggatatggatgatgtcactgacgctgtacccctgcgcgtccagtgacgccgccagtgccagtgaatggctgacagcttccggtgcaaacc gccgctgtacctgaaccaccgcaaacggctcaccaccggcattcaccagaaccagcacccagacctgagagacatcgcccggtgaacat aacgccaggtgaaaacgttcgtgggcaggcgtgaaacggacctggacggggaaaaagccgccaaactcgctgccgtactcgccgttcatc gtccagttatccggcgcatccagctcccgcatcacggcgtgagtcagctcacggcgttcatcacttccggcagcccggatatcttcatattcctgt gtgttcatggtctgtaatacattcagggtcagttcagttttcatcacggtatctcccttgctgccgccccgtctccggcggggcggtgtggttgttcatc aggcggcatcagccaggttatcggtgcgttcagtggcatccgtctgtgggcgtggtgcacacatccagcctggaacccagcggttgtctttcata tggtgctctgcatgttcagccgcatcacctttcttcatcttctccgcgtcccgtgcggcaccggacagccctgccttattcagggctgcgataatctg cggcggacgttcgcgcgtcgggcacctctcttacttaagaatcccgaaatccctgatgacatggttcatccagtacccggcaccgtcctcaaaa ttccaggtacgccagaccattgcaccgttattacggacaaccaggcgaaactgttcgccgtggtcatcctcgataaagacgttccggtactgtg cggcaacagcttcggcatgtttgcgggtaaaaggaccgtcaggcagcccctgaagtattctgaaatattcgctcatcctcatctctccggtgatg ccccgcacagaacggggcggggcggggcggttatcacagtcagcccgatcagaacgggatatcgtctgaaaacccgtaatcgtcaccctc cggcggttgcggctgaggctccggctgcgcggccttacgtccacggcctttcgtttttgcgccaccttttttcgtaccggcctccgcctgtggttccg gctgaggctgaccgctgaactgttgcccctcttccggctgagtctgagcacctgcggcacgtcccagcatctgcatggtgcccgtggtcttaaca agaatttcggtgacgtaacgggtaataccgttatcctcccagctacgggtgcgaagctgaccttcgatgtagacctgcgcgcccttgcgcagat attcgccagccacttccgcgagcttgccgaacagcaccacgcgatgccattctgtctgctcccgcatctcccctgtctgtttgtcacgccaggtttc cgacgtggcgacctgcaggtttgccactgcgcccccgttggggatgtaacggacttccggatcttttcccaggcgtcccacgagaatgaccttgt taatgccacgaactgccatgatgtgcctccttctgttgatgttttcagcccccatcatcttttgagtttcgggagccgcggttttgacgttttgcgggga cctgccttttgttccctccggtattcaccatcacctttttgagaaccttcgccgcaaggggcatcactgccacggccggaaacgactgtccgggg gcagaccgcaaaagtttttgcctgcttctgggcaaaaagttttgcgggatgtacatttcacccctggactggcggagacggttgtggctgtatcag cccccggcgacaggggcttaaacaggtgaatggcaccggacagaacaacgggcagggacccgccgcaacgacgtgaagcgcggtttg cgaaacggcgttgccccttgcatcaacgccagcccggctaagatggaggcacggcacagcccgcacacgactcgacgtccacataactg aacccccacaaggccatgctccgcatggccgggtaaagcggagccggcggcgacttcctttgtgctctgtccggggaccggcaggaaaaa ggcggggattttccccgccatcgtcatcatttccggatttttgccagggcatccgccatgctgcggatttcatcccgtgcctccaagaaaactccc gcagaatatagcggtcaaacaagcggcggggcggttcacccggtatacgcagcacatcacagaccaagccactggccaagatatgttcca cacactcactggcaggcattaatggaatcgtagtctcacgctgataatcagatcgacaatacaagtgagaccgtggtcccagactgattagca gaccaacaatacgagtgggaccgcggtcccagactgattagcagactaacaatatgagtgggaccgtggtcccagaccgataatcagacc aacaatatgagtgggaccgtggtcccagactgattagcagaccgacaatatgagtgggaccgtggtcccagactgattagcagaccgacaa tatgagtgggaccgtggtcccagactgattagcagaccaacaatacgagtgggaccgcggtcccagactgattagcagactaacaatatga gtgggcccgtggtcccagaccgataatcagaccgacaatatgagtgggaccgtggtcccagactgattatcagaccgacaatatgagtggg accgtggtcccagactgattatcagaccgacaatacaactgggaccgtggtcccagatataatattcaggccagttagttatgttttctagcctgt gctaaaggaaattaagtaaagacagataaatgtagactaaaacgtgccgcatcaaagtgccgctttttcaagttccttaagaatggcttcaattc tctctatacactcagttggaatacgagatctatctaggtttaacaccattttatcgcccttgtacaacactgtcgctccaggagcaaactgatgtcgt gagcttaagctagttcttgaggcagatgacgttttaagcacagacgttaaaagagtgatgacttcttcagcttcaaatatcactcctgcttttttctgct cgtgaaggttagatgcctgctgcttaagtaattcttctttatctgtaaaagctctttgaagtgcatcacctgaccgggcagatagttcaccgggatga 66 ME151672980v.1Attorney Docket No.: 139465-00120 gaaaaaagagcaacaactgatttaggcagtttagctgtgttgatacagcgggtaataatcttacgtgaaatattttctgcatcagccagcgcaga aatatttccagcaaattcattctgcaatcggcttgcataacgctgaccacgttcataagcacttgttggcctataatcgttacccaatctggacaatg cagccatctgctcatcatccagctcaccaaccagaactcgataatcactttctgtaagtgcagcagctttacgacggcggctcccatcggcaatt tctatgaccccagacactcttcgaccgaatgctggtgtctgctgacctgtcagtagaaaagaagggataagatcatccagggcgtcttcggtaa gcagttcctggtcacgttcattacctgaccatacccgagaggtcttctcaacactatctccccggagcacctcaagggtaaatttcacgtccctac cacatacaggcaaagtaatagcattgccacgagccatagctcctacgcgcgcaattaacgaatccaccatcggtgcagctggtgccgatga cggagtatcttcaagcggttgagtcttaatcgtatgttttggaatgacaggcgcacgcttcattatctaatctcccagcgtggtttaatcaaacgatc gaaaatttcattgcagacaggttcccaaatagaaagggcatttctccaggcacctgttgaagagcgttgatcaatagcctgttcaaaaacagttc tcatccggatctgacctttaccaacttcatccgtttcacgcacaacattttttaggaccatgcttccccaggcgtcccgaatttgctcctccatccacg gggactgagaaccattactattactgtatttggtaagcaaaatacgtacatcaggctcgaatcctttaagatctacgtttttgagcagatcacgaa gcatatcgaaaaactgcagagcggaagtgtagtcgaacaactcagcaggcgtggggacaatcaacacatcagcagcacatacaacatta atcgtgccgatacctaggttaggcgcgctgtcaatgacaatgacatcatagtcatgagcgacggtttcaatggccagtcggagcatcaggtgtg gatcggtgggcaatttaccttcatcaaatttgcccattagctcagtttcaatgcggtgcagagccaaacaggaaggaataatgtcaagcccagg ccagcaagtaggctttattgcataagtgacatcgtccttttccccaagatagaagggaaggagagtatcctctgcatgaatatgaagatctggca cccagccgtgatacatcgatgctgttccctgggggtcgttaccttccacgagcagaacacgtaatcccttcagagccagatcctgagcaagatg aacagaaacagaggttttgtaaacgccccctttatgagcagcaactccaatcaccggcggaaatacgtcttcagcacgtcgtagtctcgtacc aaacacgtcacgcatatgattaatttgttcaattgtataaccaacacgttgctcaacccgtcctcgtatttccatatccgggtgcggtagccggcct gctttctcggcatccctgatagcctgagaagatactccaactaaatccgctgcttcacctattctccagcgtcgggtgattttcctcgcttccgggct gtcatcattaaactgtgcaatggcgatagccttcgtcatttcatgaccagcgtttatgcactggttaagtgtttccatgagtctcattctgaacatccttt aatcattgctttgcatattttcatttaatcttgcattttattgcaaagcaacaacaaaattgcaaagtagtcaaaaaaccgcaaagttgtttaaaatga gagcaacacatcaaaaaaggaaacaggaagagcacatacctcagtcaataattatcactagtactcgtagccgtcgtgtaaccgagcgtag cgagctaactggcgaggacgcgcgctaactgttcacactcagtatccctagcaccaaaataactatctgatggtcaaggcggtaatgactcca acttattgatagtgttttatgttcagataatgcccgatgactttgtcatgcagctccaccgattttgaaaacgacagcgacttccgtcccagccgtgc caggtgctgcctcagattcaggttatgccgctcaattcgctgcgtatatcgcttgctgattacgtgcagctttcccttcaggcgggattcatacagcg gccagccatccgtcatccatatcaccacgtcaaagggtgacagcaggctcataagacgccccagcgtcgccatagtgcgttcaccgaatac gtgcgcaacaaccgtcttccggagactgtcatacgcgtaaaacagccagcgctggcgcgatttagccccgacatagccccactgttcgtccat ttccgcgcagacgatgacgtcactgcccggctgtatgcgcgaggttaccgactgcggcctgagttttttaagtgacgtaaaatcgtgttgaggcc aacgcccataatgcgggcagttacccggcatccaacgccattcatggccatatcaatgattttctggtgcgtaccgggttgagaagcggtgtaa gtgaactgcagttgccatgttttacggcagtgagagcagagatagcgctgatgtccggcggtgcttttgccgttacgcaccaccccgtcagtagc tgaacaggagggacagctgatagaaacagaagccactggagcacctcaaaaacaccatcatacactaaatcagtaagttggcagcatca cctttattcattataaaatgccccgtaatatttatctcacaatgattatgattagtggatacgcccctacatttccagacagctgttatcacttaatctatt actggcccgcagccgtagatattctagccacggtggtaacgagtatccggaagagattccacttacactctttcaatcctattgcaaaagctgatt tactcaacaggattaaaagagggagtttgatacaatttatttcactttttgttagaatactaattcttttcttgtatttttttgaggaaatgagcacggaac acgacataatctgtagtcagtaaagagtctatcctccctgactacagattatgtcgtgtattatatgatatcaacttggctgtcagcggccatgttgat atcagcgtacctttgttgtaaaggaataccggtcagaacagcacagagtagttcagaccaaaggttcggcccctgcctttgtagtcgtacagtg acgctgggccgtaacccgggctgtagtacagtggtgcacgctgcccccagacagtggtgtagtcacggtcgaagaggttttcaatgctgaagc tgagtgtacccaccggaagctgataactgccgagcagatccacggtggtatagccatcgaccttgtagccctgcgcgtcgctcacgtcaaag gaggtggtgctctgcacgcgcagactccacgggtccggtgcccagccaatgtaggctgtcgcttttgatgggcttgctgtcttcacatcgtatttct gccaggtaccgttcactttcgactcagttttcagcacgttgaagttcactccggtactccagtcagtatcaggaatcaggtagtccaccgcaccttc 67 ME151672980v.1Attorney Docket No.: 139465-00120 cacgccgtaaatacggcgtttgtcgtccaccacgctgatggtcagatctttattcgccaccacgctcttatcagaaatcgaatagtaggccgcga tttgggtacgcagattattgccagtaaagcgccagcccagctcataagaatcgactttcacgccttccagcttgctgtcgctgacgttcacactcttt gttagaggaagatggccgttcactgcagcaccatagatgccgcgaccatagtatttacccgggtccggcagctccacgccctgggagaagtt gagccatgcctgctggcgttcggtgatgtgcatcagcagaccggcgttgaacaggaagttgtcgtaatcgactgagccgccaggaatggcgt cggcggatgtagccttcccggcggcaatctgccgttgctgcgcgtagccgatgaaatcatcaatcttgttttcagtgtactgatagcgtacgccac cgttgagggtaaagagattattgatgtcatagcctgattgcaggaagaccgccaggttggtgatgtcatacgacggatagcgcccggtggtgta aatcttcttgttgttcagccctccggaagcgcttgcctgagccaggtcgaagaacatctggttggaggtaaagcgctcatgatcagcatccagcc cccaggtgatttgccagccgtccatcggtttgctgttcagagtcagtttcatgccgtactggtcggtgtcctgctgtgacgaagagaaagccgtca cctgtttgttcgcatttaccgtcgggaacgggtagaatcgcaacgactcatcgcggtagtaaacctgaccgaccagctcctgtcccagaaaag cgctgtcagagtactgcaggctgatcaaatgccgctcagtgccgggaatacggtcggaattcagcccgttactgacgaatggcgtgctggtcc ctctgatggcagagaagcctttcccgagattaagcccgtaatcgtcgtcgccctggcttttatagtactgtgtgatcaactgaagctgccgggattc atcgatgttcagcgtaccagttcccatgatgtccagccgatcggagtactgcaggccggtctgggtgttatcaagcaaggtggcatcgccgttac cgtcaaaccagccgccaaatttctgatatgccacggaaagacgtccggagatatgctcatttccgccggagacagctccggcaatgcgttcat cgtgatctttactgctgctaaagccacttttggtgccagcctcaaactccatcatggtttccggctggccttttttggtcacgatgttgatcaggccacc ggtactgccgccgccgtacagggacgtcgcaccggagatcacttcaatatggtcgatattaaaaggatctatagagtccagttgtcggctgtcg gtacgtgaagagttgagacgcacgccgtcaaccagcacgaccagcgggcggccacgcacattcataccgtagttggtgcggctccggctgc tgacgtcaaggccagggatcagctgagccagtgcgtctttaagctctttgccgccctgaatctgctgttccagttcggcgttttcgataacccaggt ggtttgcgccatctccgctacggtgcgattgctgcggttggcagacaccacgaacgtttcatcatcggtttgttgagcgactgctggcgccatcat ggtaagaagcagtgggttgagagcccaaagcgtatactttttgcttatcatcattattcctccatacgggcggaaaaaatcagcccaaagatgct gtgtagcgagttaaagaagcagcctccggctgcgttttccctaggtgccgctgcgccactgaatcagggcgggcggcatactgagatcgaata aatcacgtcccattacgcgattaagaatacgtgcagatctccatgccatcaggctgagctggggttcggcgatgccatgggtttgcatactggcg ttgaccacgaagatgttgttctctttcgggccactccattccagagtgaagtcatcacgcactttaaaggtgttcttatcgtgcatggtgatcagggg catcagtgagggaagtatttgtggcaacgcagaacggtaacctgtggcgaaaatcaccacatcactttccaggctctccctgccctgatccag atgatgctccatcagcaacttccagccaggaccactactttccagagttgttaccgagcggctgggtagcagacggatatttcttggttttctcaga acttcaaaacggtggtacaactcacgataaatggtcagtaaagaatcggcagtgatgccatccgatgtcattttctgctcatccagtaactgatg gcgaatatcttcctccagtccggagaagcctgaaatatattcaggtgtaaaatactcatcagcaaaagcagcctcatccagtgcgttaaaattat tacgccgggagacccagtttatttccgccgcttctccccattccccgcgtaatgcattaaggaacaggtctgcaccactctgtcctccaccaacc acggttatccgttttccactaaggtccggccgacgaagattcatttcactggcatggaaacaggattgtgtcatatgcttcacacagggtggtaaa taaggttgttttcctgtaccaaggcagatattgcgggcaaaatattctccctggctggtttgcaccagaaacaatcgacgttttttatcgaaatcaat gttttcaacggtatgactgaaatacaggttattcatatcttcagcagcccagcggaggtagtcagaaaactcttcacgggatactgtacgtagtct gcttgtaaggaagcgatagaactttttgtgcttcaccagatagttaacaaaactgtagggatttgtaggtgcaacagcactgaccagatctttcag aaagacggtctgcatatgacaatccggtaccagcatacccggatgccaggaaaaatgaggatgttcatcaaagaagagacagtccagttctt cgatctgatgtgacaacgcagcaatgctgagattaaatggccctgtccctacaccaataaaatcgacactttttttcatgattcatattcctgagtta ccagccacagcggattttgcagatcctcaaggtaattcggcagcatgcggctgccgccatccagatcaggccaggtcagttttaccgggttca gcaccacgcgaatgatttgtggcctgaaaagtgagaaaagcgcaaaacgctctgacatttgtggatgttttttcatgtaatcactcaacactgctg ccagcagttgataaaatcgcctttcaggtacgccaagacgaaccatcagtggcgaaataaaacgcagtactgtcacgaagtgacccgtctgc aaatcatggattaagtagtccgcactcaggcgggatgtaacatcacgaacctcctgaggcaaagagtccatttcggggaactcttctttcacca gtcgcatatcgccctggaagtctttcagcagaacacgctgtggaaccccctctttcatggcgagagttatattttgtccatgtgcaataagcgcga caccgtagcggcaaagcaggtgatacagaggaaccacgaccacccggaacagttgcgtaagccaggtttcagcgtccagcccggagcg 68 ME151672980v.1Attorney Docket No.: 139465-00120 gtctatatatgcgcctgccagcggctgattgttttcgtcgcactccatcagtgttgccatcagaaccggactttcatccggtttcagccagcggcac ggattctcccgccagataacaccaagcatttcctggtagcgatagggagcccgggcaagcgcggcatagccttcatgggagacatagcctg cagccggttcaccaagtatcactgcgccgctttgcactagggtggcgtcggtcgcaaaaacctgttgtagccagcgtgaagccagtggtccgg cagcgatgtatctgccagggatcccccggtagcatgaggtgttgtagatggtcagcggcagcttgatatccagcccgccccggcgactggcgt tggtcagggtacgcagcgactgctgggcgagccactggtcgccaaactcgccgagagacaccatcctgccttcggcaaaatcagcgatga agtcggtagcgattttttgctgccactgccacggatgtaccggcagcggcagccagttatgatccagtccgttttcctgccagacctgactgaag cgggcaaactcctgcggatccattgcggccgtcaacaactgatgaatatccatctcgttatcacagcgccagatcatatgttcacgttttaccgcc agccagtgcagtctgaaggtgttggcatactctggcgcatatcgttccagcgcctctttaccccagccacggcgacctttattaaaaacgaattta ggatgaccgctcagcaggcattgcaggcggtcggcattaagattaatcaggtcactggcgctcagcccgcgacgggctttcagtagttgcagg tcgcccagcagcgtggcatacaaatcctgcatatgctcagcaacggttgcatcgctcattgacagtacctgctttagctgcatcagcagcgtctg agccagtactggctcgtccgcgcagcgcagagtttgagcatcaatccagagccagccccagataccacgttcagcgatgaagcgccattgtg ctcccggcaggttaatgcagtagcggtcatcgccctgagattccgcgtggaaaacctgctcatactccagctcagacaacatttttgccaccag gcggcggttgaccaaatcccagtctttgtgattcattacagaccgacctccgtgaagaagtgatgacgatccgccatcaccatgcgcgagcgtt tatgtgggaagtcgaactctttaattgtccggtatcccgcaggctcaagatggcggaacaggcgctggttatcggtgcgtggctccagtactgtg cgctgcgtacgtggctcattcagcagcaggtaatgtgtcaccccgcgcagccagctttgcacatagtgggctccgcgccattgctgttcaccaa ccagcagatgcaggccacggtcaaagggctgccacgaatagtggcgaccaatgcggtcttccgccgcccagtagatttcgaaataactgaa cggccgatcgtcgaaacaaccaatcagcgggaatgcatgtttactggtgagctggcgttccagataggcgatctgtacctccagtgagccactt tgctcccagaaatactcaacgcgcggatcgttcatccagcgagtgaaacgttctgcatcagaaacgggatcggcaatgcggaaactcagcat cctgcgaatgcgtggatcgtaacgacgataaacttcaccctgcggacggggggggcgctgcgggaaataaatctcacgctctgcatcaaaa accattttaccggaggcccggtttgccggagaactcagccataacggtaactgccagaaagcggaacgatggataatgtcgctttgtacctgtc cgaaaagcgtcagcgcctgtggctcctcacaccattcggaccagggaagaaccactgctgaaagttgtggtgcggcgataaatatctgatcc agcgcgtcgcacagccaccctgttggcagctccccgggccagtgcaacaccgcgctattgtccagcccccagccaagattcagaggcttgtc gagtttttcacagcgcagtccatatccgctgtgaacaatgtttgccccagacattcagacctcctgagcctgcagcgggttagcgaaatcaaaat agatcaccgaagggtcaacgatggtgttctcgttatgatcgttcagataacagaaaaagttaccttttacgttccagtacggactttccagaacat agttgagacaggttttatgagtcacctggtcacgcacttcagccagcaatgttcgtacacgagccatcagattcgcttcgctgtccagcccggca gcacccagcgcagcagttacggcaaaagtggagttaaccagcaggtaataagggaaatagcgcagcaactgctcacgggtgaaaatatttt ccgcctgcgcctcgtcaatggtatcgagccactctgtcgcgtgaggcataaacgcgctgccctggcagtcacggtaaataaatccgaccggc agatccccaagcatctgcacaagaatattttgctgatgcgccagcagcaccaggccgtaatccgcttcagccgtaaacagaggcttcagcac ctgctgacagtaagcatcgacccatgcgtgggcggcctgttgcacagtgataccgaggcgatcgctcaggcgttttaccgccgaaaccagca gcgaatcaccgccatccggtgcggcctgagtcagggaaaccagaacgttggtctggctttgcggctgctccagcagcaggttttcacgcagg gaaaacaaactttcctgcatgatgttgccgttaagatcgcgcagcccggcccagccgtcctcctgcattacccggaaagtagggaagcgggc ctgtagcatctgccagccgtcggtttgcgccagacgtgccaggcgcattcctcgctccacctctttcacagacagagtacggacggagttggtc agccgaacgctcagggagaacttgatcatatcgcggctggtagcacagtagagggagcgggaagaggtggtcggcagccacgacgtgcc ggcctcgcctaagtctctgataagtcctttagcaaaaagtgcctggcaccacacttgctgcaaaagatattctccctgccacgggtgcagcggg aacagccattgattgtcacttaattcgttgagtaactggggcgcattttctgcggcaaatcgcgtcaaccgctgttgaaggttaagatgcagacttt caccagcgatttgcgttttatccaccgaaaaccaccgcagcgggaagtgaggtgccatgtcaggcaggtatcgttcagcttcctggcggttaaa cggttcatgagacttaggtgcagggtggaaagcgtgtcctgtcagcagtgcctgctcagcctcgccaaaattcaacgctttttcacgcaggatgg cccagtcatggcgggcatcaattgtctgttgcgtatgggcgtagctttccagcacgcgttggtggaaagtttcgcagaggggaactggcagaaa aagttgatgccggagcttatcaataataagccgggagagagtggtaaagtcgacaggataactgcctgatgccgtgaccagatgagcagga 69 ME151672980v.1Attorney Docket No.: 139465-00120 aatgcaaagcggtgatgctgggttggggagaaataagccactctgaaatggagcgacttctgttcgtccagcgggataatcaattcgtctggc gggtattccgccagttgccagtctttggtttcacgaatcaaggcattcaggaagcactgcgcagccacatctgtggcggatttttcagagggcag gatcataatcaaacagctcctacacagtaaaataataacaattatgtcaataatgattctcattatcaatgagaaatgttattgtcagaaaaattaa ttttacaaataaacacaattaatacatggtgttgaaacattacaatgtcatctaagatcgaagatacgccccaaaaaaccctgtcctgctggcca ctggcgttcagtgccggtcttctcggtatcggacagaacggtctgctggttgtactccctgttctggtcatacagacaaatctgagtctgtctgtatgg gctgccctgctgatgctgggctcaatgctgtttctgccatcttccccatggtggggaaagcaaatttcccttactggcagtaagactgtggtgctgtg ggctctgggaggatatggcgtaagctttaccctgcttgggctgggaagtgtgctgatggctaccggtgccgtaacaacagcggtggggttggg aatattaatcatcgcccggatcgtctacggtctgaccgtgtcagcaatggtgccagcctgtcaggtctgggcattgcagagagcgggagaagg gaatcgcatggccgctctggcaaccatcagctccggcctgagctgcggcaggctattcgggccgctgtgcgcggcggcaatgttggtcattca ccctctggcgccagtgtggatgctgatggcagctccggtgtgggctgtggtgatgcttctgcggttgcccggcacaccaccacagcccacacc ggagcgcaagagcgtcagcctgaagcgggattgcctgccttatctgctttgcgcaatgttactggctgcggcaatgagcatgatgcagcttgga ctttcgccagcccttactcgccagttcgatactgataccaccactattagccaacaggtggcgtggttgttggggctgtccgcaatagctgcgctta tcgcgcagttcgtggtactccgtccacagcgcctgactccagtggctctgctcctgagtgccggggtgttgatgagtagtggtctggctatcatgct cactgaacagctatggttgttttacctgggctgtgcagtgctgtcatttggggccgctctggcaacacccgcttatcaacttttactgaatgataagct ggccgacggcgcaggcgcgggctggcttgcggccagtcacacacttggctatgggctgtgcgcattgttggtgccactggcgtcgaaaacag gtgtctcaatcgcgctgattgtgacggcattatttgccgctgtattatttaccatagtgtctgcatgtatttggcattaccgtacaatcaagtaaggctat gatgttgtactcacagccggcataacgtcctgtgcaatttcgctgtcgaaaccgcctgtgctgaccgcccagtgcactcacctgaagcacatcg cagcatgccaccaggatgccagatcgtcttctgaaatgggtggatacattggcaacgttaaccgcaacaatttttcagagagaattttggggggt gagggccagtgatgtaaaaaatacgccaagaggttaattcattgtttatactgatatgtcaggtaatgcgtttgcagtcattacagtcgtcatggag actgtaatgactgcaaacgcataccatggagcgaaattctatcctaagcgtcacattagttgataaacctgtaacgttaatgaattctcatgcgaa aagtatggttatgataaaaaagcctaaagctgtcatgatgagggagccaatgacgtgaacaagtacagacgtcaaagcccatatataatttcc cgcctggagtaatgcgaatacttcaacagaaaatgtagagaaagtactcagacctccacaaagtcccgttgttatcattaatttccaaaaagga tcaaggtgtggttgtcgtaaaaagtacgccaacgcagtaccaataattaacccggcaaggaggtttaccaccagtgtacctggaggtagatttg gaaaaagactgttaaagcgtgtggacagtaaccagcgcagagtacatccaacagaaccgccgataatgacggcaaaaagtgatttaatca tgtacccctcataaaataattttttatcgtaacaatatcagaatatgttctcatttgactgatattgttaatttatatcctttgactacatgcaagataagg gaaggttctatttattgacattaataccctcgacaattgctgagcagaagcccccacgacaagtgacagaatgacaacatctacagtggggag cgttttatcgatgaacaagcaaccaagaagtaacacggcataggtgacgacaatgccagtaagcatagaaataacaaaaacgtgagctttc atcattcctccagaataaccccgacgtgtaagcctgggggattggcacaataatgtcatttaaaggcagactttccagcaaattgcgccgatga acccagttcatcctcaattctcataagctggttatatttttcaatacgctcaccccggcatggagctcccgttttgagatgacctgcgcgcattgcaa ctgtcatatcggcgataaagctgtccactgtctcaccactgcggtgtgagataaaggtgccccagttgttgtcatggcaaagttgtaccgcatcaa aggtttcactcagggaaccaatctggttgagtttaatcaatgctgaattggcaaggttttcgtcaatgccgcgctgaatatactttacgttggtcaca aacagatcgtcgccaaccaattctatctgctctcccaatgcaccatgcaggattttccatccagcccagtcatcttcagctaccgttaagggcag gaagatcaactaggtgctctggggctggtgaccaatgcggttgtgctgtgaaacacactttacatgcaggaggcgttgtcacatcttcgcaaatc gggagaaactcctgaggagtagcatccggcaagattatcgctgctgatacatagccatataaatatgctggggcattatacgtcttcgttaccgg aagatattcttaagggtgagttaagatcattaaacttcaatacaaacaattaattaaactcttagcgtggttttttacatcactggccctcgcccccc aattttctctatgttttgccactacttccctcattttactgcgctgtttgctacccctgagaatcgacattacttgctatccagtccgcagcaactctctgg caacaaaacttatcagaatcataagtctgtgcattattcgttcaagcgcatcgccaataagcggatgttgctggccgagtgttctgctaatcggat gataacaaatcccgtggttcggcattttcctttgtgcgtgcgttgatgctggtaacggtaaacgtcatgatgaacatgaccgcgaataagacaac aattgtcggagctggcgcgctgtcgataaaaaaggacagccagacaccagtcattgagaccaatatcgatatgccagtcgccagcaggag 70 ME151672980v.1Attorney Docket No.: 139465-00120 cgcaatatgaaagcgttgtgtcaggagtaccgcaatggcaccaggcgcaatgagaagagaaatagagagaatgatacctaccgctttcagc gtcgccacaatggtcagggagaccatgcacagcagcccataatgcagccatctcgtatgcaaaccgcttgcctgcgcctgttgataatcaaa gctgaacagcaaaaaatcccgccattttacgctaataacaagtgtaaccagtccggcaataatcaccgtctggattatatcgccgatggttatcc cgagcatgtcgccgaaaagaatatgatcaagatgtacgtctggtttgactgcaatatacaagatcagacctgcggcaaacatgccagaaaaa acaatccccatcactgtatcttgcttgattcggctgttgtctttcaggtatcccgtcgccaccgcacagaatactccggcgacgaatgcccctgtag ccagaggtaaccccagaatccaggcaagcacgatgccaggaaaaaccgcatgactcatcgcgtcacccatcagtgcccaacttttcagga ccagaaagaccgacagcagcgcacagggtatggaaactaccagtgaaatcagcagtgcgtgactcataaaggtgaactgcagtggctcc agtaataatgccatcatgagcgatcctccagcgctttatgcgcacgccgacgatttgccaaaagcccatgggtgggcgcaaagacaaatgct aaaagaaacagcagtgtctgcgccaccacgatgattccacctgtcgcgccgtccagaaagtaactgacccacgcgccgaggaaactggtc acactgccaatagttacagcaatcatgagcaggcggggaaaacgatcggttaacaaccatgctgttgcaccgggagtcaccaccagacag atcaccagaaaggcaccaaccgtttgcagcgccgcaacggtcgaaaccgacagcagggtgaagaagaggatttttaatctgcccggatgc aatccaatagcacgggcgtgattctcgtcgaaaaaggtcaccataagatctttccatttgaaaaaaagcacgatgatcgagagaatgccaatg atagtgagttgcaggatatccgccggatcgattgccagaatattgcccaggacgatggtctgaatgttcacggatgtcgggttcaatgacaccat aaacagccccaggccaaaaaaggacgagaagatcaggccgatgatggcatcttcttttaagcgagtgagttggttaagaaacagcatgctg cccgctgccagtccgccggaaaagaaggctcccagagaaaacggaagccccagcatatatgcccccgcaacacccgggacaatagagt gcgacagcgcatcgccaatcagcgaccagccttttagcatcagatagcatgacaggaaagcgcagagaccgccgaccatcgccgagacc cacatcgcattgagcatatattcatagctgaagggttccagtagcacgttcatctttcttccctctgcaccgctgacggtcgatgtgccacaaaag ggcgttcatcgtcagtaataatgctttcttccgagccgttaagtgtgacatggcgcagtacgccgctaaaagccagctccaggttctcggctgtaa aagtggtgtctgtcggcccgcttgccaggacggtgcctttgaccatgaccgtataatcgcaaaatgtcgtgacggatccgagattgtgcgttgag acaagcatcgttttgccttccgcgcgtaactccctcaacaggctgatgattttcgcttcggttttcacatcaacgccggtaaaaggttcatccagca ggatcacatcaccctgctgtgcaatcgctctcgccagaaagacgcgtttcttttgtccaccggaaagctcgccgatttgtcgatggcgaaaatcc accatatcgacacgttccagcgcatcggtgacaatctgacgatccctctttttggcgatacgcaacatacccatatgcccatagcggcccatcat caccacatcttccaccagtacaggaaatgaccagtcaacctcttctgactgagggacgtaggcaaccaggtttttctgtagcgcctgtcgtgtgg gaatacccagaacagatatcttcccgctggtcagacgcacaaatcccataatcgctttaaacaacgtcgattttccggaaccgtttaccccaac cagagcggcaatagatccgcctggtactgtgaaggatgcatcgcgcagtgctgtgtgcccgttacgccaggtgacagtaacatcattaacgac aatgcctgcagattgcatcattatttctccctttttccggctttaattccctgtaccagcgtactggtagtaaccttcagaaggtcgatatacgtaggta ccgggccgttttctgtgctcagggaatcgacatagagcacaccaccgtagtgcgcgccggtttcacgcgcaacctgacgcgctggtttatcgga aatcgtactctcgctaaagactgccgggatatgatttttcttaactatatcaacaaccttacgtacctgctgcggtgttccttgttgatcggcattaatc ggccacagataaagctcttttagccccaaatcgcgtgcgagataagaaaaagccccttcactggtgaccatccatcgctgattctcagggagtt ccgcaatctgcttacgcaggggggcaagggtttgggtaatcttggctttataagtatcggcattacgttggtaggtttgtgcatttgccggatcgtatt ttataaacgcatcacgaatattatcgacgtaaatcagagcattatctggcgacatccaggcatgggggttaggtttgccctcatagggcccttcg gtgatccctactggcgtcacacccgaagagacaattacttctggaaccccattgagatgctggtaaaagcgttggaaccacaattccagattca taccattggcgagaatcagttgtgccccctgcgcacgtttaatatcgccaggggtaggctgatactcatgaatttctgcaccaggcttggttatgg atgagacttctgcagcatctccagccacgtttttggccatatctgcgatgatggtaaaagttgtaatgaccttgaatttttcagctgcactcgcctga aatgctatcgagcaggtgaggactaggcaccccaagagcatggttactttttttattgagagcatgatttagtacctatagtgagcaaaattagaa taaacatagcaactactatgttttatagcagaagctataaaatgtaaataattatcattagcgattataagatcccaacacagggattaggttatttc ctgctgacgatgtggtattgaggataaaagttgggcagcgctttggctttaaaatgaacttgttcaggacgattggtaatggcaaagcaaaaata atgtaggttattggcaagttctctaggacgcaggagagcatgaaaaacccaggggcagtcactaaatgaagggatcggtgatgctgccaact tactgatttagtgtatgatggtgtttttgaggtgctccagtggcttctgtttctatcagctgtccctcctgttcagctactgacggggtggtgcgtaacgg 71 ME151672980v.1Attorney Docket No.: 139465-00120 caaaagcaccgccggacatcagcgctatctctgctctcactgccgtaaaacatggcaactgcagttcacttacaccgcttctcaacccggtacg caccagaaaatcattgatatggccatgaatggcgttggatgccgggtaactgcccgcattatgggcgttggcctcaacacgattttacgtcactt aaaaaactcaggccgcagtcggtaacctcgcgcatacagccgggcagtgacgtcatcgtctgcgcggaaatggacgaacagtggggctat gtcggggctaaatcgcgccagcgctggctgttttacgcgtatgacagtctccggaagacggttgttgcgcacgtattcggtgaacgcactatggc gacgctggggcgtcttatgagcctgctgtcaccctttgacgtggtgatatggatgacggatggctggccgctgtatgaatcccgcctgaaggga aagctgcacgtaatcagcaagcgatatacgcagcgaattgagcggcataacctgaatctgaggcagcacctggcacggctgggacggaa gtcgctgtcgttctcaaaatcggtggagctgcatgacaaagtcatcgggcattatctgaacataaaacactatcaataagttggagtcattacctg ctttttcggttgaaaagaaattgattgaattactggaaaaacaatctggtctccatacgtctgtggattgcagtactccccttacagagcagagtctg tcgagaaatgacggtgaagaccagaccagaagaaacgctgaaaatatgttcggagatgactgaactaacccagcctggccaggggaata tctcgccatttagttgtataacactgactcaacatttcccgtttcatttgccattcaggggcggttcctcgccccgcaaaccacactttcccttttcccg actggttcagttcatcgagtgttttcattaacttttcactgtttttacggggctggatttcatcaaataatcccggctgcgctatacccgatggtgtgaaa tcagccagcatgactcctgccttcatatagcggtacccttcacgccagacatggtttaaggctctgcatgcggcggcaataatgtcccggctgtc ctgtgtgggcaatgaaagcttttccacagcggcattgctgtaacagggttcagggggatgtccgtacaaatgtcgtcacctgccggcaatactg acgctccccacgtagtttctctgcggcccgctctgcatactgaacaacagcctggtgcatggcatctttgtctgtgattcgttcaccaaaactgcga ctatgtcaacgccacgatgtttgaccgttatttgccattttcatgcccgctaaatactttcgttactgatgctgtccgctttgcaccatttcatcatcactg attggcgttgtgtttttggtcagcactccagctttacgtttctcttttaaccggtaactctctccttgttctttttgacggacgcattttacgtttgggctggat gtagtaacgcagcatggaacgtccgcccgtataacccattgccttaatctccgcgaagataacctcgctattccagacattctctgccaggcgc atgtcgatgtaatccataaacggtttcagcttaaccattttgtggcgggtctttctggctggcggttcagggtatttgaggtagcgtctgaccgtccgtt cagagcaaccaatctgagccgcaatatcgacaatgtacgcgccctgctggcgcatttgctttatcatgtaaaagtcctctctgctcagcatgttga tgtcctttctggtgtgagaaccttaaggaaacaacatgttgggtggagcggacaatccaaatggtgaattaccgtcttatatcactggcgctgaca cctgtagtttgtatacacaatccctggcattgaaacacacatatgaagtgtacttcggatatgaccgtacgctcaggaagtacggcagattccgt caaaaattacctgtaaagacagacatcagaaaaaacagtgctcgccgcagaccggtgaccgggcgaagcccgcgaaccggcgaggaa gcgcaatgtatctgtagttacagggagcaggtacgccctgaagccctttcataaaacggttgtgtgcgctgtccttctggaatgatttatccacata tccggaggcccgatccagtaaacagatccatgaatgatcaacaaaggatccattaaagatccccatatcgctgcaaaccttgtcactcatggg ccgggaccacgatcacataagctgtggcatgttactgataaactgtaacatgctaatgataagctgtattcagtaatccatatactgaagtaagtt aatgacataaactatggtcagtatgccagactcagttgttaaatacaggctgcaggtttttcttcagtcagttagcggagttctgacacacgattttct gtttattcttttactgtccacaggctggaggctttctggaaaacgaaaattcagacatcaaaaaactgttcggcgaggtggataagtcctccggtg agctggtgacactgacaccaaacaataacaacaccgtacaacctgtggcgctgatgcgtctgggcgtttttgtaccgacccttaaatcactgaa gaacagtaaaaaaaatacactgtcacgtactgatgccacggaagagctgacacgtctttccctggcccgtgctgagggattcgataaggttga gatcaccggcccccgcctggatatggataatgatttcaagacctgggtggggatcattcattcctttgcccgccataacgtgattggtgacaaagt tgaactgccttttgttgagtttgcaaaactgtgtggtataccttcaagccagtcatcccgcaggctgcgtgagcgcatcagcccttccctgaagcg cattgccggtaccgtgatttcgttttcccgtaccgatgagaagcacacccgggaatatatcacccatctggtacagtcagcttactacgatactga gcgggatattgtgcagttacaggccgatccccgcctgtttgaactgtaccagtttgacagaaaagtccttctccagcttaaggcgattaatgccct gaagcgacgggagtccgcccaggcactctacacctttatagagagcctgccccgggatccggcaccgatatcgctggcgcggctgcgtgca cgcctcaatctgaagtctcctgtattttcccagaaccagacggtcagacgggcaatggagcagttgcgcgagattggatatcttgattacacgg agatccagcgggggcggacaaaattcttctgtattcactaccggcgtccccggttaaaagcgccgaatgacgagagtaaggaaaatccgttg caaccttcacctgcggaaaaagtcagtccggagatggcggagaagcttgccctgcttgagaaactgggcatcacgctggatgacctggaaa aactcttcaaatcccgctgaacataaactgtagtcagtgaagagcgctcctttactgactacagcttatattaaccggtgcagtgagtggtctgctc actgcagtttatattctgtttcctgcagtgctgtctgtagctgagctgccatctgcctgtcccttacgtaagtgatcccgtaacctgatcctgaggcatt 72 ME151672980v.1Attorney Docket No.: 139465-00120 gctcccttcataaaacatgacttactcactacagcttatatacatgctccagcttatgttatgtctgttctgctgaccacagcttatataaggaagcgc gtatgccgtcacctcaggggaggcagtgtacgcaggatctccgcagcatcccgcccgtcacctgtgaaaggcactgccagcgtggcagcca tatccagtgcaaacactctggtataaacctccatcgaacgtggatccctgtgaccaaccagtgcctggatgactttccggggctggcggtgata gagcatgtgcatgatatagctgtgccggaaggtgtgtggtgtgaccggaatcgaaaagtgtactccgtcagcttcggcccgtctgacagcctgc ttcagccagttgcgcatggtttcgtcggtcacggcccataatggttcacgacgacggggccgggtggtgatcatccagctttccatctgcctgac atagcttatatctgtcagcggaaccaggcgcacttcatcttttggcgggcgtccgcgtcgcgcacgcactttttcggacaggatccgcacaaacg gtcttactccattcaggtcaaatgattccggcgtcagcatccgggcttcgccaatacgcattccggtattccagagggtggcgaacagcatatgg tgacgctgatccggcatatagaagagaagggcactcacttccggggccagcaggtaggccggtgtggcccccgtggaagtggccattcttct cagggagagcgctgttgcaaaatccacccccggcgcaatgggtaacagggagacgcgttcaggtgaattctgcaggggaatgacattgttc atgagttatttttcacgtagcacacgcactgtttatggatacaggtattctgacctgaacgcccggatccttccacgacaataaatcgcagcgcgc ggatccttttgtttgattatcacgcattattgcgggtaaaaccaaatacataaaggcgcattttgccgctgggatcccgtcttaagccagtcatggtg cgggctgagggctggtttaccgtttttgggagaattggggtaatgccaggacaggcagaaacggaaaaaaaatcctgcagacgcccttaaa agacaactgaaaatttctgactccatattcctctggtttcagactgcttaatcacatcatcaacctgatacataataaacagattctgttcacagaat gaacaattaagtaaatggttagattatgcgaaaaagtacagatatttctcaccatcgttattgaataatatgtcttcacacaaacaatattaatatttt gagtgtaagccctgactcaaagaaagaaatgtctgttttcggaatcaaaaatatatagttatcaaaatacattaatttcaaatttgaaattaatgta aaacgatttagtaaatgttatgacattataaatctactcctccatgtttagaggagtatttgcagactggacgctcttatatagttacttacctttataca tattctaacataagcatttaccaaccttacagggaacagaccatagtagtttatatttccattcaggtctgaatgcaccgatagaatatatcatcttttt attattatcctgacaataccttcgtgctttatctatattcagccacataaggacacttcccggacttagtttcatacaattctcatcattcagaactgca ccatttggaacgtcataataaacattacatgagctttcagatttcagtactatatcaatagcacacggtttattatcccagaatagcacatggccaa acataagttctcgtaagtgagaaataaacattaacagattatcatattcgtagcaaggtagggtacctccaaaccgggaatgaaaaagtgatat ataagagctggatatttctttatcggataactggcttatacacttaatctctccgccattcctgataaatctctggatttcatttcggcgagttttttcaaat ttaggtgaaaaagattcctttataatacaattttgtttttttctggctattttccatgttgcattgataatattttgtttatttataattgataatttattagttttatcc ggaaaaaaacatcttgcatgtggggaaaaaggaattaatacctcatcagatgaaagaggataagaccttcttgccattatcccaatttgttttcc attgcacagaaaatatgagcctttgatatcaccatctttttcatagtgaaaaaaatcaaacttccagtcatgatgtatcataaagtatgaaacaac atccggatgcatgtttacactaccaccgtatttttcaaaagtttttttgtacgtttcgaatgtagatggtttccaggccgtaataatgcgctttatattttta attgacaaagtcatatcctcggcgtttttatatgttatgagtgcaacaccaacaataattctgattatgataaatattatttaaaatcaacttccatttgtt gttgaattgaaaggtgttttgtcgtttctatacagtatttaatataatcagtaaactttggcggtttaggcattccgatactcagtagcttgtcgttactga aacaaacattgagcatactgaacgctccatataaacgaatggcttttaacataaggcgttcgttacagggaccaaaaatatttttaaaatcatga cggctcattgccagtatgtcataactgactttagtatatctgtctccaacaggatcacagttcaaagcacgggctacagcttcgtcaatagcagag aacgtcacactactttctttacctgctgatatatgaacaatctcaccattaattaacgaactttcaagcaacattagcaatgcatcagcacaataat ctacagggataacatctattttatcatccagagagcacataaatttttgcaacattaaccccattctgaatacccagaaaatactggttgaaggta agcaccctaaacgactgtggccaacaataattgatggtcgggcaaccaacaaaggtaaatcaggacactgcttacgcatcagatattctattg ttgcttttgaatgcgtatactccactaaatgttcacctgtttctgatgaagcagactcttccttaactagcgaccccgtatgaggtgtacaagacata gcagtaccaacatgaaggaagcgtttcagtcctgccacttttgccattcttcttgcaaaagcaagtgtacctgtaacattcacattccatataaaag gattattaccaaaagaagctatagccgcacagtttataacatgagtgacttcatcaagacgaggatccatgagaaaggcttccggattatttag atccccaggcaagatgttatcattagttaatgcatgcaacctttcctcacaaacattaaatttacgcatattttctttaattctttccagtcccgcttgtgg agtaggtgctcgtactaaaagtagcaaatttatattattacagttatccagcagcttatccaggaccgcgccaccaagaaatcctgttgcacctgt aagtaataacaatttcatatctaacctcttttgttttcatgtaatcattataaaaataaattatcttacataaactatgattaaacaaaatcaagagag attaaatgtaaaaacaatgtttatttttatacgacaggatccttcaggttattgcgagtgtttttattggatggtcttcttattattgacgagtaatagaatt 73 ME151672980v.1Attorney Docket No.: 139465-00120 ataataaaattcatattattaacatcgcttatcaattatttttatgtcgataacactgtgtcgttatgcatgaagagtggcagagcagcatctttaaaaa tacaagagttaacagaaacacttttatcgttgagcacatataccctccacatattataaaaaatcaaaagaccaccatcagaagtgctggccttt tgtttatcatatttaaagtgtcatgaaaaagttaaaaacaggcatgcttcaggttaaaaataatacttcagaccagctgtggtcataaagttatagc tttcaatgcctgcaccatttttggtgtgatcagatatattcaaatttctggagtacagagaagtatcacctttcttattagtaatacggttccatgttccttc gacataaactttagctgcaggtgtaatataataaccagcatgtagtgagacagagtaatagttctgattatttacatcgctacgataggtgatccttt tctcagggttgtaatgctcatcattatccgatgccttcacccatccgctatatttgaaactccccccgaactcaaagctatcataccgataattaccg gttaaccctatatagggcattttaaagtgttgtttgtagcctatagctctttcaccatctggaaatgatcccgtttcatcccggaagcctccctcactac tatagatataagagcctccttttgctgtaaagctataacgattttcctggtaaccagccataagacctaactgatagtctggttgattcaggagccat cccttaatattcaggtcgaattcattagcaaaatttagacgggtattcggatgtttactctcatcagtccaggttccgggattgctcgtatccagcca gtccctgtccaccatattgcctccacgaccagctaatgttgtccaccctgaggcaccgacagatactctgggtaacagatcccagttaaatgcc cccttgacaattggcgcattactgtatttccagtccagctggctggctttgcgccctttttcttcaggcagataaacccgttctttggccttgccacttaa agtgccaaaatcaatctccgtactgattttctccggtgtaaaagaaagcccagtatcagaagccagtgcagcaaacgctacaggagctgaga gtgcagtagcaagaatctttaagtacatagacaagttaactccattccactcgtttgtttttgataaatatttttttatttactcgtagtagtcaactgaaa aatgctgttttgtttaacgtttgtttacaatgtggccgcatgtcgctttatttattcccttttatgtcagaatatcaggggagtcagtcaaacccggtatga ttactgccccggtcatccatgttccaggaaatatcagggcatacagggtgctggtaactgcgaataaggccctggtcggacactttatgccggg aacgtctgtaacatggaaaccggtaccttcttcctgggtttattttgttaactcttatctaaagggagacaaaatggccaaatcctgcgattatgaag caatcagggtttttattagcgttaatgtcggcaaatataccaataacgccattacagttaattattcagatagcgtttgtgcgacaaggcattaccat tcgacgaaaataaactcaggtagttgatatcagaaatcaaccgtggctccacctacaaaaaaagttgctggtttataaatccctcctttctgatta ataagaaaactaataacatatgagtataattacttgttcagataagttaattatcttcgtgattgatatttatttggtttcagtaaaaatgatgtttttattat aaagtttataaataccaagtcggtggtgtttattagtgtaatggaggggagtgcgtaaatcgttacatgtaacacttccttccaataatactttataa atcaataaatgtatgattacttatttcctgaaatatcacatatattgctatatttcttaacgagatattcttattttattgatgattgcatctcttttatttaatgt aaagctttcttattaatcttctaatgtattgcaaaatggtagatgctgagtaatcaacagacttgtatgtaaagtcaagattaacgttgctaaagcac gcatcaattttctgataaaaggatgtggttgtttttttcgataaaaaaaattcttgtctaaccgctggtgaaataagattattattaccccggattaatac tgagcctaacaatagaatacaaaaaaaacaaaatgctttaaataaggggagcactataagttatcctctttactttatgcttaccaactcattttgtt actttataacataactcatcttgcgttgtaaacagactcgttttagttccatgcaccttttgagatcgtgaccaaataatggcgttagtgatgttcttcca gtgtcatatggttaagtggttgatatggcgttcactgttatattctgacaaccagtttgccgccagttcccgcttttcatccagcgttctgaacagaca aaaaccgagtatttttgttcggtatgtccggttaaaactctcgataaaagcgttctgcgccaacttacctgattgggtaatctccagttttactacatgt ttttctgcgcattacgtccgtgttggataaaaaaaatcctggccataatctatgcgaatcatagctggatagacatggttttccgcaatccttgaacc gccccgggaatcctggagactaaactccctgagaaagaggtaaacaggatgactaaaaatactcgtttttcccccgaagtccgtcagagggc gattcgtatggttctggaaagtcagggcgaatatgactcacagtgggcggcaatttgttccattgccccaaagattggctgtacaccggagactc tgcgtgtctgggtacgccagcatgagcgggataccggaggcggtgatggcgggctcaccaccgctgaacgtcagcgtctgaaagagctgg aacgtgaaaatcgtgaactgcgccgcagtaacgatatccttcgccaggcttccgcttattttgcgaaggcggagttcgaccgcctctggaaaaa atgatgccactgctggataagctgcgtgagcagtacggggtcggaccgctatgcagcgaactgcatattgccccgtcaacgtattaccactgtc agcaacagcgacatcatccggataaacgcagtgcccgtgcgcagcgcgatgactggctgaagagagagatacagcgcgtatacgatgta cggtgtgcgtaaagtctggcgtcagttgttacgggaaggaatcagggtggccagatgtacagtggcgcgcctcatggcggttatgggactttcc ggtgttctccggggtaaaaaggtccgcactaccgtcagccggaaagccgttgtcgcaggcgaccgcgtaaaccgtcagttcgtggcagaac gtcctgaccagttgtgggtggctgattctacttacgtcagcacatggcagggggtcgtctatgtggcgttcatcattgatgtgtttgccggatacatc gtggggtggcgggtctcatcgtctatggaaacgacatttgtgctggatgctctggagcaggcgttatgggcccgtcgaccgtccggcacagtcc atcacagtgataaaggttctcagtatgtatcgctggcctacacacagcggcttaaggaagccggattactggcatcaacaggaagtacaggc 74 ME151672980v.1Attorney Docket No.: 139465-00120 gactcgtatgacaacgcgatggcggagagcatcaatggcctttacaaagcggaggtaatacaccgtaagagctggaaaaaccgtgcagaa gtggaactggccacactcacgtgggtggactggtataacaatcgacgattgctggaaaggctgggccatactcctccggcagaagcagaaa aagcttattatgcttccatcggaaacgatgatctggcagcctgagttcacagataaaacactctccaggaaacccggggcggttcacctacag agtaccgctaccacttgtgaagctggcagattcagattgttttcaatcgacaacgcctcatagttaaagtcatcaacgacattgaacgtgtgaagt ggtcaacaaaaactggccaccgagttagagtttttccagtatcgattttccgattcgtttggggataacccaccgttatattcgtgcggtcttagtgc gctgtaatatccaacgatatagaccgttatggcgtgagcggcctcgcagaagcttcgtaacccaccaccggcatccattcgttcttcagactcct gaagaagcgttccattgggctgttatcccagcagtttccgcgccggctcatactctgcctgatctggtatcgccacaataactgccggaactgcct gctcgtatcgctgtggaacatcaccccgccgggcttacccgtccagatataggtcacatcaccgcaccacacctgtatagcggcgtaatcccc aaaaacgatgctggttgtcagtaaacaagccatgaatgacagtcgttcctgaaatcataagacctggaggagtgtatgctccgtcgcagtaatt accacgatacgcttgcagaattttttccagttactgaagcgtgaacggataaagaaaaagctcttcggaacgcgggaagacgcctccaacga tatttttgaacatcgaaatgttttataacaataagcgtcggcatggttcctgcgaacagatgtcaacaatagaatataaaaacaattattatcaaca acttggaaggagtaagactactcgtggcgattcaatattagatcattgatggagatataaatggctcactttttatcggttgttgacgatacgtttatc aagtatacatgaaaatttcttcataccagctatatgagacgttaagactaataatcttactcccaaaatgactatgttgttgcagtttggtttattgact cttgtctatagttcaccgaatgccaagatgagggagcgaggaactttactgtctacgcctctcttcctggttacaaagacaagcgtaaaactcac catccattagaactaaaggcactttcctgagcagagttttcatctgcaagataaaagctattttacagccgtaaagtccagtgaaacgaaagag aggttagtgaattaattacttgatgtattaaagtataatgggcgtagataacaccttaatacgaaatcttgtcaggctttcccggtaattagtaatact gttgaaacaaagcgagagggggttagcccaatttacgcaaataaataataatcttgattatgattaattccaaataagtatatttttccagtagatg ctgggcattaactaagaaaacccagaataccactttcattgttatggaaaacatggacaatgttaatgaaatatatttctcaccgcgccatcatttg taccttgacactgttgatcattattatcaccgttttgttcacctttctgagaagctctgatgtgccagaatatattactgccccggtgcgcaaaggcgat atagaaaattctgttctggcgacaggccgtattgatgcaattgaacgggtaaacgtaggggcacaggtatcagggcaattaaaatcactgaaa gtgaaacagggcgaccatgtgactaaagggcagttgattgccgaaattgacgacctacctcagtgtaacgatttgcgtaatgccgaagctgca ctcaacgaagtgaaagcagaacttcaatccaagcaggcgctcctgaaacaggctgaactgcgttttaaacgccagctccggatgctaagag aaaatgccagctcacacgaagattttgagtctgcggaggcaatgcttgcgaccactcgcgctgaattacattctctgaatgccaaactggtcca ggcacagatagaagtagataagaaaaagcttgcccttgaatatacgcgagtcgtagcgccaatggatggaattgttattgccattgtcactcag caagggcaaaccgttaactcaaaccagagtgccccaacaattattaaactcgctcgtctggatgtcatgaccattaaagcacagatctccgag gctgatataacccgtatttctgtggggcaaaaagcccgcttctctatcttctccgaaccggacaagcactacagcgcaacactgcgtgccgttga actggcaccagaatcggtaatgaaagatgactctctcgccagcaacacttcagcatcaggctccggaacttcaaatgcgtccgtttattacaat gctctattcgatgtgcctaacccagaaaatcgacttcgtattgccatgactgctcaggttaccctgattactgatgaagcccaaaatacactgctg gtacccattcaggcagtgcacagaaatgaggggaagaagcaacaggtcctggtgctggcagcagatggcaggctggaaccccggaatgt gaaaactggtatcacgaacagcgtggatattcagatcctcgaaggtttaaatgtcggagaaaatgtcgtgctgtcgctgccggataagaaaga gcccgaagaaaggattatgctgtgaaaaaacttattgagttaaaaggggttagccgaacttatggcaacggagatcaaacgcggaccgtcct gaaaaatgtcgatttgacaatcgttgctggagaaatggtggcaatcattggagcctctggctcaggcaaatcaaccttaatgaacataatgggtt gccttgacgtacccaatagaggtgattactacatcgacggacaaaatgccgcctgtctttcacccgatgagctggctagagtgcgccgggaac acattggctttatattccagcgctaccatttgatacctgatcttagcgcacttggtaacgtggagatcccggccatttacgccaacagcgaacgcg acagccgtcggcaacgtgcaacggcgttgctgggcagactgggactggaaggacgtgagcatcataaaccttgcgaactttcaggcggtca gcagcaacgcgtcagtattgcccgagcactgatcaacggtggaaagataattttggcggatgagccaactggtgctctggattcgcaatccgg gcaagaagtgttagccattctgaatgaacttaatcgacgtggtcatactgtagtaatggtgacccacgatatgaaggtcgcccggcacgcaaa gcgaatcattgagctgtgtgatggcgaaatcatcgctgatagtggaggttgcgtatctgcgacggaaacactaccaaaaaccaacagaatcc ggcaaagctattggaaaacactgctcgatcgaacgcgtgaatcgatgcaaatggcgctaaaagcgatgaagacgcatcgcttacgcacca 75 ME151672980v.1Attorney Docket No.: 139465-00120 cgcttaccatgatcgggattgtttttggtatcgcctccgttgtcactgttgtggcattgggtgaaggagccaggcaagaaacactggaagagatta aaagtctggggacaaacgtggtcagcatttatcccgggcaggatttgttcgatgacagcattgagagtattagaacattggtacctgctgacgc aaatgcactggcgaagcaagggtttatcgatagcgtcagcccggaagttagcgcttcagacaacattcgttttctgggtaaatctgcgatagcgt ccattaacggcgttgggcgggaacatttccgggtaaaaggtattgagttgttgcaagggactacttttagagacgatcgcaacgctctacagga agttattatcgatgagaatactcgtaaggccattttcgacaacacagggttacaggctctggggcaaatagtctttcttggttctgtaccagcacga gtggttggcatcgctaaaagtaacaaccgtagtgatgcatcaaatcgcattactgtgtggatgccttacagcacggtgatgtaccgtattgtgggt aaaccagttctgaccggtattagcgtcaggctaaaagacaatgtggataacgaggctgcaatcagcgctatctcccaactattaacccggcgt cacggcataaaagacttccagctttataactttgaacaaataagaaagtctatcgaacacacctcaatgaccttcagtattttaattcttatggtcg catgtatctcactaatgatcggcagtatcggggtgatgaacatcatgttgatttctgtaacggaacgaactcatgaaattggcgtacgaatggcg gttggcgcgagacgcagcgacattatgcaacagtttattattgaggccgtattggtttgcctgattggtggcgcactcggtattgcactgtcgtatat cacaggtgcgctctttaacgctctggcggacgggatattcgcagctatatattcgtggcaggctgctgttgctgcatttttttgctcaaccttaattggc ataatcttcgggtatctcccagcccgcaaggcagcaaggatggacccggttatttcactggccagcgaataatatatgaaaattttatcatcgttg actttatgccttatcaccgcactctgttcaggttgcacaaacgtgctgaaaagtgactatcgcgctcccgaagtgaactatcccatcaactggacc aagggcgatgtggacggaaatacatccccgtttgactgggaagaatttaacgatcctaatctcgataactggcttcacctggtgatgacaagta ataacgacatagctatagctgcactgcggatacatcgggcgcagctggatgcggaaagaacaggtatcaccaatactcctgcactaaaggc ggcactgagtatggacggaaaaaaacagctgaataactcgtccggctgggcaaaaagcggttctgccagccttggcaccagctatgagctg gatctctggggaaaaattgcccgtcagcgtgatgttgctgagtgggcagtccatgccagtgaagaagattttcgctccgcgcgcctgatgctgct ttcagaggccagcaataattactggcgtatcggttttgtaaatcaacaaattaccaccctccagcagagtattgactatgcaaaagagacgctg cgtctggccgaggttcgctatcgtgcaggaaatatctcgtcgctggatgtgattgacgcgcagcaaaacctgctcacgcaagagaaccagctt acagggctacaacgagaacattcacagctactcaatcagcaggccgttttgctcggcaccgtgcccggttgccagatagtcgagcccacaac gctgccaaaaggaagcctgcctaaggtcaatgcgaatattcctgccagcatactgatgcgccggcctgacataagtgctaaagagtggcagt tgcgtgaggcgctagctacggttgatatcaagcgtagcgaatattaccccaccttcaacctgacaggcgctttgggtaccagcagcgcttcgct actggcattactgcataatccggtcggttccgtaggcgctaacctgacgctaccgttcctggagtggcgacagcgggacatagaggtaaaaat tgcccgtaacgattacgaacagcgagtgctggagtttaaacagctactttataaggcaatgagcagtatagaggacgcgctttcatttcgtaacc agttgctgcttcaggagacaagactcagggaagaactggaacttgcacgtaagtcagagtggttaaacgaagttcgttaccggcatggtgca gtacgtatttcattctggcttgatgctcaggaaaaacgtcgtcaggcggagctgcgactggatgaaaaccgcttcaatcagttgcagaatctagc aaaaatttatcttgagtttggcggggcatcaacctttccttaagtctaattggggacaaccaggagcaataaggagtgaaaaatagccacagct gaaaacaccatagccattgacatttgagctttacctgctcttgaaaacaaaaaatcactgttaagcgcaatgctattcacttcagcagagcagta ttgcgtgtcacgagcatgtttaccattctgtgtaaatatcttttcttaaaagtgatcatcaaagcagtcatcaaactcgataaatatccaccggcata gcgtaatgccgagcaattaaggatcaattgaccgctccttaaactgcggcactataacggcttctacaacagggagccgttttcttgtgcataga aaacccccagctaggctgggggttccggaaagctttcagctttgagccagttattaaaaccccttttgatttattaaaacaccttgcggtctggcaa ctgcaagtgtcaaacaagaaatcaaaagggggtcccaatggggaacgaaaagagcttagcgcacacccgatggaactgtaaatatcacat agtatttgcgccaaaataccgaagacaggtgttctacagagagaagcgtagagcaataggctgtattttgagaaagctgtgtgagtggaaaa gtgtacggattctggaagctgaatgctgtgcagatcatatccatatgctacaatgtttgtcaaagctcagtgaaaatgtctaatcactggctcattat aaatgtccatatattgaaaaggccgctgcgaaaatccctcccgcagcggcctctttagcataacgtcattatgcgaaccatcacttaatgttcttct cgcattttaagagaaatagtaaatttgtagcagtaagatgaagcatatagcttaccaattcttccggaacctctttaatatcagctccttgcccatga cctgcatttttgtttctgatggttggaattcctgtttccagcattgttttcagacttgtgaactggctttgtaaatatgccggaattaaattttgacttaaaca gctatttattagttttgatgctgtatcattaggactatatttccagttattcttatcatgtattgctttcattatactttcaaatgatttcagacaatcatttagac attctttataacgcttatgcctgtaatgctcatgcgcagcaaggaattcatcattggcgccttcaaacaagggttctccacttaaaagaatcagggt 76 ME151672980v.1Attorney Docket No.: 139465-00120 tggctttacgacatcagcatgaatcaattgagagtctatccttattatctcttcagattcaaactgataaccaaccccatgttctctaaacctttcattta gttcaataactgcatctccaggactttgacttgttatgtctttgaattcataatggtttttcgcaacgtggctcactagtatctggaagcatatttctatga aatctaggcatttctctgtatttctctctttaaggaaaaagtcgacaagagcgtcaaagtcattctttgcaaattccttcaaagaaaaaacaccatat tccttagaaagtatctcatggatatatgcatatactttgtcagcttcatttctatacgatgtgtagcgttcattagaagaggggaagccgatagaatc agttattattttaataatttgcaccctgagtgcatgtggaatattgttatattgatatacgtcatttacttcgccacgaatttttctttgtctcttagaataaat attaataattgccatgaaatagcctcaagatttttttacatgctgttctgtgatacttgctgtttggctctttgcaccgcgaagcggttcggggttctgtga cacccgaattttacctcggtttcctttgtatataatatcattgaagttatttgcatttctcagaaatcttctctggctttgattgcaatagccgaaagatag cgcacacatcgccgttggtcagttagtacgaggtttccgccaagttcaccttttaccaaagatggcctcagagcttccctgaagctttatcatggct tcaagtaggtcatcttcgttcagtgagcgctgagatttgttgatgctaggggcgtgccgtctgcgggaaggctcatcaccagaacaaggtaacg attgtgagcgattattgtcccgttttctctggacctgacgggcaacatccaacacatggccaagtctcttgttatctacaatggcgacgggatcaac ctcagaaagccggtcataggtggagtagggcagcagcacgccatttagtcgcagctccttatgtccgtcaggataatgccagacatcaatata cttgccgatggctcttcgactgtactcgttgtcctcaatcagatagagcattttatcgtattgtacagtcagattttttgatacccggcgaggctctcgct aggtaaattctgcatcaaggtcatcgtctaatgccagcggccggtgcgcgttaaaatcatgcaagggttctttggcaaagcgccggttataatcc ttctgcaaatgcattggccgcttctattgtacagatccccttaagcctgagctccttaacgagtcggtcctgaagtgtcaaatgagcacgttcaacg cggccttttgcctggctggtttcagcacacaaaggagtaatattaaggcagcgcatggccctggcaaactgggtttcgccggggccggtggtg gcgtgcttattattgactctgaatacgctggctttatcgctgtagaggatcatcggtttaccgtatttctcgatatagccacgtgtagcttcaaaatagg taaatgtggactcagatttcacaaacagcaggtgcatcaggcgactggtggcatcgtcaacatagaccagggcagaacagggtcgacccc ggttttcaaaccagtggtggtcgcagccatcaatttgtatcagctcgccagtacaaggacgccggtaacgaggctgatgaattttcggtgcgcgt tgtctgcggggaatccagagtccggcctttatcatgagacgtcggatggtttcctttccaaggaccagaccgtgaacttcctccagcttttcccgcg ctagcgtggggccaaaatcacggtagtgctcccggattatactgagtgcctgatctgtcagtgacgctggcaacagacggttaccagtgcggc cacgactctggttgttcatgccaagcggcccatactggcgataacgtttgagcagacggctgcaatgacgtggcgtgatgcccagcatttcggc agcctgtccaggacggatattgcgatcgatgacatcctgaaggattttgaggcgattgatttctttcacggtgaacattccggcgtcatttatagcc atagaaaactcctcatcaggaaaaagtgagtcgttgtctagacatagggatctgagaaactatagcctggacattcctaatgagccacaaata gacattagcattgagcctctacaatgtttgtgcgcacaatctatcttatgttaaataacattgaatattaacttacttcatgaagcttactgcttctatttc cacttctttttcttattaatcgctcccctaatccacgcattcatctgctctacaggctctaaaatattttttctgagtactgagcacaaactgctccataaa aaacagaccacaagtttgggcatgatactgatgtagtcacgttttattgttttcatgaagctctctgctattccattactcaccggactccgcaccgc cgtgttcttcgatttaagtcccaacatccgagcgaactggcgtgtttcattaacccggtagtcagagtcagcggtcgaaagacctcccaggtagc tactctgcatacccactataatgcaactattctcgtgacgtgtgtttcagctctcccccggctatgatacgagatggtatggagagcccttctgggg ccactgcctgcaccagatgcaacaggaagcctgatatgaatgcaatttctgtcaggacaggcgggtaactttctgtggccatggatcagggaa tactccacccgacatccaccactgaatttcttcttcagtcaacaagcaaagggataaggcatgtgtaaaatcattaacttcagtggcattgccaat gatagtcaactgacagcagcgatcgccaaatcgcccagaggctgtgtcgatgtgtcgctgtagcgctgaacgttcttcacgagttaaattattgt cagtatgttccagtactccagctttccagtagccaatgagcgccagactgatgcttcctgctgactgattccagagcagggcaagatcatctctcc cggggagccaaaaaaaacctttactgcgataaacgcccatgcccataaagcggtgacaggtatcccataatctttgcggatggaatggcctg tcgtccctgatgacgcgccatataagttcctcattgttgcacggagtatccatgtttcctacaagtgcttccagttcactgatgagtaactcaacacg gtcaaaattgtagtccgggatcgccagtaatgtgctcagttcaatatttccccatgataccgcgagcacatccacggaggggttaagtgggtgta tggcttttgccacagcggagataatgtcaaagggcaggcggtcatttctggtcagcagcagcctgttgcagaacatgatttgctcggccagcaa attctccaggccgcgtttgttatgccggagattttcctgaagagtggggatcagttttttcccatcattgtaatcttcatggagcatgacagtatcaac gagtgttaaaaacgccttcaggcgtacacgtgaatggcgacgcaggtatttgaccagaggaagcggatgactgcttccggaggtttcaatcag catcacatccggcttgttgtgatcaagcaggtggttaattgcgagatccatctttttgaggcctgcggggctgctgatgctgtctgcggtgatactta 77 ME151672980v.1Attorney Docket No.: 139465-00120 cgaaattattgtcttttgcatctacaatatctgtgttggcgatcagcacgccgtccacatccagttcgctcatgtcattgactatgacagagactgaa agctgtttttcgtgtgcctgtatgagcaggtttttcagcagagtggttttccctgcgccgaggaagccattgagaatgatgacagatgttgtttccata tcagcagtacaccagttagttagcgaggaaggcgcttggtggtacaaagagcatccggattgaatcgcataacgtcgacgggagttgttgtcg tttttcctgtgaggtctcaacctgccacagaacgcaccggatttattggtatttagtctccatcagcataattatcaccatttttatctccgggggaaga tttgttctgaacaggagtgggaggtgtgacctcctgaaagtaattacagattgctagttgttgtatttttattcgaatatgtctcttcgtatcagtgatagc acctagataaagtatattccgggagcgttatctgttctagatatcagagagtcagtcatttgtaaaaaagagagctgcatcattctatttttcctgtct attcaccctgttgtcgtatctaaaagacatccgttccgaggatgtcgggcacccgttctgtactaagatacatagcttttgggtctgcactatttgga accgacagaagtctgtctgtgatatccgcatttttctgtgtgcgatatattccgaaaaacttcaaaccacttaaaagaggtgtttccggaggataag ttcgaccagggaatagtgtttcctggtaagtgtggcggagccagttcaccaggagtatctttcttatccagttatactggattttattagatgatcacc ctaccctgcagcagtacgctgacggtgtttttttgggagaaccattatgaataacgtcatctttgattggtatttatctaggcaaaaattcattcaacgt ccactgccaagatgcaacaggcagagtccttaactgtcagaatccaagtgctcgaaattattcgaattccttactaattcctcctacacaactatt gttatggaagcttgcgaagatgctcactatatggcacggaaattttcggaaatgcgtcataaaacaaagctaatacctcctcattttgtccaaccc ttcgttaaaattaataaaaatgatttctttgaggctgaaaccatatgtgaagttacatcaagccatcaatgagatttgtagcattaggagctgaatcc cagcagaggattccacttttcatccgtcgttgacaatggccttgaaaaacgccgttactcaccgttctccttcacggtaatacgcagttatagttgc agagcaatgtaatggatcttttatattaagaaattgtgtgagtggagaggtgcatagatttgcccctatatttccagacacctgttatcacttaaccc attacaagcccgctgccgtagatattcccgtggcgagcgataacccagtgcactatgcggatgccattcgttataatgctcgaacgcctctgca aggttctttgctgccgttaacccgtctggtttgggcatgacactgatgtagtcacgctttatcgttttcacgaagctctctgctatgccgttactctccgg actccgcaccgccgtgctcttcggttcaagccccaacatccgggcaaactgccgtgtttcattagcccggtagcatgaaccattatccgtcagcc actccactggagacgccggaagctcgttgccgaagcggcgttccaccgctcccagcatgacgtcctgtactgtttcactgttgaagccgccggt agtgaccgcccagtgcagtgcctcacgatcacagcagtccagcgcgaacgtgacacgcagtctctctccgttatcacagcagaactcgaac ccgtcagagcaccatcgctgattgctttctttcacggccactctgcctgtatgtgcccgtttcgatggcggtacagcaggttttcgctcaagcaaca gcgcattctggcgcatgatccggtaaacacgtttggcattgatcgcaggcataccatcaagttttgcctgtctgcgaagcagcgcccatacccg acgataaccatacgtgggcagctctctgagcttacccagcaatagtggacacgcggctaagtgagtaaactctcagtcagaggtgactcacat gacaaaaacagtatcaaccagtaaaaaaccccgtaaacagcattcgcctgaatttcgcagtgaagccctgaagcttgctgaacgcatcggtg ttactgccgcagcccgtgaactcagcctgtatgaatcacagctctacaactggcgcagtaaacagcaaaatcagcagacgtcttctgaacgtg aactggagatgtctaccgagattgcacgtctcaaacgccagctggcagaacgggatgaagagctggctatcctccaaaaggccgcgacata cttcgcgaagcgcctgaaatgaagtatgtctttattgaaaaacatcaggctgagttcagcatcaaagcaatgtgccgcgtgctccgggtggccc gcagcggctggtatacgtggtgtcagcggcggacaaggataagcacgcgtcagcagttccgccaacactgcgacagcgttgtcctcgcggc ttttacccggtcaaaacagcgttacggtgccccacgcctgacggatgaactgcgtgctcagggttacccctttaacgtaaaaaccgtggcggc aagcctgcgccgtcagggactgatggcaaaggcctcccggaagttcagcccggtcagctaccgcgcacacggcctgcctgtgtcagaaaat ctgttggagcaggatttttacgccagtggcccgaaccagaagtgggcaggagacatcacgtacttacgtacagatgaaggctggctgtatctg gcagtggtcattgacctgtggtcacgtgccgttattggctggtcaatgtcgccacgcatgacggcgcaactggcctgcgatgccctgcagatgg cgctgtggcggcgtaagaggccccggaacgttatcgttcacacggaccgtggaggccagtactgttcagcagattatcaggcgcaactgaag cggcataatctgcgtggaagtatgagcgcaaaaggttgctgctacgataatgcctgcgtggaaagcttctttcattcgctgaaagtggaatgtat ccatggagaacactttatcagccgggaaataatgcgggcaacggtgtttaattatatcgaatgtgattacaatcggtggcggcggcacagttgg tgtggcggcctcagtccggaacaatttgaaaaccagaacctcgcttaggcctgtgtccatattacgtgggtaggatcactccgataacatggtgt atacggagaagcacatccgtatcatcagtgtgacgactgcggcggccatccatccagtcatcggttcgtctgagaatgacgtgcaactgcgca cgcgacacccggagacaacggctgactaagcttactccccatccccgggcaataagggcgcgtgcgctatccacttttttgcccgtccatattc aacggcttctttgaggagttcattttccatcgttttcttgccgagcaggcgctggagttctttaatctgcttcatggcggcagcaagttcagaggcag 78 ME151672980v.1Attorney Docket No.: 139465-00120 gaacaacctgttctccggcggcgacagcagtaagacttccttcctggtattgcttacgccagagaaataactggctggctgctacaccatgttgc cgggcaacgagggagaccgtcatccccggttcaaagctctgctgaacaattgcgatcttttcctgtgtggtacgccgtctgcgtttctccggccct aagacatcaatcatctgttctccaatgactagtctaaaaactagtattaagactatcacttatttaagtgatactggttgtctggagattcagggggc cagtctagtgcacgaatcctagaggcagaatgtttctccacatgcttgtggagaaaccgccgcattttgcccgttctgtacgatactccgtagcca gagataagatattctgttgctgtatgctacatacattacactatacgaaatcaggtctgttcaatcagtaaggaaataaacggacaaacgttatgt aacccagacagagatatattgtctgggaaaatagtggcttttgtggcgaaaactagtaaaacagcaacccgaaccacttgatgtgcatcgttttt gattattcccgtatactcttgcagaaggagttctccgtcgggctactgtcatggttaatgcggggaatatggcgacaatacaacacacctaaaag agtaatggacagatgaagcggtttattcatttcccatgattctgagtacctaccaagtctgagtaaccacttttatacttttaattttcgttcatttagctat cgtttaattattatcacataggattctgccgtttttaacaatgcaggataataagatgaaaaaaatgttattttctgccgctctggcaatgcttattaca ggatgtgctcaacaaacgtttactgttggaaacaaaccgacagcagtaacaccaaaggaaaccatcactcatcatttcttcgtttcgggaattg gacaagagaaaactgttgatgcagccaaaatttgtggcggtgcagaaaatgttgttaaaacagaaactcagcaaacattcgtaaatggattgc tcggttttatcacttttggcatctatactccgctggaagcccgggtatattgctcacaatagttgcccatcgatatggggagctcatctgcactgttcat taatatacttctgggctccctacagttgtttttgcatagtgataagcctctctctgagggaggaaataatcctgttcagcgatgtctgccagtcgggg gggctgcattatccacgcccgaggcggtggtggcttcacgcggggatgggcagattgatctgatatgcaaccgacgacgaccagcggcaac atcatcacgcagagcttcattttcagatttgggccaccttttgatttctcgtttaacttttgcagtcaccagactgtgatttattttaacaaatcaatagctt tcataaaattctcgaagctgaaagcttaactgaaccaaccagcctggctggtgttttctaaagacaaaaaggccatcagcaaaacactgatag tctgaatcacctttatcaatcatgtatgaagaaaatatgcgcaattagttgacagtgattatcattttcattaaaaagagcgcgtagcattcttatttca tgaggaaatttacccgccaacaacctgagtagcgaaagctgttcatccccaatatttttgctggcgggttcttttttctactgtcctcctccccagag gaagcattcccattaatcattactggcgaggaggcaagagaaaaagtcggttctttgtccctaaataacagcatcatttcagtccatgcaaaatc tgatttaccagctcacataataacctgatgatacacagtgtaagttcacagagatattgcaattgcctccggataagtaaggggagattgcacta tgcaaatgcagcacctgaggttggctatcctaagcagccgattatgcgttgaggctttcagtgatgggatatagcaacaatgagaatgaaggct ctgcacttctgggataaacacggtatttctgcagcttctgaagcgtttggcgtatcctgccgcacgctttactggtggcgtcagttactgaacaagg gaggacctgaggggctaatcccgcacagtaaggcacctctggtgcgacgaaaaaagcactggcatcccgatgtgctgaaagagattcgac gcctcaggacagagctgccgaacctcggtaaagagcagatttttgttcgcctgaagccctggtgcgcataacgccatcagacctgcccgagt gtttccaccatcggcagaatgatcgccaccgcacacgataaaatgcggatgataccagtacgtctgagctcgcggggtaaggctctgcttgtc aaaaagcgaaccaccaaacccccgcaggccaaagcactaccgtccggtaaagacaggagagctcattgggatggacgccattgagctc agaatgggtgaaatgcgtcgctatatcattaccatgatcgacgaacacagcgattacgtactggcgctggctgtgccgtcgctcaacagtgata tcgtcaatcactttttcagccgtgcagcccggctgttccctgtcggtatcagccagatcatcacagataacgggaaagagttcctgggaagctttg acaaaacgctgcaggaagccgccatcaaacacctctggacctatccctacacgccaaaaatgaacgctatctgtgaacgttttaaccgggca ttaagagagcaattcattgaatttaatgaaattttactctttgaggatcgcacctgagg

[0186] Nucleic acid sequence of pAPEC-2 ΔAMR plasmid (SEQ ID NO: 2): gtgcgaatgcctccggcgcgggccggattattctgaggagatcactttcagggagaagctgtggccagccggctgtaattacggttacgtgac agaatcatgcgctccttcacacgacgctccacttcgcgttttaccgcctcaccattagcagtgaagcgtccttccgagatttcacgcgtcagctgc cgtttcaccagggtgacgatatcctgacgttccctgttcgcatcacgatgcgcacgggcacgttttattccacgggacttaagctctgtctggtaac tgcggaaacgctcacgcacaaaacgccaggctttcgctatcagctcatccatacccagggtatccagcccctgctttttgcgctgtttgttttccca ttcaacacgactgcggcgcgcagctgccactgcatcctcagacacatcaagggcagcaaacagagccagtgtgaacgtgatgtcggtcgg aatgtagcacccgataagcgggtcatattccgtctggtaggtaatcaaccccagctctgacaggaacgtcagggcacgggtggcacgggtg atggagagttttccggcagcagactccgtcgccagtccgcactcaatggccagcgtggtgatggagcactggacgcggttggccagcgggtc atagtggaaacacagcccctgcaggagcgcatcaatagcccgtcgacgcagcaccggtggcatgcgtcgacgcagaccacgcgaacgg 79 ME151672980v.1Attorney Docket No.: 139465-00120 gcatgcgccacatgaatggcgaaatcaaaacgggaagtgaagcccaccgccttttccatcagtttttcgcagaacttcagcgttccggcacctt tacggggtgtaaacaccggattcgggttctttacctggcggtaatacgtttgttgaagatcagtcacaccatcctgcacttacaatgcgcagaag gagtgagcacagaaagaagtcttgaacttttccgggcatataactatactccccgcatagctgaattgttggctatacggtttttagatggccccg gtaatctttcatgtcgccaaacttgaagaagattatcggggttttcgcttttctggctcctgtaaatccacatcagaaccagttccctgccaccttacg gcgtggccagccacaaaattccttaaacgatcagcaatctatcacgcacaccagaaacaggcaagaatgtgatgtcgctacattttagttagct aaaagcactcttctgctttatcagttcggcaatcatctcagcctgggtaacgccctcagcttcacacatcacctgcaattgcgcttttaacgcagca ggaatgaacactctgagttctttatgtgtagctcgttttcttgccagggatgcctgctgacgttctgcaagcgtcagaggattaccttttctgtatgctc gctttgtacctgatgaggaagtcactgcattttctgtctgcgacatctcgcctcctcaatactcaaacagggatcgtttcgcagaggatactacagtt ttttgaaatcagcgacttgagaattgtgacgaagatctgtgttggcgcacaaatcaacggggattactgtcgtttaatgtgatttaaactgtgaaat agtatggttttcagttatggaaacgccgggagcggggaaaacttgctttttcccgtttccggggttggacaactgagcaacgcgaaggcgtcag ctacgatgttccggggactgctgatcagcccctgttgtattcagccattctccggtcatctgcagcagttttctggcatcgtggcttctgagcaacgg aataccagcctgcctcagaacctcttccagaagaatatcccggcgtctgcgttccggtcgtaaatggctggcatcatccagctctactgctgcaa caatactgaacgaacgccgctcaacgataaccacatcaacatgccactgagacaccatcctgaataactgccaccactggcgtgatcgtgg ccggatattcccgttcagctggacgatatccgcgacccgtacctgcggacacagataccaccgcttcatatccacaattctgaacagtcctttca taaaactgacttccctttctgtcagcagcgtcttctgacgttgccagtattcaccgccggacagtattcgctctgcatcaccggtccgtatacctgcc gcatgcaactgcatcagcaaaggagaatgctgatttacacttctgaggataatccggataagtaacgctgcaataatcagaacaatgagaat aaaaataatcagcttcattgtttatctgctcctggcaattaattattgctgaattattaccttacatatttccatatctcttttggtatcctgtcatataactttc cggtgattaattccgctaaccggtggtctgctgctgaattaaatgattcacgttcactgtttaataaactgtctctgtttttatgaataattttttccagcgt attgactgatgaacatcgccgtaactgatacagccattcctgttttgtttttgccatttttattcccgctgagaacagatgaatgataagattgccagc gctttatttacggtgtctccatatccaggggggaaccggatcagcatctgaccagagcccacgtttttgttgtctggcttcgttctgaaagacaggt agcacgggatctgtgttgtactgctcatatacccaggcagccccggcccggaccataaagtgattaacattcattccatctggtgcataaacctg cccgagaatacgaccataacgatctcgctgaaaatatgtgacggtaaccgtttttcctgcaaccagtgatttcatcatgtccgttgaccaccggc cataatcctgtttcttttccggtgcatcaatatttaccagcctgatccgcaccgccttccgtgagtccatgacctcgatggtatcaccgtccagtaca cgaacaacccgtccatgaatatcttcagacaataccggccatgaaaagataaataataccagtggaatatatttcatcatgttattctccatatca gataaatggcacctgtatttcaggcatcctgaacattgcagcacacaatgaataacaccttattttgtgaaataaatcacaaaatataaaatcac gacttaatacggaaaaaactctgtgctactcagaactgtccggtcatactgcccgaccggatattaaaaaaagcaaatctcatgcattattcag ccttctgaatccctggagtaatgaaggctgctttttcgttacacccttccccggcagcgccggtggcattgttcagccctttttcgggcaaactgctg ccagtctgtccggggtgtaacaccggcagcccgtcgccatgcttctgccgcttccagaataacggcccggcttccggcactctggtaacgcca gacctgaccgttccgagaactgtttctgaatccattcattttcctgtcccccctttagttgtcagtattattgtgataataaaaataatcagtttcaccata ctctttcccttttttatctgataaacggatactgaattaataaattcattttccttgccaccgacttattctgtttcttatcaatgcatttcctcacagaagaat taatgctgccgggatattcatacacaccacatatactgctgatgtttcagtgattattcccgatgctgaaacggaaaggcgcttttctttcccgtcttct taagcgctgagcacccgaatctgtgattcaggcgatgtttttctcctccatatattcacttttttcaccatgtctggcaaagtgaataatgctcatatca gatcctgctcagggaactggctaaatggaccggaccggggaagcaattccccggtttttctttgccgcgcacattttttgaattaaatcacacatttt attgttcctgcttgtcctgccaggaaaatatgttacaggtattgttacaggtattatgtcgtttagtccgattcagcttacagacagggataaccttctg gttcaccataataacaatcatccttaattccccttttcagccccttcgctccgcaaccgcaacgggggctgtttttatctgagtacagctgcgatacc tcatgctttattctcctttttcgcctttttccagattatctttttctccctgacttctgtgactccccgacgaaccagttcattccttatcgccactgcccgggtt ttgttccattcgtcacgcatgtgtgactttaaataattattaaatgtttcgacggacacaggcatattaccggctccgctgccggctatctgctgcagc tttcctgcccaacggctgacgatatcactttcgggaacagaaggttgtttcaggcagttacttttgtctgcaccagactcaccggcagcagcaata ctttccggtacatcacgacggatgcaggtaataccggcttctttcagcgtcctgataacaccgtcatatcccttatccgatgaaatgatgtggtatgt 80 ME151672980v.1Attorney Docket No.: 139465-00120 tttatctttccctgctgcacaacagcttatccgccccagctccagaacaagatgaaaatcgacattattcctggacacaccactcacctgcctgat agtgattctcgcaccttcaggaaaagagtctgcaggaagaacaacgacgttctgctgtggcccgacaaaaataatcagttccccatactgctc aagggaaatatttttcagactgccacagttttcataatcaataagggctgtcagggtgtttttatctgtactcgtattattttcttccatgtttttaccttttatt cataaccgggtgagcaataacacaaaatattttatttccgggaacaccatataaaaagaaagacttccctcgtgaatattcaggcgcatattaa acgacgtcttgttttatgaaatcaatcacaaaacagcacgttattacttaatacagcaataatcctgtgataatgaatgctgactggtcatactgcc aggccagatataaaaaagaaaaatctcatgcatacttcagccttctgaatccctggagtaatgaaggctgcttttttcgttacacccttccccggct gcgccggtggcagtgttcagccctttttcgggcaaactgctgccagtctgtccggggcgcaacaccggcagcccgtcgccatgcttctgccgctt ccatccagcgtccactgacctccagaataacggcccggcttccggcacgctgccagcgccagacctgaccgttccgtgaactgtttctgaatc cattcatctttatcactcctctttaacccgtcagtattatggtgttaaataatctactgaattggcttcctttcgggtacggaagttatttccttttaagcctt atttccctgcctcctctctgatggaggctttttttatttctcatcaagcacggcctgaagttctgcctttatccggttctggcggcggattttatccagacg cgctgccgcatacgcttcctcctcctgagaaatatgctccgtcacatacccttccgtgtcataccggcaggcaccggctttcatggcacacagat agctttctgaacgggtgatggccttcagcgccctgcgcagttttttatgcgagagcgggatattccgctgcgccacgtcttccagtaacacgtccc ggataccgcaggccagaagccggggcgtgtcaccgtcaaacaatcccggccaccagggtttcagggtattcacggcctcatccagcgtggg caggttcagataaatggacagtgcctgtctggcctgcgctttttttgccgccagctctgcttcccgggctgctttctcggccagcttctgctttttcacct tccactttggtggcgtggtgacattgatgatggttttccggctgcggacaggcgtctccccttccgttttccgcttcagtgtcagtaccggtcgcttctg ctctgtcatgctgtgttctgcctctgacgaatcatcctgttacggtcaaagataccacacccgtcacagcgccagaacgcccagcacagccag atgacaggcgtagaataccgggaaaaaatccccgggccagaaacggggtaatgattttcctgcacaggacaccagaccaacggtcagca ccgtcatcacaagaccagccaccgccgctgcatcactgctggcaaggttaagcgccggaataacagccagcaggcaggcgaccagcgc cagacgttccgctctgtcttccgcccggtacagccggtggctgactgccagcatcagcagcccggcaatgccgtaactggtgccggacaagg gcccccacagggccatcagaagaatggccgctgctgtacgccacccactgcgcgtttcacaccacgtcagcacctgtgccgccactgcaaa ggcaaacaggatattcccctcataccagggaaagccggccagataatatgcgaactgggcaataatcccccatccccacagccggttaatg gccggttgccggatattcgcatgacgggacagattcagcccccacaccagggcaaacagcggaaaggctcctcgcccggccagaaacat ccattcctgcttcagctggaatatcagattgatgtggtccagtaccatcagcaccagtgccaccgttttgatgatatcccgctgtccgggtgacca gaccagaaatgactgcaaccaggtatcggtccgtgcagccagtgaatcgttacgtgtcgtgttcgtgttatccgttgtcatcagtgtttgtcttcctgt atcagtctccgcccagggttttctctttctgcaggtcacgaaccatatcccgctccatctgctgcagacgctcctgcagcagattttcccgggcaac ctccctgaccgcttcctgctcccgttgtgattcacgcagcacactctccggcagcgcggcttcccgttcagttatgtcagcccggtcacgctcctgc ccggcaatttccctgacagcctgctctgttttaccatccggcaggtcaggtttgttctctgccatttttctgacaatttcatctgcgcggcgttccgtttca cggcggatggcctcttcagcctgccgctgtgccagcacctctgccgtgaccggttcgccatttccggctcccggctggacgctgctgtccgggat atccccccacacgcgaccaccggtgatggcaccgggattccacggacgaccttcaccggcaattcttaccaccacaccgctgtcaggattat cacgggcaatccggacaccatcctgcatattatcagccagcaggctctctccgttacggctgccctgcagggccacaaactgtgcatcttcact gcccttcacccggccttcaccgctgaatccccgcagcccgtttccgtcatccgtggtcagtgggttcagccagataccggcggacctgccgtta cggtcaaacgccggcagtgccacatacggctgcggatatttacgtcccggagcaataaaccgtgcaggactgtctcccccggccagccccg cctgacgaagaacggctcggcctgccgccacgtcccgcagctcccgcgccgtactgaacagccgctgcgcattcatgacctcccggtccggt ttcggctcaaacacatcgtgggcagttcctttctgtacggcattgttaatggcatccgtccagccctgacggttatcggtgtacacctgcacatgct gcttcatacgcgacagggccacgtaggctgactcaaagccggccatcagtttccggttaccttccgtgccttcaagcgcgatggcaaaggtttc actcgcaccctgcgcaccgtgggcggtgatggcataggccaggtcaatatgttgctctgcccgctcctgtccggggcgaatcacccgggtctg ctgtccgtccgacagcgtgacactgtcaccggaaaccgctgtcaccgtccagacgctgttggccacataaccgcgctcccggtcactcttcgtg aagcgcatccggtcaccggtccccacccggatggtgtccggggtgtacagtgtgacgccttcagccaccgcctcccggggcgaaatcagcc gcgtgttaccttcagcatcctgcagggttatcagcctgtcatccttgctgataccggcaatccggtgatacacattatccaccagggcaagggca 81 ME151672980v.1Attorney Docket No.: 139465-00120 tccgggttattctcccaggtggagagacgacgcagctccccgtcacgtatattcgctgtgttcaggacaggcaccatgacctgctctttccccag ctcaccggccttttcccgtgcatcatgaatcatgctgttcagtacgcgccggtcctcattcaggtgcgtgacaatcagcgtctgctcccgtgcttccg gcgtcctgccggtatagtcgcggacaatggcttcatacagtgtcatggggacatccgggaaagcctcgcctttcagcatcgccttctgctgcgctt ctgccagtttcgcttcctggctgtgactgaactccgtcacggagtgctccggtgcccatgcgccctccagacgtggcacctgagacggtttcaca ctctcaagcccggacagtgccctttccacattccggttaatcaggctgtataccgcctcccgcagttccggcgtctgacgcacaatctccttcatg atgaccacatcggcagcactgcgcgtctgctggagacggaaaggctgaccgggcgcgatggcctgcagctggtccgtgtcaccgctggcca cagcacgaccgccaccggccgcaatcagggcgtatgcccgtgccatgtcggtattgcccaccattgagctctcatcgagcaggaacagcgt gttgctgaaatccggcgtttctccgctgcgctgctgcagctgcgtgtcatgcagaaaggacgccagtgtctgtgcatccacgccggcgctgcgc atctcaccgaccgcacggtgcgtgggccccagccccacaacccggggacgctcactcgccggcagcatgttcacggctgacatcaccgcc cggaactgtgtggtcttacccacaccggcataaccctgtaccaccgtgaaacggtcggacgtttccagtatcatacgggtggcggcacgctgtc ccgacgttaacgtctccatgagttcgccgggtactctctccatcagcggcgtgaccgcctccttaccttcgagaatatggcgaagaatacttttttct gcctcatacgacgcacgggaaaccagcaggcctgtgccatagccttttgccacatccacatacagtaagtcaccgcgttttatctgcgcattgat ttcccctcccagttcagtaaaaccggttccttctgcagcaaacgacttcgcctctgtcagcaggtccaccatgctgaaggccaggtttttactttcc agcacaggaagggccagatgaatggcctgctgtgccggcgtgtgcagcccggctttctgcagactgatagcggtttccagcgatgtctcaccg gcccgcgcctttatctgctcagaaaccaccgtaaaggagggatggcgggcaagtttttccgcagtacgggtttcatccagtgaggaatacagc cggacatcacgaccgctgcgggccagaccgttcagggtggcgttgtccattgccatctgtgtgacggaggcaaacaccgtcgcactgtcactg acggaatgcccgggcgtttccacccagccgttctccagcttcagtgccgtgaacggtgaatcaaccacaggcagggtggccggttcagcccg ccccggcacaacaaccgtcatcgcctcttcactgacggatgccacctgcaggcggtcaccgccggagacgcggagtccgggaattttccctg tcaccctcagtcgctcgccgtctgccaccggcattttttccggccggaacagcgaccagctgctgtccagggaggaaatacgcaccacctgcg tttcgccctgcgcatcccgcagggtcaggctgtgactctgcgctgtcacccggtctatcacatagcggtcatgactgcgtgtctccgggttccact gctccatcaccatccccggtcggtacatatcccgcagataacggctccggctgtccagccagaccggtgacagtgcagtcatggtcacctcag ggtgtccgagcacgccctgcgttttcagctcactgcgaatggcctgtgtcagtatggcctgttcccgtaccccgctgacctgtgccacgctctcttct ccggctttcacgctggccgcaaaatctccggccagccgggcatagcggacattacggtccggttcactgatgatggtggccggtcgctgttctc cgccctgccagcgatatgtgttcaccccggcatccttcatggccatcagcgcactgcctgtaccggttcgctgcccgctgtcggttatcaggacct gtacgttatggcgtgcggcaccatccagcagggttaacgtctctttcagggagagtttttcgccctggtcgacgataacggtactgcccggcgtg aaggccatgccttccagcagctgacgacgtccggttatcagttcaccggacagccgttcatcctgcttcaggttcatctgcgagcgacggtcag cagcgataatctgcacctcccgcccctgttcccgggccatcatgaccagttcagccacccgctcacgctgcccggctgcaccgccctgcccgg acacaatggccagcgacgggcgatcctgtgccagaacactgacggcatcgctgtaaccggccgtccgggggacacttttctccggatgtacg gtcacccggttctgtttcatgatgtcacgactgagtgcccggactgacagctcatcgagcacatgaatcccggacgtgaacagccccttctcac gatcgagggggataagctgctcacggctgatggcctcatcgataccggcgcgtgcccgttcaatcacaccattttccggcggcagtatgccga ccgtcctggccagcacgtccgtgtacgtgaactgcacttttcgttcacttaatccggcaatcgcctgtgtcaccgcctgctgcacatccggcccgt cctgtgaagctggcccgggcgcctgcgtgcggatctccgtacgctgatccgccgcgtcacgatatgcccggatgtcgaacccggtttccttcag cgtctgcatccattcagccattctgatttccggatcgacgtgctgtttggatttacgcgtatccagtgccgccacatcccgggatttcagcgaggcgt cttccccgacggcctcccggatagcctgtgagcgtccggaaaaggcctccaccggtacgcccggcatctcccacataccgtgcttacccacc acttcggtttcatagcccagcgcctcaacctgctctttcagtttttcccggtagagcctgccaaaggcaatctgattagcgtacacattctcaatgaa ccccgttttccccactttgtcactgctcagtgtcttccactcaccgttatgctgcgtgacattagccaccaccgcatgcgtgtgtaactgtggttcctga tcgcgactggtgtcgtggttaaacagtgccatcaccagattaccggtcagcaccgtttctgactgtccgtccgtcatcacccgtgtggaggccag cgcctccacctgacgaacagcaaagtccacggcctggttatgtgcatcaatcaggcgcttatcgccacctaacatggccatcatggagacact tttgggggcggagaaggtcaggtcgtagccgggacgatgcttattactgccatcctgcatgcggcttagatccgctccgtccggcagccttccct 82 ME151672980v.1Attorney Docket No.: 139465-00120 ccagaagacgggtaaaaacatccttatcgacgctgccctgcagcccaagctgttcagcccccttgccggcccagcgttctcccatgctgccca gtacatagtaattatccttgtcggtgtaatagttaccggcacttccggccgatctgacctgcgcgatactcatcatcagaaatcatctcccggctca acatcctccccacgctcccggtgcacattaatgttcacctcttcacgacgctgcatctgctgctggatgtccggatgattttcctgttgccatgcctca taagcggccatatccaccacttcaccggattcactgatcccgggtggcagttgctgttccatctcttcttccggtttcattttcagctcctgctcgatcc cccctgccggaatattcacacctgaatctgacttcttatcgttgatggcaggagacaccggctgttgcggctgttgcggctgttgcggctgttgcgg ctgttgcggctgttcagcctgagtcacgtcttcttcggaaacaacttccggcacatccggttcgaagaggctggccatctgacgaccttctgcttcc cttgcggcaagtacggcactcagacggttctccatttccgggttgatgtcacgcggaataaactcaggggcaaccttcggacgtgcctgatattt cagtgagagttttactgccggatacggtccgggcagggtgacataacaggtcagatccggcagagactgaatgtcggaataactgaccagc gtctggcgctccatatccttaccggtcgataccccgtcacgcacggggtcagcaccgtaggaatactgcagactggctttcaggtgctctttctca ccaatctcaccggcagcgaactctgcaatcttatggctgggagaacggaaaaaggcacgggtgttcatgacgtcaaacagcgtggcagccg ctttctcaccgtagatatcttccagctgggcataggactggataccaaacacataacagccaccgaacttacgggcttccggcaggatctccac caggtccggcagtttgtgtaacgtgggtaactcgtcacagaaaaaccacacacgacggttacgattttctcccattgccagcagaccacgaat ggcaatggacagccacatggagatcaccggtttcagggaggcatgggtgtcggcattcgacgaaataaacagccagccgtttttctgatcttc ccggacaccccgcatccagtcacggatggtgaagggctcaccgttatgctcaatcccctgcaggtaacggatagctttcacgtagttggtcagc acagcacggatggaaattgccgttttctcaattttctcttccaccaggttggccgccggtgaattacgcaggaaggtacgcagtttttcaattttgat ggaaagcagtgtgtccaccagtttgctgtagctgcggttggggtcattacgcatcaggtacgccgcttccgcaaaaatggtacgtcctgaaccct gccagaacgggtcttcttttgttcccatagggatcagagtatttgcggtattatcaaaatccggctgtgtcaggcactccttccacaaatcccatgc ggcacagcgggcatccagcggattcaggattttatcgatggagggatcatagtaacttttaacaaattcccctgaacggtcataaatcaccacc atatcaccgcgctgacgggcgtagttggccagacgacggataacctccgacttaccggccccgaccgtgccgtgcagacagaagttctggat ttcggaatcccggataatcggcaggtcgccaatccggatatcggagtccttgccgtcttttttcagcatccgggcaacgtctttcggattgtctgtca gctgacgaccacctgtgacttcattttcgctctgttgtttaccctgacgccccagaatccaggagacaacaaagaaggtgatcaggcaaataac cagggccacgacagacgccagaacgaatgcagaccatagctgctcaccgcaccagatcatgtatttatcatgcagtacctgagccgcgttca tccggaaggttttgccgtaatactggatctcatatactggctgggacttgattaaatcccgcatgccttccagcgtggtacaccaccagtaaatac agccgttcacaaacgtctgccagcttatttttacccataaaaccagaccaacgagtatccagaaaaaaataaacaggcagtaaagcatgata ttggcgatctggctgaacatgcggatacgcatggacgcaatctgaccgccctgggtcatatcctttgcgttaaaactcatgatgatattccgaag aaaaaaagtcagtcggactgattattaatgatgaaaagattaataaccgttaaattccggcttcgccggcaacacttcacggtcgcacaaaga atttgcacaaccgctcagtgttttcatatttatttcggaaacgccggggagaaataaaatttattttatttctttccggggttggacaacgagcaaag cgaggcgtcagtcaggaggccggtcaggccagcctccggaagataatcagagaatatttgcgattgatttggccagctggtcttcgagaaca ggcttcgcctcttcaaatttcaggttaaccttgttcgcatttgagaccacacgggtctggtatttatgctggttacctgtttcagtactggtctgaattttc gcaccggatgtgccctgacgcagggcggcaacattatccgttgtcaccgttgccttagtacgctctgcaatctgcacatccgtgatcatggtata gttcacatcttccaccatcgcatctgcagccatacccaccagaccggcagcaaggcccacaccgagtgtggcaccggcagaatttgagttat atccggtaataccggcacctaacgctgcaccaacagctgcgccttcataaccacggttcagccatccctgagactcccgcagatccatcttatc ggccttcagcacatttgcctgaatccagtagtaggctttatccggagaagtcaccacctgatatccttttgctttcacagcatcagcaattttgccct gcagcccactcatgtctttatcagacgtgtttttgatctgcagaaataccgtgcgttcgctgtcgggtttaagccagatggtttcactcatctgagtctt cacctcaaggttacgcttcttgattgctgtgctcatcgcaccacaccctgaaagggccagagtggaactgaccagtgcaaccatcattaattttttt gttttcatcaaattacctttattacaaaccacagttttcttatcgcctgtcatcatagtcctgccaacaagtcccaaaagtatttcctgctgaatcaaat cctccatgcatagctaatcctgttgccggattagtacgataaaaatccacataatcaaaattcacatcatcaaaatccagatgcaaatcttcatta gtttgttcatttacctgagattgcatccattctcttacacttacacaaccactaattcgccttaattcgtatagtgtaccaatgataaaaaccatcaggc ttaataacggagagagtaaggaagataatggcatcagtatataccaaacgcctttactgaatttaaactcatccgacacatgccgtttttttcgtat 83 ME151672980v.1Attorney Docket No.: 139465-00120 ttgagaaaacaaccatataaacaatccctgccatagtacaataaagcacaggataaaaactgtttcgtcactcccccaatcagccagataac aggcaatgaactgtatcaccgtcaccagagcaatatgagctaaatttttcattatcatctccccatacgatattattgcttgtgcttatcctgatactcc tttgctcttttcatcagttcctgtggactgtcagcaccaggcattctttcctgacccgatttttcttcattatatttattattctgagataatgcctctgttttatg gtgattttgtagttcagaatactgtcttcctacaatattttcttcgccacgtatgctgctctgagtatctccgatattgccacgatattctgtgaccatatta tcaacctgatgttttacatcattacggatattactgtcctgcgttctttgatcaataatggcctgatgcccctgatggtcagcaataatatcctggctgc caccacccgaaggtacgctctccattcctttaccaatatccccacgggattcacgccaggcattatcaacaccaggctgcacctgttcctgaac aaaagaccacgccatagcccgacggcgttctgcaatctccggcgaactggtatttgtcagaatagcttctgcatcctgcggtgcgcgtttcatca catactgtgcaaactgttgcgacaggtcttcactcatctgcccgctcatgctttcagtacgggaggccatttccgcatattcatggctgcgggtcat attcgtcgtgtactggtcgtaactctgtttcgctgagttcagggctgcagacagctggtccacacgggaatcggcattattgtccgtatggctgccg gattcactgaccttgcgactggtaaagtaatcgctggcctctttaaagtcttgtgtagccctggcatcgatatcatggcgagcatcatggctggccc gtgaaccactgctggcctgatgcgcatcctcatcactccagtcaatacttgcccgaccaccacctctaacacctgcgccaccaccaacaccaa gaattttaggtttaaccccccattcagcatgaacatcgccatacatccccgcagaacctctcgtacttcttgatgccagttctcttgttgcctgctcatt actgatattatgcgccttcgcatagctttccactgcactgcgcatacggctggccatcatggagtcctgtgcactcatggtgctgtcagcgccacc ggtgacagaatcactgctcccccggttagaaccaaactgactgagcgagttccaggcactggcaatactgctgctgaacccatgcagggca ctttctgctctgttcgatgcttcccgggccatttcctgttgtgccgctgcaatctgacgcgttgcattgataccgaccggtaaacgggacatcgctcc gcttgcatccatcaccatattaccgtcacgggtctgtgtggcagttgcgccactgccggtctggtacatcatctgaccaaacgacgtggtgctgtt ggtgctccagctgaagccgttcacgttttccgtctgcatgttgccgtaggagtaattaccgtcaaccacactgccagctgcactggccgtcggact gatagaggaagaggcgaagtggctgtacacgctggaaaaacctgcgccaaggccttttaccatcatccatgacaacggggggatcatggct gaaagatacccggcagtagaggccaggtcagagtatttcagctgtatctgagagagttcagacagcacgaccggcgcaccattcatcttcgc atagaatgtcatggcactgttcagaatggcatacaacaacggccaggtctgcagccacatcagggcaaacacatagcctttcaggacggac agtgtcagtttgttgaagacagctgccagtaccagaagcgggaaaataccaatggcaattcccgtcaggatagtctgtaccattggcaggtttc gcattgtcacatgaccgatagagacatgcgccagacgttgcttctccatcgatgacgtggtggccagattcaccaggctggcggtgtcaccatt acgggcggcattactggtgataccctctttcagggcattcatggtgacgttctggcgcataatctggctggcagactgactggagccgtagaaat aactgtaactgtcactgaccagttgtctgaataacaggtccgggtccggtctgccgccaaatatctgctgcacataataatgccaggtcttgcctc ccgtctttgtatcgagattcagcctgtctttcagcgtgaccgacgcatccttacaggttatgaaattattattgttgtcataaacgccccgtagcgggc tgggtcgggaaaaaatcagcgtgtaggggtcagcggaggccatcagatcttccagcgtgtatttatgattcaggtaaatatcacccagcacac agttctggacgtagtcctggaacagattgatgatttccggattccgggacaggaaatcggtgcttttcacaatcagattcgccccgaacagcatg cctgtcttgctgtaggtgacgctgtccggttgggtgaatatcatctcgtagctgaccaccatcgcatgcccgatacgggtcgtaagagaaagtgg catcgccagaccgaccggaacattatccacccgatggactttgaccaggtcactgttatcaataatctgcaccgatgtgcggacattaaccagc aggctgataagcacaaacacggccacccagcccagcaaatccatcacgttatgccgctgtacccacataaaagccacagcaacaacaga cagggtgagcgcgattttttcaattgaatcccaggtccgggtattcataaatgctgcaatggcattcaggttgttacgcaaccactcaccaccggc aatcacataaacttcattcacagtgaattccctccgaagtgatagttgttctggtaacggctgagcatacgggcggaaagctgctgacgcatgta gctcatctgacgttcgacaaccagcagcgcatcctgctggacctgtacctgactctggaaaatggcaatctgttgctgtgcctgattcagattctcc agaacattgtccatcacagcctgcggatagttcccggtggcaatcatggcccgggcctgctgaagcagttcctgaatgtactggagcatgatgt cgtagccgatatagtccgtcagctggtaaacgacgctgttggacacaccgagcatctgcgggtcaatcagatatttaaaaacgggaatggtgg tgctggaaataaacccctgttcccgggcgtcaagcggagtgtcactcactgctttattctgaatactggtgagcagttttgtaatctgtgacttcagg gctttatcccgggcgatggtcacatttgcatccgccaccaccttcagacacttgtccgaatcgttgcagtggtaaatttttgccgttcccccctccat aatcgcccggaggatatcgcggtcagtggtacgcgccggcaggggggtaatctcgctgtcttccccaaaaatcagcgtaccggtgagcgtca tgataaattctttcaactctttgttgccgtcaaacattctgttttttttcagcgcctcccatatgatattctggtttttcagcacccgctccttgtccttgtcgct 84 ME151672980v.1Attorney Docket No.: 139465-00120 ggctttgtcacgaacagatgaggactgccctccgaccgtgcacccctgacgagatgcggcccagtcggcgaagatattgctttcactgccaat gtcctgacaaactttctgctgagattcctgcgtctgagggaacagcccccccacaagcccctgtgccacctgacaggaactgaggttcatactg ttaatgtcacttgccattttctgcaggaagtcttttgcggtttttatttccggcaccgttgtctgcagggcaaggtcaaaaaagtaaccggcagcattg ctcataatctgcttaacaaaacgctgcaactgatcgccattaataaaactgaacgagccgagataggcgtcgatgccgccacatccggcattg atatccggcagcgtcatggaaatcagctggatatttttgacctgggtgcgggcatacagggagccaccataggcatacccggcggcctgccc ctgccagacgcccgcctgggtgacattggacgcaaatcccagcttattaaagaactgattcatgtcactgttcacatccgctgacgctgcgggt gtgaccgtcagtaaaacggcacaaagaaccagaagaggtttaatgcgaggcatcgttattcccctccctgaaactgttctcccggcgcatacg gcgtttcagccggaatatcctgatccccccgtttattaattcatttaaagggttcctgacgtatacgataagaaatccacctgcaaacgcccataa cgtatacggaatgacatacatcaggatgttccacatataacgaaaattatttaattccgggtcagctttccacgaatcaatacaggcatcaaaaa tactgtgcataatcacccataaaaaagttccggcagcaacgaacacaaaaaacagatcaaaatatatatcagaaaccctgcgttcataatca taattccgtttagtttttctgttttttagttccgcatcatttcttttttctttcattataagtttcctctgatatttctgatgattcattcagacaggttatgcggctgta atatgacaccgaaatccatcccacaattgcccccacaaaaaacacaacagaaacagaaaaggtaagacacagcgaaacgacaatttcc acagggctggcgggataaaaaagcccctgacggataaaggggatattcaggctgacgattattcccgtcgcgatgccggatattccggcaat gagtaacaccacctgcttatttcgcacctttttcctctccgtatatctgcagaacggtatccatccgcgccataaaactggcggcatccgttgcccc ctgcaaaagcggtaatgcctcaagtgtgttgacgttgaccaggaaggtggtcggtgtggccaccgggatattcgggaagaaggtctgcatcac gtccggtggcaccggtaatgcttccggaaaggcagtatccccctgaccatccagggtgtagggaaaaacggaaaagccgtactgctgtgcc agctgcttcagtaccgggtcaaactggtgacagtaagggcaacgcccctgcataaacagcaccaccttccagtcggccaggttaacctgcct gccgttgctgaggcggaaccagcggggagccggcttttctgttctggctgatgtgtctgctgccggttgtgcaggctgcacggccataccctgcg gattccagagtcgttcaatttcatcccgggtggaggcctgcacacctgtggccatcatcaacaaaatgaccggcagtaatttagtgagagacat gtccgccctcctgctgcccttccatcatggcctgttgcctggcttccagtgcctcacgctcttccaggctgacacgtgcaatccatgcatccagcac ctggtatgcgccgaacagcacaaggatggccgcaatcaactccgccagaaaaaaagccatccagggtttgctgtaaacaagacgggcga cgatatgaaaccagacacccagggaaaatacccacatacccgtaatatcaagccggggtaaagagaatctgcatttacttatcatgttaaact ccggttattatttacatgagaatattcatgtctgtaatattcgcgtttgtgtgatattaatcagatcaacctcattattttgtatggtttttattaccgttttatat gactcagcctgtcataagccccttataaaaaaccacatcactccggaccatgctgatgacaacataatacagataaatattcttctgaacctgtc gaagctcgtaacgttcggtaatgagccaatagccattggaacaaccattgctgcgccctgattcataattgcaccacacagaaaccagagact ggcggtaagcacatataaacacgcgatattaataacaaggaaaaccagtgaactcaggagaaacattttcctgtcattacgctcagaaaag aaataaccggcaataatgacaaatattaacagaacaacacctgtaaacattttcaaataatcttcactcataaatcaaatccgcgattaaaatttt ggtttaaaatcttcagaaacgttcaggaattgttttgccaggtcgtcctgcgaaataaagccgtatgataacgggcgaacactgccctgtttcggg tcaaccagcatcatggccgggaaatatttcacgccgaggcgctgcgcctgcccctggtcagtccgggaatccggcaacagcggattaatcac gccatccacggaaacgggaataacactcagaccatacgtatcccggaagccattaatgacctgcgccagctgcccgtcgatggggtcctgc ccccggtaaaaaaacatgatgccgtagtgttcagccagtttcgcaatggcctgtcgctgctgcgcctggtctgccgccagctggttccgtaccgt gccgttgtaatggctgtactgcaggttatagtccagttcgggatgtgccagcatggcctttttagcactcatggtgaaaagcccggcctgctgagt ccagtaattctgcagccggaaatatttcacaaaattatccacgccgggatacagaatggcttcgtacagcgcccgctttgttgccgtctgcagcg cggccagtttctgcataatatccggctcctgacggggggctgcaggtacaggtttattttctttttcctttggacttattttctcgttataccactgccagc ctgcatctttccctgacgccggaaaaataaagcagcagagtaacagtggcagtaatgctttattcatcttcattcctttcatcattttttctttcatcagc tttctgcaggccagtcagggcagaaaaacaaaaatacagaataaacaggagcacaccggagtaaaggataacgaaagcagcggcaag catcgcgtctgccgtggtttcttttaccgtatcccacgaccagataattaccggagaaatgacaatcgctgtaatcaaaagccggataagaaaa tcaacaagacgattagaagtgaatatgactttcatcatttcccccggttattttccatttcttactgtcctgcctgtttcagctggtcagcaatctgctcttt cactttctgcgtcagaacgccactgtccgggattttctggttattcatcaggtcttcgtagaagttagtgaaatccagtctgtcgaactgaattttctgc 85 ME151672980v.1Attorney Docket No.: 139465-00120 agctcatcaaccgtaatcccccgacagtcaggatgcttcgcactgccaaaactgatacgcaactgcccgttacgcccctgctgctggactatct gcgccagcttcgaatcaaactggcagtagctgcgctttttctgcagacagacacccagcactttccgggagcagaactcgcccacactcacc gtcagtttgttgtcctttgcctttcccagcgccttttcttcactgttgcacttcgccagccccacatcctgaccccagccgccatccttacagcagttact gaagccggctgcggccttacggcagaatttcgcctcacccgtgaaagccctgacatccacaccattcagggcggcaacatccttacctgcgg cggcaagcgcagccagctgtgatactgcttcaccaaaatcactgctttttccgctctgtgccttatcgcattcaccgtcaaggcagaagacatca ccaccacagaccatcacttttcctgatgtttttgactcgcaggagtacgtggcgtactcatgcagacaggctccctcatcggaataaaacgcgca ctgacgtgaggccagcgtacaggccggattatcgacatatttctgacaggtgccgttatctgcggactgagtgacatatttatcccggtatgccca gcatgcctgtgtgatgttccagggtttaccttccatcactccggttttcgtaccacccggctctgaacactcagtgcctgtcagcacgccatccgcttt attaaacggacaactctccacccactccacccggggaacccattctttatcacggaccttcatcctgagtttcagcgtaaaggtggaagcacca ctgacaagcgattcatagactttcctgtcaccattcccgtgcgggaggcaattaccgttagcagtacagccactaccggtcagaacctgcccct gtgtcacctgcaaacctgtcgccccggacagggggtaagtatccgactgcccgtttgccaggccgacattaaaggttgtattcatgaaggtata acgggagttcaggaagaaaaaagcggcgtatacagacaatgatgcactttccactgtaccggttaccggagccgtgacactgaacaccagt ttcagaccgttcatactgaaccggaactgtgagtgcggtatcgtcacctcacgatattcatccgtgtatttccagtccccggtgatactggcagtcc gtgtgcagtactcctccaccatcgtgtcccgctcacaggtatagtttgtgaactcactgcggtttatctgctgcgcactgcactgctgcccggtgtttc ccacaatgctgtcagcccggttcaccacatccctgccggtctggataaacggtgcatccggtgaaagaatgtctttcggcttgttcatgaatgact ctgtgatagtttttccggtttcaccggtcgcccattccgtggtgccgtcatttttcaggccgccatccccgccggcagtcacgccaccgtaatattttg tctcgtccggattcgcgttatagccggggatactctcctgtggcttaaagccctgaatactgcctgtaccctgcccctgaatctgacgggcaaaat cagaacccgcccggtaatcactgttgctgtcagcctgtgccacactgactgccagccccagcagcaatggcagaagacgtttcatacctcact ctccctctgatattgctcaaaccagaacacgactggtttgttcaccacctgaatcagcccgaaccgttctgcggtcctgaaattcacgctgcttctg cgcccccgttcaacctgcagggacacctgcgttctgaacatctccagcgaatatgttgttttcagcaggttgcacggcgcacaggccgggacg aggttttccggacagtcggcctccggtctgaaaacatcaccggtggatttcagcctgaaaatgcgcttttctgcagccttcatgcactgctctgact tgcggagcaccgcctgaacatggtcggcatgccatcctttctccggcagttcacagccgcagtacgcacaacgaccgccgaatttcatacgc agtttttcccgctgtttctttgtcagcgccatcaatgcctccttacgggaacagatttacggattcagtaagcggaccagcgtcctgaagtctgcac gcagtgagcgacgacagtaccggtgataccagtgatgagggccttcaggctgaagccggaaacagagctgaaggtcactgttaaaagaa gccccgacggaccagccccacagcaaccggggcatcatggactccgcatcctgccgcagtcgctgctcaagaagagccgccttttttaccg gacagacaaaggcaatataaggcggcagtttcgtaacagggaaaccatgcccctgacaggacgcatatgtacaaaatccgtgaacgttcat cgcggccaccagtgcacggatgtccgggtcaatccggtaatagcctgcaggtaaactcattgcgacttatccccggtgccgtccagcagtccc tgagccacctgccggcagtctcctgcggtggccactttttccagcgcctgcctgacgcgcagatttccccggatgatgtcgtgcccctggctgca gaacaccaccagggccggaacgctgcgaatgccgtactgtgaaaacagcgtcgggtcgatctgaacaccatcagttgcgccgtctttcacca gggacaacacggcgtcagccgtggccttcaggtcgttattcaccatgccccgcagcgtggcgggaataccgtagtgccgggtttcgcccagc attcgtttcagcccctcttcgggaatggaaaacgacacaaaatacagggcaccctgccggggttttccgtcctgcgaagcctgctgtttacgga ccagttcatcgagaaactggttatcggaatgctgaaggggattcgccctgacttgctgctctgcccatgcctgcaactgtgtgtccggcttttcacg taactgctgactcagtgtttcctgctgcttcaggaactggcggttttcagttgtgttcatattttctgaagcatgaacagagataaacagtcctgctgtc agggcgaataaaatgtatatttttttgttcatataattcttctgcaaacaggcagtaattaatcactacaaccaatatctgatatcaacaaccagcttc tcacggtatttcagggattattaatcccgggattacgcgcccggtcgcgttgtggtaaaatatttcagcctgatatatcgattgcaagagatgacag gctaaaaacataccaacaaggagttacaatgaataataacacatcagaagaccgccctgatttttcattcagttacatggacaatcagagagc agatgaaataaaggctttaaaaatattattattcagcatcatcagaaatatgcctcatgatacagccatcagtattattaataacatcgtaaatact gataataatgaggcgctcaatagattatgcctggagataactgacgccatgttaaacacaacgagaatcgttgattagcttctttccccatttatctt tttcctgagactggctgcaattaattgccagtctctgtattgctctgctgttattacctttttctccatactgaaagtgtcacggggtacagggagagat 86 ME151672980v.1Attorney Docket No.: 139465-00120 acatcctgatgcaaggggagctgcaggccacattgtttctgttctttttccatcatttatccttttcttgtcagtgttataaaaatacacagttacgttttcg cgtagttgtctgcttaaattttcctgctgcttcaggaattgtcagttttcaggagtgttcacattgtctgaggcatgaacagtgatatgcatccctgcag ccagggcaaaaagtacaaatattttccggctcatatcatctcccgacacagtgatataatttaattaccggcgcacttttctttcagtctctcagaca atgcatataattcatctatttcttcctgagttcagcccattctctgaccacatcaaaagcactcaccctttccagcagggagaaaaaacgctcctca ctgataaaaatttctttttctttattacttatctttcgtaaaaatgataaaaaactcataacacacttttatttatattttacatatgcacacacggttatattt atgagaaccgacagactcatttcctgcgcttaacagacataaacttatctgacgaaagagacaagacaacaacgcttaccactgtcagaata agaaccgtcttcagatacagtttgacaagcagtattttttccagtgtatctgcatcctgccaggcgttaacaatgtctgagataatatgttcagggca aagataaaaccacaccacagggcatatcaccatagcaaaaggaaacagttttaatacattgcctgtcaaacgaatgaacactcgggatata ctcatatacatatgaatgtatttactgcttaatttcttttccatttaaaatattcctctttttgtttaagttacagaaatacacagttacgtttacgccacatca ggtagccgaagtttttctttgtgttgggcggattttttcccgtctcccagcgcatcacactgcgcccgaacggatggcactgcccgctgtccggata catattcaccatctgataacgccagcgttctttgggcaggattggggacggatattcattacagacggcgttatttttcccgatggtttccataatcat gccctgacggtgtagtttgaacgccatgcgttcactgaccagcagggaggactgcaacggactggactcattactcacccagccattgaacg ggtacatacttccctgcgaaccggcacaccagaacagaacatcgagaggcatattaaaggcgctggcaatggcatctgctgcacaggccc cctgtgctatcggattggcaaagatgacagcttccggattgagaatggtggtcaggctgctgtccgtccaggtggggtcaatttcagaaagata agcgatatccaggtcaccaccttccagacagcccagcgatgtgatgatgttcagccagtacgtcagcgggtatttgtaccagtgaacgtgatag aacgccccgttcacctgcttctcatcctttttcgccgttccctgtgccgttttaccaaaagccggcaggctgaagcccaggttaaccatgcatcccg gtgaccgggtgacgtccgtcagcgccatcggctcccagtaaccaatggccagcccgatacgcctgaacagcggcggcggtgccggacaa atctgaatgggcatcgacgggttcgccgtgtcggggaccttaccctgactgactttgatactgcccagcgagagcgggaaaatacagctccag cagatatccgtgatcgggtttacaaaacgcccctcacaggcagaatccgcagatgccgcagggacatgaccggcgagaaggaataacatc agcagccacaggcttcgcttcattttctgccctcctctgccggaataaattccaccttcaggaagcgatcgccgggaacggcgctcacccgtgc cgggacctgatcaatgccgaggcgctggcagagcacaccgttctggtcaaagtaaatacggctgtccagcgctttctgcatctccgggatact gccctgcacgaggataattttgctctccagtgtgggcggcgtctggcgtttcatccaggccacctgcgccggatcatcgccattgatgaaataca gcgtctggttaaaaggaacatactgcagcggattcatcacttcaccctgacgggcaaacacccgcccttcgttatcccggatatccgcagcca gtctgacggacgggtcaaacaaccggctgctgtatttctccgtgcgtccgataccgggcacggcagggggccgcagactgttacggatcacc cgctccttaaaggcatccattttccggcccatctcaccggactgctccagtgccgtcaggcgctgcatgatcaccgtcagcatgtccggctccttt accggccacagatcgccccaggtgccaagatcggcggcggccacactctgcccccatatcagcagggcaatcagcccccggcatctcatg gttccgccctcattcgccgggcaatatcctgctggatttcccgggtgatatccggtgccccctgtaccacggcaggcgacaccagaatgaccgc gtggtgtttctgctgccatgcctgcaggctggcttccagtgccgtgttaaaacgcgctgaaagggcttttgactgtgcttctgacagttgtttctgact ggcgctgtcaaaaaacgcatccacggtctgtttcatgttgaaggcggcagtcaccggtgcgttcagtctgacaatgccataactgatggctgcgt tcagtaacaccattgccaggcatcccggcacggtccaccaccagtggctgcgtcggggggctgtaacgtcagtcgttttttgcgttgtggtcatg caacactcctgtatttctcatgttcttccagccaggcctccagcgaagccatctccggccccgatttcttccacgccagctgccacaccgcctcat gaatactcataccctctctgcgtttctgctgcacaaactcaaaatccgggccgtcagagctgtacatggcacggcttaacgggtccacaaacag acggtgccaggaggaatggttttccacctgcagcaggaaggaactgaaccactggtctttggccgcaccaaacttaccaatcatgtcgcgctg cagtggctggaactgatccgggaataactggttatatttcgcgaactccttcgcactctgtttgaggataattttgtaggaggagttaccccatgcc gcacgggctgcgctggatgccttgtcagagtcaaagtcgacgatgttctgtgtgatggtgatataggcaccggtatgacggcgacaggtgcggt aacctttttgaataaactcaccaactttacgatttttgaagtccagcagacgccagccttcatcaatgacgttcagtttcttgagggtacgcggcgtg cggtacatccggttctcgatgtaaatgataaggctgaacatcaccgcaaccagcagtgacggacggtcttccagtccgcccagctccagcac caccatttttgcgtcatcccgcagggacggctcatcagagttaaaataccggccgtaagtgccgttggcagtgtactggtcaagcagcacaatc atttcgtccagacggctgcggatggtcggtgactcgacatactggtcgttatcacgtgcgtttttcaggaaatccaccacatcatcaatgcgcgcc 87 ME151672980v.1Attorney Docket No.: 139465-00120 tgcttctttttagccagccaggaggctctgaccgcctgcagcaacagaccttcatgcacttcatccaggttaccgttggggctggccatcaccga caactggtcacggacacgctccgctgactggtcaatatcagtgatgttcgcaaacgggttaaagcgcagggtttcaccgtccagatatacgcc ccccatgttctcacacaaagacttgtagccatcccccatgtcgaacaccacggcaaagcccccggagtccagcacgctgcggataagtggct gtatcaacccggttttaccggccccggatgttccacagaccgccatgttgtagttggtgttattcatccccttaaagaagatatcgataaacgcca gctggttacggtaggtgggtgccagcaggcctgccggtgtcagggggttatccgccactaacggcatcaggttggccacattaaagctctctgc tcgctgaaccacaccggcctctttcagctgtttaaacagccctttcccggccataaagggcaggcaggtcaggaaattgcgcatgtggttaaag cgcggtgaaatcagctcaaaaccgtttttacgaaagctgttcagaatgtcctgctccacttccagcgccgtttcattgttgtccttgcagaatgctgt gatgttaaggaagtaggacacgacagaagactgaccggagcccagccgctggcgcagttccccccactccttagcttctttctccacggagg gaaaccatttggcataggaggtcttcgactttttctccaggtccatgtacttcaggttggcttcactgtgggttttcacctgatcttccaccaccagtgt cagcgtcaggatgaacgggcaggaaatggacatttccgggttcagcagattgctgtagttatcggccatgttccacaggaaggcgatttccgg gttacgggccagatggaaattcaggatacgggctgtgctgttcctgccgttctcacgcagccccagcgtcaggtaatccgcccggaccttcaga tcaaaactgtcctccacacactgataattcagatcagaatacgggtccaactgacgtcttttcgggtacagagagtccggattatggttaatcatc tccccgacaatctcgatgaaagcctgtgcatccactgtctgtgtggtgatttttgcgccgtgaaaagaggcacggatgattttcaccagattttccat ttccagaatgtcggcccggctttttttcttcgagggagaacagtacgagataaacacccggtaatgacgcagggtcaggggcagattcatcccc tccggcagcgggaactgtgtcgctgccgctttcatataataggcacgggtaatggcgttaaaccgttcagcctgttcacctgaccaggagaact cacgcagcccgtattcaatcctgtcaccaaccagctgactggacatcagatggatacacaacggaataccgcgcggcagtttggtgcgcagc atatgatcgagcgcctccacaatggactcattcgcaccattgatgggaatggcttccagcataaagcccatcgtggtgtcattcatgaacaggc cggactcctggttgtaatcacggtacggcagcagacggctgaactgcgggaagctcatttcgcccagaacttcattggcctttgcggattcgtca ggcagtttcagtgctgtgacgagggagttaaccgcctgagtgacggcctcaagtgggttattcacatgaattccttagttgaaaaataaagcga gaaatgcggtagccatgaagggaatacctgtaattaacgcgccatttcccggcgcatatacaaagatttcggttttctgaatatcaactcatgttc atccggaattaaatcaatatgttctgcgactgacttatgcataatgttttctctgcctttcctgatacgcctgacattcaacacataacgtcacaccgg gaaatattttccgccgggcttccggaataggattaccgcatgcttcacagagataaacaggaataccatgagcatttattttctggcgtatccggtt tattcctgtcatggtcagttgttctgttactgaatatgcttcatcgacttcatcactcacggaatattcctcccgacatgttctgacacgctaccgcccat cccttttcacgcacagggcactcgttttacaggcagattttgaatacagaaagcccgccggcattgccggctgatatacaacttcagggccagtt aattaatacgtggttttccccacgcagacggtttaataacaaaaaacacactggatggctgatgataaacatcctgattatcaatataaggcgct atccataatgcagccgtctgctcacccgttcttagcgggcggggaggtgttaccggcgcaactgaactgaccggaacaatcgttttcacttcccg ggcagccgtaaacaacggacgtggtgccagcagtttctgctccggaagtgccgtcaccgcaggtctgctgccggacggagtggtcgcggtg atggtctgcaccggcattgtccggaaattcccttcagccaggcgcggcagtgatgccgcaaccggctttgcttctgaggactgctccagttttttg gccttctcattggcctgctccatcgtcatacaggtatcggatgtggtggcgttacactcaaattccgtactggttccggcacagccggaaagtaat aaggtccccagcagaggaataaataaagaaatctgtttcattgtccctctcctttctcatcgcagttcacaaaatcattcttcaaatccctgaccgt acaggtttttacaatgtcaggattttcaaaaaccgtattccgtgatgatttataaacatatgccacaatgcgtttcccccccgcatcaggttctggtgc actgacttcacataacggaggtgttattccggattcatacagcaccggataatttatttttttgacgctgccatcagaaaccagcttattcatcgaag ggttccttatatttctgaataattctgtcgattagttctttcaccggaacagattttccgccaccggattgtccggcaaccacaatatgccccggaga agaaaataagctcatcgacattcccttcttcatcacaccgacagcagccatattaaaattgtgcggcaatgattcagaactgaattccttcggtgc aggaagtacgattcttgtggtgcagttactgagaacagcatgcataaaatcatctgatacgcttttatccggtacagagcgggcagtaataacat taatattacgcatgttcatttttatctccgttgtttctgtttgttctgtcatcaggaaaataaacagggcatttcctgattttttccggcagggaagcgtcttc cgtataaacttccgtcagacgttcatctgcggttaccccccggatacatttcatttttccgggcagcaccggatatttcccggagataactgccagt gagacatacatcaatgccagtgttgcccataatgcggtaaccagttgcatcagtgaccggtcagtctccctgacataatctgcagcatcagcag cacggtaacggcagattttctcttctgctcctggaagatcatgcagagaaggcaaacactctcttacaggaaactgcccggcaggggctgtttgt 88 ME151672980v.1Attorney Docket No.: 139465-00120 cttacgaagtcaatttcctgaaccagtaactgccctggcgtggtgttgtccttccataaaacaaacagagccatcagggccattgtggccagtgg cagaatgacggtgtacttcagacaccagaacagtcctcgcagaacccgggcgacaacataccgggcggcatggcacgcctgacgggac gacatattatttgccatcgttgccccctccctggctgccggacataaactgctgcggattcaggttgcccagctgcttcagcatctggtcagtattaa agccgttaagatgactttcagccgatggcggaacaggaaccggactttcctgattctcttcttccgctctgctctgcgtgcgttccagcgccatctct tccacggttttcagctggaagccgtcctggaacaccacggtcacttcgttgcccgcaccaatcggtatcaccgggtgatactgttcggcacgtttg atgtagtagtcactgagcgtctgtgcggctttcgatgcgccgccaccgatacccattttcaggacatcaccagccccgtaagcggctgtggcac ccagcccgacagccggctgggaggcgcgctccattccctgaccgataccgtcaacaaatcccgcgccccatgcccagccgaggattttgcc gttacgcatcaccacttcgcccttgataccgtttttaccccggaagctgacatgccccttaatcggcatatcaatggttttgccgtccttcaggcagc tgatattgcgggtgcgtacaatggcacgttcactggacacatccccccaggcttccagccccacaaaacatcccgttgcgtcatacgttttgctgt tcggcatttccaccagaccggtgatacgcagctgcatcggcaccgtggattcattaccggtgactgaggcattggcatccgctccttcaatcagc atggctttcgcaaaacttcctgacggaatgtacggcagcgatggtccctgttttccctcattacgggtaaacaccttacgctgtatccggttaggca ccggcacagactggtacgtcacctgtggtggtggcgtgacaccattcccgggataaaacgccgtcggcggtggaacagcaacgctgccctg cggcgggaaggatacgggggtgtttcctggctgtggttcgccttccggcccgggcggtgaggctggcgtctgcggtacaggctcaccggtcgc ggtgacgggatttgcacccagggcttttacctgctctgccagggcagcattatcctgtcccagcttttcaatacgacgctggtcatcaccgcgctgt ttgttcaggacatccagttcacgacggatttcctcatactgcttctgcatctgcgctgccgtcacctgcatctctgtggtggcatgctgacgcaccttg tcatcaaaggtcgtatccaccacaccggtcatatccggcacaggctcctgttcagccaccgcctcaccgttaccggccatgtccacatcagac agatacagtgcaccaccaatcgcggaggctgcaccgacaaccacaatccccagccacaggtactgcttgcgtttcacaatggtattgatactg gccatcagttgccctccccgtcacggatgacatacacatccatgcgcgcacctgccagtaactggctggcaggctggctgaacatcacggca cgggttcccggctgccagaaatcactttcccgaaggttattcaatgggcagacagcgttttattttccacggcaaagcggaccatcttcaggtgat tccccgtccatacggcatctgccactgaagacagccccgccggggcctgcagggtttcctttgtcacaggaacctgataccagcctgcaggta attttccgccacggacagcctggctgatggtcacaagcagggattcgtaaggcgtggacgtttcccacgcgccggcttcttctccggttccgcgc aggtcactgaccagctgaatggtacgccccgcaccttcacggggaacggcctgaatggaaaggttcagaccacgttctgtttccagaatgaa cgtaaagggctttttgttgacggtggcaaccaccacaccgccggaggcggtctgctcattattggtcagggcaccatccagactgttcacggcg ataatccggtcaccgggaacggtaaacagattcgggctggtattgctgatggcaatactggcctgaccaccgttcaccatggcacacacgtgg gggccagcgtaccgtttgcggccatcacgctgcangaaaaaaacagctgccgaatattattgcccgtatatttttctcatcgtctgtttcccgaaat tctcagcagtcccccgttttcccgctcagaataggaagtaatgttggattcaggaaaggtttgatccccatccggttttaagccccccagaatttca cacggcatcggaggcatacccggccctgttggaaaaagctgagttcagttatcgttttaatacgcttcgcttcttcagccagattaatttcatctggtt ctgtgcgcccgggcggatatattgcagaagcgcttgatgtgaggcatcgacggtttctgatgaaacattcagacggagggcaataaatgaca gcgccatctgctgtaaataagaggcgtcggcactgttctgtgacacggcaaagggcacattaaatcccattggggtcacggcagtgcgttgctc attctgaagacggtagttattcaccccctgaatgacgttaacagagaggctgagaacaataagcactgacataaatataaaggcgatggcca ttacacgactggtacttaaacgggcaccgtgttccatataacaatcctggtatcagttctatttaatccactgccggaaacacgaatcgggaacat tatgaaaaataccgcgcagcagggctgttggcatataccagtaaatcaggtcacgtaaccagtaactgcctcgcccttttttcagttttttaatccc gaaataaaccagaaccgctgcaccaataccgaacagatatttcgatgttgtgataccccagccaatacagattgcggcggggatcagttcatc cagtggcagggcaaaccagcggctctggttggtcagtgtttccgggaaacgatatttcttaagtttattctcgtctcccgacatcgttttatttcctgtc agaggccaacgacggccatacccacagcaataaatacagagatgatggcaaaaccggccaggaacttgacgtttttggtcatcatgtacat gaccgcaccgaccagaacttcagccagaacaacccatttaacaacactggagtccttaccgaaggtcgccttaaccgtggagttaccactct gcatcaggtcctgacctgccccctgggcggccagtgccagttgcgggaagaacatcatcagaacagcagcagggatcacagcgcgagcc aggcgaagcatattcagacgagtgaatttagaaaaaacgatttctttttgacgggcgcagaagcaccctgaacacttaaaacagtattcatatg attcctctgtttaatattgatagataaaaactaaagttctttaaatgttgactcgttctcttcgatcacctccctgaatatttcttcattatagaaatcagga 89 ME151672980v.1Attorney Docket No.: 139465-00120 aaccgtttcagatgatctctgagacggatctgaacctctatagttttacttctaccgctacgatctttggcccgaataagtcggttatttgtatcttcatc gagatacactgagactgttctgcttatcccgcctctcgcgttacgcctcctcactcaattcacctctgtttcccgactaataaaatcatgattgcaca gaaaccgacgaaaaaaaacacgccaaaattggcgtgccttatttaacatttacttaacaccataaaattcacgaattatacttaatagcgaatct atttctcttttatagatacatgacactctgaataaatcaaagtgttcaaaaaattcttttattttccgtttaactggtttaattcgatcctcagatgctctgtct gtaatacccaactctctggcaattgttgcgtgaccaacacccagcatataaaaagagaaaacaaccaatgtccctggagaaagatcattccat atcgttaatttatcagaagtatttgtattacttccactctgatttaacgcaaaaaatggataatcaggaaacggattaatttgccataaaacaacag attcgtcattataaaaagaaacattctccatccttatctgatagatttcgccatgaaaattaaaacgacgacaaaatgcagagccatgtccaaga gcctggcattccagttcaagtgatgaaagaaaaatttcaacctgcagacgtatataactctctccggataagggtttatcttctctggaaaagagtt caataaaagccccattggcataaaggattttccggtttttgttacggacacaaaccggaacgggagaatttctgtagatattctgaacgttaagg atgtatttatttacagattgactgttaagtggcctttctctacgatccagcgcacacataggaacctccgttaaggattctatggacagtcgatgcag ggagttcagacctccctgcatcggcgattttcagtttcaatttttaacgtggcattaaccacgtaattaatataacctttatgcggttaattgtcatcaa attgaaccagatcaaaatcctgttatttgaagcaaatcaacttttaattcttttttattcctcatcattttctggaaagaacctctccatctcagatgaca ccttatccctgatatcttcgaccatattcgcatattcaaactttggattcccggaaacatgagggcttagtgactcaatacctaaaatcttagcaact gttgactgtgtttttaccgcacattcaagaagtaccttattaaactcagcctgattgaacgctgactctttacgctccatctgagcctcatatacacga agccctaattcgagaagcatagtggatatgcttgagaaacttacatctgtacttttagctccttctgcacgtcttctttccacaattttatttattttataga caatttcgtcactgacataagcctgcactttcgccatatgtatacctctttattttcttatcgcatcaaatttatttcacatataaaaaaatgacaattcat cgatgaatcgaatcgtgacgcaagttacgaaaattacgattcatgcagcaaatcacatcacgaattgaatctagagtcaattcgctttaactcgtt gttttattattactgattcatctatgagttgcgttaaattcattggtgaatcatatgcgattcaccaatgaatcctttttataatgtaaaataaattaaaata cattatttaaaacataagttaatgattcaaatagcaaatcagcaaaaacttgtttttgcgtggggtgtggtgcttttggtggtgagaaccaccaacct gttgagcctttttgtggagtgggttaaattatttacggataaagtcaccagaggtggaaaaatgaaaaaatggatgttagcaatctgcctgatgttt ataaatgggatctgcgaagccgccgattgctttgatcttgcaggtcgggattacaaaatagacccggatttactgagagcgatatcatggaaag aatcccgttaccgggttaatgccattggtattaatccggtaacgggatatggcagcggactgatgcaggtagattcccagcattttaacgaactg gcccgctatggaattaagccggaacatctgacaacagatccctgcatgaacatttataccggtgcttattatctggcaatagcctttaaaaaatg gggagtctcctgggaggccgttggtgcatacaatgccggattcaggaagaccgaacgccagaaccagagacgtcttgcctacgcatcagag gtttaccggatttataccgggataaaaagcagtaaaggcatccgggttccgaccacgaagaaatcactttcccaaattaacagtgtgcagaac aattaatattgtatacattgtcaaataccggttatgccgcgacagcggcatggaaggccacacacaaaaatatcatatgagtgataatgctgac tggccgaagccccgcaaagcgggaccccatcttatatatatctggtaactacgcagcctcctgccagaccatttttcgcgggcacaatatttaca gactaccgaaaagccagttgatgaaattgaggcatatcagacgacacagcaatgccgcaggatgataatataagtaaagccccgtaaaaa ttttcggggctttacttatattatcatgacttccactccctgaactgtccggcaatcatccataatgctttgttgattcggatatcaccgtcaatgcctgta atggctcttgttctggtatttttaccggaggcacttcgccccgataatccaccttttatcatattctcctgaacccgctgccaggttgtccagagatcgtt ctgtttatcctcccaacggcggggagtaattatttgttctggcgtcactggcgttttattttcatcttcgtacctggccatcaatgcagctctgccaaata aatgttgttcgtcactgttaagatgtatctctttcattgcctccatattttcagtgactttatcaaagacaccgagcacttcatacgctccctcaataacc tgcccgacaatatcacctttatgtggaacgcggatttcgccaaaattattcccacacaccaggccatttgtgcagacaaaacgaaaaattcctg ggatcatctgataactggatgaaccatcatgtgaattaagcaggataatttcaggaacttctttcccgttaatatgcccttccctgcgaagacgtaa catatgtttactgtattcgcggcgtcctaaatcacgaacccgactctgacaggcaaagaacggctggaaaccttcatcacgtaacttattgatgat attgatggttgggatatatgtataacgctcactccgggattcatgtttatcaccagaaaagacagatggaacaaactgcattaattcgtcatccgtt aacgggcgctcacggcgaatagaattatatcgaccaaaacgggaagctaatcgcataacagtaactcctttatgaaggcataaaattaataa aagttttttcgcttaatatcatccccctcggttatcgttaacaaaaagacaacaattccgaggggaaggaccatcaaatcaggtcagttcgggac tgaccaacctgccgagtgaaaagcagtaacagcgcgatcaactcatcagcgttttttctgacaagaggagtatgattctccaggctatccagta 90 ME151672980v.1Attorney Docket No.: 139465-00120 agtcatgcagctcattattgacctggtggagcaccttaaagagtgcaccagaatcccggcagctctgatatgcaagatttgcctgaacagcacg attctgcatgatcccgatttgtgcgattgcgtctttctgctgatcgcctgttggctgataagtggtgttaaaagaaaatgcgctcataattcctcctgtat cgatgttttcagcccccatcatctgttgagtttcgggagccgcggttttgacgttttgcggggacctgccttttgttccctccggtattcaccatcacctg tttgaaaaccttcgccgcaaggggcatcactgccacggcctgacgcacctgtcctggggagaaagcaaaaatttttaccgggttttgggtgaa aaaatttgtgggctgttcatttaccctggacagtcagtgcggcaggttgtggctgtctatgcccccggcgacaggggcttaaacaggtgaatggc accggacagaacaacgggcagggacccgccgcaacgacgtgaagcgcggtttgcgaaacggcgttgcgccttacatcccggcggcctttt gccgggatgccgggagcgcaggccacggcgcgacagaggcggcgcgaccggaacggtcgctgcaagcccggggttttcacgcatcgg accgtagcgacagcccgaaaccgcgccagcgggttcggcggagactggcgggtgcgccagcagctgcctgtcggagtcgggcctgacga ccggatgccgccaggcagccgggagcgaaggtgcgcccggcagacaggacaacactgcagtagtcagaatcagagcggcaggaaaa agaagagcagtagcagtagcagtagcagtagcagtagcaggaacaacaacatcatcagtaacagacaaatgacaggcatgaaaagac agaagcacacagcagaacatccgcgtaatgaaaggggcggcgcgtaagcgcggcccctttcagcgccaccggcgtgctgtcttttcccggc gcgggcagacgggcatttatcctcccgcctgcccggcgtcttcaggcccgcagacaaataatcatctcggggcacgggtataagcaaacatg ccgcgcgacagctccccgggaatgcagaagacgccagcaacgtcttctgctggcgtgatttgttcagacctctgtcgaaccgatccatatacc attcagataagcatcaaatgcatcattcccctcctgcacttcctgcggctgaacaaggttcaggctttcgtgtgtccagccggcttcaggtgcaga cactttcagcagaagatgcccatcagaatcacaaatctccagatcagcaaacgtgttcccggtcaggatatctggttcaccgggtcgttgatctg taacatatgtgatatatcccatccattctctccacattaaatatttcagcactatccgggacgacggtgcggcccgattttcagtattccagagcaa gttcagccatcagagcatctttcgcttcccgtgccagcctggtttcttccatcgtcagaaaaagcccctttcgtgcagcagcaccaatgaataccg gacgatccttcagaggaagccgggaaagatgattcatcgccatatcaaacaaattcgcttcataaatttcatagtgcagaccacatccccaatg agcctgcttttccagcctgctgatcaccacatcagcccggataacatcaaagggattgcgggaatgaatattacgaataaagccatcggtacgt gctgctgacatatttctgcccatctttaatacacctctgttttttgcccccatcatcttttgagtttcgggagccgcggttttgacgttttgcggggacctg ccttttattccctccggtattcaccatcacctttttgagaaccttcgccgcaaggggcatcactgacacgacttgacgcacctgtcctgggcagaa cgcaaaattttttaccgggttttaggtaaaaaattttgtgggctgttcatttaccctggacagtcagtgcggcaggttgtggctgtctatgcccccggc gacaggggcttaaacaggtgaatggcaccggacagaacaacgggcagggacccgccgcaacgacgtgaagcgcggtttgcgaaacgg cgttgcgccttacatcccggcggccttttgccgggatgcagggagcgcaggccacggcgcgacagaggcggcgcgaccggaacggtcgct gcaagcccggggtttcgcgtcagggatggaagcccgccagggggacgcccggaaaagacacagccaggagaacaacaaaacagaca ataatcagggcagcagtaatcagagtcagaacaggaggaaaaagagaataatcaaagggaagggatgtgatttctgacaggtcataaac agagaaggcgggggaaaagaacgggcgccgcaggcgcgcgttcttccccgttcccggagggaaacggccttcctggcagtctggttgttca tgcccgcacaatgccagcaaggagaaaggggccaccggcgaaccagcagcccctttataaaggcgcttcagtgcagtcagaccagcatc agccctgaaaaggcgggcctgcgcccgcctccaggttgctacttaccgccggattcgtaagccattgttgccgtaacttccctgtatccgtccttc agccggacttcgcacaggcgattccgggtcaggagtgtgaatatcaacaacgtgcagcacacgattaatacgcaccagataagggcgtttcc cggtagtttcatggtttactcctccttgcaatttgcacggtaagaggccatactggtgttggttagacatacagtgggcctcgtgggttaatgaaaat taactacggggctttctgctttctgccacacaacacggtaacaaaccaccttcacgtcatgaggcaaaaagcctcaagcgccgggcacatcat agcccatatgtctgcaccctgtcagcatctgcaccgttctccgtcacgccccggatttccagccgacgcagagcactgaacagataatcgcatt cagcctgtttgtttgccctgaacggtttcgcgaccgtaaccacccactgatgatttccgggccagccatgtactttcctgacccgcccgttaatcgc atacagcaacccccagcccgccggtaaatcggccacggtaatcacccccggctcgcagagataaaaacgccagtctcccatgcccttttcc ggctctgaccggaacacctttttccggtcagccaggaaatcagaccgtgagcatttcacctcaatgagacatgacgctccgttacggaagccc agggcatcaggatgttcaccgcaggacgtccatgcgtgaaaacggtcactgaacaccaccttaaagccgttgttgcggagaaaacggcag gccagttcacaaagttccctgtgtgttttcgggtgtctggcgttctgcatcacgccacctcccggagtttgttcgggaccatccaccgctccatctca cggacagcgtgaccggcctcccttgccgccataccagcctctgcgatgacctgccagaatggcatatcctgccactgccagcgttccatcccg 91 ME151672980v.1Attorney Docket No.: 139465-00120 gtcagtgacggaaaacgctcgccccactgccagaacagggtttccaggtcatgctctgccacccggtcactccagcgtgcatgccagcttccg actgtttccggcgaatggaatgccagttcgatttcagcctgagcgaccagcgcctggtacgcacgggctttctgctctgcctccctcgcctgctga cggctgtacttctgcatgaccagtgcggatttatggcgactgcgttccctgacacgaaaccgcaccgacggaaacaaccgctctgctgcatca ccgggaaggggaaggaggcagacctcgccctgtgtttcaatcagcacatcgacggcatacagccactgaagacggttgccgtgcagttcttc tgcggtcagggacggcatgaaaaatcgcaggtcagacagcgatatcctgcggcttccgttcagacagcggaaaaacgcccccgcatacgg atattccgccagcgtgttaccctgatggcggcgtttttctgtctccgccaccgcctgccatgcggcctgttgctctgtgcagagaggaaccagtgc ctgtgaacgtacgctcatgcctgacttccttccgccatcaggatatggatgatgtcattaacgctgtacccctgcgcatccagtgacgccgccagt gccagtgaatggctgacagcttccggtgcaaaccgccgctgcacctgaaccaccgcaaacggctcaccaccggcattcaccagaaccagc acccagacctgagagacatcgcccggtgaacataacgccaggtgaaaacgttcgtgggcaggcgtgaaacggacctggacggggaaaa atccgccaaactcgctgccgtactcgccgttcatcgtccagttgtccggcgcatccagttcccgcatcacggcgtgtgtcagctcacgacgttcat cacttccggcagcccggatatcttcatattcctgtgcgctcatggtctgtaatacattcagggtcagttcagttttcatcacagtatctcccttgctgcc gccccgtctccggcggggcggtgtggttgttcatcaggcggcatcagccaggttatcagtgcgttcagtggtatctgtctgtggatgtggtgcaca catccagccaggcacccagcggttgtctttcatatggtgctctgcatgttcagccgcatcacctttcttcatcttctctgcatcccgtgcggcaccgg acagtccggcatcattcagggctgcgataatctgcggctttttcaggtgtccgaagaagtttgcttttgtcggctgccaccagtctcgcatgtgaaa gccgatggcggtttccagcgtgtcaagcggactgcgtgacgtgtgaccacactcacgggtctggacaccgttaaggctgcatgcggtgcaga aactgagcagggataacagcacctcctgactgagtgacaggaatgtcgtcatgtcccgtgaccatccctccggtaacagggcagcaagacg ggctttttctgccatcagcgccatgaatgcggcaccttccttccccgatggcgcatcgctggtcagcgaataatgtttacattccaggctgatttgtg ctggtttactgtacgctccgctgccaaacacattcaggcagagcgtccatgccagcagtgtcagggatttgtccggttgctgcatcagtgctgcct ggactgccagcgtgcgttcggaagacattttcgtcagcagtggcagactgatttcctccactgacgctttctcctgcacctgttccgtacggtcagc gtcgtcagccacgtcatcctcactgcgcaactgcacaccacgctggacacacacgttgccataacgccaggacaccaccactccacctccg gcacgcatctccggcgtccacgccctgaccttcgccatgcagcgcatcagcttcatttctgcttccagcaggtcagattcctcacactggttttcca gcgcgtcgtaacgcgtcatcagttcgttcagacgttcttcttccgcctccgtcagcaccgcttccggctccggcagacagcggtatgttccggcat cctcacggcactcaccgacaggctccatgcgtccggcgcaccattcccagccttcggcttcccgaaggaactctgcgacagcctggagtttttc cagcagggcggcatcgagcgccacgcagtccacatagccaccttcgtcgtcgctgaacaaatcggtgcgcagttcgtctggcgagaaggca tcagcccccacgaagcggaatttactgttccccgccaccgccacttcactttcggtcaccagacgacgaatggtctgtacttccggtttaccgcc ccatcccgactgacaggcggcttcaaacacctgcacctgacgcgcggtgtcgttctccagcgccagcgcctgacagtgttcggtggtgatgcg gtcttctgccagcgcatcgaggatgacaggcgcaaggtcagccagtttcagcattcgctgaacgtggcggggtgaatagcccagcaaatcac cgatttgtgcaggtgttttgccttcctgcgccattgcgcggaatccggcaatctgttcggcagggtgcatatcccgacgctgaccgttttcagtcatt gatgcagccgtcgccagctcctgcggaataaccttcacgcgtacaggccagtcagccggaaggatgccacgctctgccagcatgttgagtgc tgccagtcggcgaccacctgcggcgacaccgtggcggtcaccaggcagggcatgaacaaccagattctgcaacaggccaacgcccttaat ggactccgccagttcgctgacggactctgcggaatacggcaccgtgcgcacattcagcggtgatttaatcagcgaggccagcgacacgctca cttcctccgtctgctccagcagggcagacaggactgtttcctgcgtttttgcaggtttacggctggatttacgctccgtttttgtcttagactctgtaact gacatatatttctcctttgaccgggatggttgtatgtctgccggacgcgttcccgtccggcacctctcttacttaagaatgccgaaattccggatgac atggttcatccagtacccggcaccgtcctcaaaattccaggtacgccagaccaggccactgttattgcggacaaccaggcgaaaatgcgttcc ctgatcatcctcgataaagacattccggtactgtgcggcaacggcttcagcctgttcgcgggtaaaggagccgtcaggcagcccctgaaatatt ctgaaatattcgctcatcctcatatctccggtgatgccccgcactggacggggcggttgtcactgtctttcgttagaacggaatgtcgtcatcgaag tcatacgcgggttcagacggctgctgaggctccggctgcgcggccttacgtccacggcctttcgttttcgcgccaccttttttcggttccggctgtga ttccggctgaggctgaccgctgaactgttgcgcctcttccggctgagtctgagtacctgcggcacgtcccagcatctgcatggtgcccgtggtctt aacaagaatttcagtgacgtaacgggtgataccgttatcttcccagctacgggtacggagttgcccctcgatgtagacctgcgcgcccttgcgc 92 ME151672980v.1Attorney Docket No.: 139465-00120 agatattcaccagccacttccgcgagcttgccgaacagcaccacgcgatgccattctgtctgctcccgcatctcccccgtctgtttgtcacgcca gctttctgacgtggccacctgcaggtttgccactgcgcccccgtttgggatgtaacggacttccggatcttttcccagacgaccgacaagaatga ccttgttaatgccacgaactgccatgatgtgtctccttctgttgatgttttcagcccccatcatctgttgagtttcgggagccgcggttttgacgttttgcg gggacctgccttttgttccctccggtattcaccatcacctgtttgagaaccttcgccgcaaggggcatcactgccacggcctgacgcacctgtcct gggcagaacgcaaaattttttaccgggttttgggtaaaaaattttgtgggctgttcatttaccctggacagtcagtgcggcaggttgtggctgtctat gcccccggcgacaggggcttaaacaggtgaatggcaccggacagaacaacgggcagggacccgccgcaacgacgtgaagcgcggttt gcgaaacggcgttgcgccttacatcccggcggccttttgccgggatgcagggagcgcaggccacggcgcgacagaggcggcgcgaccgg aacggtcgctgcaagcccggggttttcacgcatcggaccgtagcgacagcccgaaaccgcgccagcgggttcggcggagactggcgggt gcgccagcagctgcctgtcggagtcgggcctgacgaccggatgccgccaggcagccgggagccgaaggtgcgcccggcagacaggac aacactgcagtagtcagaatcagagcggcaggaaaaagaagagcagtagcagtagcagtagcagtagcagtagcagtagcagtagcag gaacaacaacatcatcagtaacagacaaatgacaggcatgaaaagacagaagcacacagcagaacatccgcgtaatgaaaggggcgg cgcgtaagcgcggcccctttcagcgccaccggcgtgctgtcttttcccggcgcggacagacgggcatttatcctcccgccagccctgtgtcatc aggcacgcaaacgaataatcatcgtcgggacatcggtataagcaaacgtgccgcgcggcagctcctcgcggatgtcagaaaacggtaaca gcttctcctgctggcgcggcccgttcagacagacggcaaccagcacgccccccggacgcaacagggaaaaagcccgcaggatatgccg gatatcctgcccgtggatgaacggcggattcataatgatccggctgtaatactgcaccggctgccattccatgaagtcgccacactgtaccctg acaccgttaaaattttcccgcagataccggaccagcccgctgttaatctccaccgcatcacacatggcctccggcgcggtatcccgaatcgcc cgcagtatggccccggtgccggcagagggttccagaatgtgatcacggttgctgatttccgccagcgtcaccaggcgatcacagaccgggg caggagtcacaaaaagctgagattcagcccggatttcagcaaccggcgcggcgttatccagccttgcgcgcatcagccggatacgttctctta actgactgtgcatgtcattatctccgtcatcccccggggatttcccggggggatataccttatttctgtgggatttccgtgattttcatgtcggtaatata cacattcaccaggcggaaattattgtccattgtgcggcggtagcggtatgccccgtggtcttctgtctctgccacactgcgcacggccttacagtc ccggggcagggcggcccactgcgcctttgtcatttcccggaaaccgtcccccggatagttgactaccggcggacgcttcgcggcctgactggc gacggcagcctcttctgccgatggtgctttgtagtccgtgatgcgatcgggcgtcactttcatcgtgccgctgtacccgagaaaactgtaattcgg cgttgtgactgaactcaccgccccgttgcttttgttcacgcggatgatggtcagccactcgccccggctgaaaacctgtccgcccggctcaaaat cctgtgtccgggtaaccacgccgccgctttcgtccagcatggcacgctcatagatcaggcggttctgatagtggttaacccagcgttgctgatgct gaatctgccgttcatgacagcggatcgcaatttcgcgggcctgctccgtggtgatgatgtcatcatccagggccgaccataacgatcgacttcct tcatactggcttttttctgccgggcgcggatacctgtccagcgggaaacaggcgctgatatggtcatgactgctgatgagttttgccatattcagat ccagcgattcagcccgccacatcgtcagatatttctgcgactgtgcgatggtcttttccgccttgcgcaaatcagcttcgatttttttgatacgacgcc agcgaacgtccggacgttctttatacttcgcgtgaagcagtgccgaccgcgcccgctcttcccagtattccgcacgttcaaagagcatcacagc acgtttcataccgttttcaatacgctgtgcatcacgacgggcgcggcgttcgctgtgatgccccacaagaatgggctgaccgggcgggatcatc gcggccagtctttccacttcatcaagtgcctgtgcggattcactggcccgctttccgctgtatccggtaaaccgctccgcccgtgcttcctgacgttc agcgagcgtgctgtcttcatcctcaatctctccggcaagtgagaggagcacgtcttcccggcccggtgtccaggctggcgcgacaaacagcg cctgtttgggtgcccagcgaaaaccggcatcatgcacttttttatacgtctccgggtcaagacgtgacgcggcatacaggcgcagtttgttgtcat ccggtgagtaggtcgcgcggtacatctgcacagggctggcggcttccggggcggtcatggtgattgctgctaatgtcataaaatgttctccttccg tctgttgccgcccctcgcggggcggctcacctcatcagttaaaagcgcgtttcagttcgagtgtgcggagcacctgcccgaaggccgtgtttttca tggcggggatgtgtacaccacgcccccggtgaaaactccgggctatgtgtcgcccgaaggctttcagcacggggtcaatttccggcgcgggg atgtcttcgcgtctgaaacggggatgacggcggggctttacagccgctgcggaaggtgaaacaatacttattgtcataatttagcctcttatgacg attgactggagccgcaaaaactgttcgtgcagtctctgcggcatggattaagcaggttcctgacctgcttttttcatatctgcatctgacgctttccgg tgttttcctccttctgttcctgcgttctcattacctgccagccggatgactgacagaacacataaatgtgtattgccggagcctgaaataaatcctgat ttttaaagaacatcacccgttgagtttcgggagccgcggttttgacgtttcgcgggaacctgctgctgtattccctgtggtatgaacttcacccgtttg 93 ME151672980v.1Attorney Docket No.: 139465-00120 agaaccttcgccgcaaggggcatcactgccacggcctgacgcacctgtcctgggcagaacgcaaaattttttaccgggttttgggtaaaaaatt ttgtgggctgttcatttaccctggacagtcagtgcggcaggttgtggctgtctatgcccccggcgacagggactcatacgggtgtgtggcacccg gcaggcaatgagcaggggcccgctgcaacgacgaagagcgcgtgcgcgaaacggcgttgcgccttacattccggcggccttttgccggga tgccgggagcgcaggccacggcgcgacagaggcggcgcgaccggaacggtcgctgcaagccgggggttttcacgcatcggaccgtagc gacagcccgaaaccgcgccagcgggttcggcggagactggcgggtgcgccagcagctgcctgtcggagtcgggcctgacga...

Claims

Attorney Docket No.: 139465-00120 CLAIMS 1. A recombinant Salmonella bacterium, wherein the bacterium expresses lipopolysaccharide (LPS) O78 antigen of avian pathogenic Escherichia coli (APEC) and does not express Salmonella LPS O4 antigen.

2. The recombinant Salmonella bacterium of claim 1, wherein the bacterium comprises APEC’s rfbO78gene cluster.

3. The recombinant Salmonella bacterium of claim 1 or 2, further comprising a deletion mutation in the Salmonella rfbO4gene cluster.

4. The recombinant Salmonella bacterium of claim 2 or 3, wherein the APEC’s rfbO78 gene cluster replaces the entire Salmonella rfbO4gene cluster in the genome of the Salmonella bacterium.

5. The recombinant Salmonella bacterium of any one of claims 2-4, wherein the rfbO4gene cluster comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 4 or wherein the rfbO4gene cluster comprises the nucleic acid sequence of SEQ ID NO:

4.

6. The recombinant Salmonella bacterium of any one of the previous claims, wherein the recombinant bacterium further expresses one or more APEC virulence factors.

7. The recombinant Salmonella bacterium of any one of the previous claims, comprising one or more nucleotides encoding one or more APEC virulence factors.

8. The recombinant Salmonella bacterium any one of the previous claims, comprising one or more APEC plasmids that comprises nucleotides encoding one or more APEC virulence factors.

9. The recombinant Salmonella bacterium of claim 8, wherein the APEC plasmid is plasmid pAPEC-1, pAPEC-2, pAPEC-3, or a combination thereof, derived from APEC strain χ7122.

10. The recombinant Salmonella bacterium of claim 9, comprising all three plasmids pAPEC-1, pAPEC-2 and pAPEC-3.

11. The recombinant Salmonella bacterium of claim 9 or 10, wherein: (i) the pAPEC-1 plasmid comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 1; (ii) the pAPEC-2 plasmid comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 2; and / or (iii) the pAPEC-3 plasmid comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO:

3.

12. The recombinant Salmonella bacterium of any one of the previous claims, further comprising a deletion mutation in the lrp gene (Δlrp). 118 ME151672980v.1Attorney Docket No.: 139465-00120 13. The recombinant Salmonella bacterium of any one of the previous claims, comprising an asd gene (asd+).

14. The recombinant Salmonella bacterium of claim 13, wherein the asd gene is inserted into the pAPEC-1 plasmid.

15. The recombinant Salmonella bacterium of claim 13 or 14, wherein the asd gene comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 12, or the asd gene comprises the nucleic acid sequence of SEQ ID NO:

12.

16. The recombinant Salmonella bacterium of any one of the previous claims, wherein the bacterium comprises a pmi gene (pmi+).

17. The recombinant Salmonella bacterium of claim 16, wherein the pmi gene comprises a nucleic acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 11, or the pmi gene comprises the nucleic acid sequence of SEQ ID NO:

11.

18. The recombinant Salmonella bacterium of any one of the previous claims, wherein the bacterium further comprises, or is derived from a parent Salmonella bacterium strain comprising: a araC ParaBAD-regulated murA gene (ΔPmurA::TT araC ParaBADmurA deletion-insertion mutation); an insertion of a c2 gene operably linked to an arabinose regulatable promoter araC ParaBAD(araC ParaBADc2); a deletion mutation in the gmd and a deletion in the fcl genes (Δ(gmd-fcl));and a deletion-insertion mutation that inactivates the expression of a relA gene and inserts a lacI gene (ΔrelA::araC ParaBADlacI TT deletion-insertion mutation).

19. The recombinant Salmonella bacterium of any one of claims 1-17, wherein the bacterium further comprises: an araC ParaBAD-regulated murA gene (ΔPmurA::TT araC ParaBADmurA deletion- insertion mutation); an insertion of a c2 gene operably linked to an arabinose regulatable promoter araC ParaBAD(araC ParaBADc2); a deletion mutation of the gene cluster wza-wcaM (Δ(wza-wcaM)); a deletion-insertion mutation that inactivates the expression of a relA gene and inserts a lacI gene (ΔrelA::araC ParaBADlacI TT deletion-insertion mutation) a deletion mutation in the recF gene (ΔrecF); a deletion mutation in the sifA gene (ΔsifA); a deletion mutation in the waaL gene (ΔwaaL46); and 119 ME151672980v.1Attorney Docket No.: 139465-00120 a deletion in a pagL gene and an insertion of a rhaRS PrhaBAD-regulated waaL gene (ΔpagL::TT rhaRS PrhaBADwaaL).

20. The recombinant Salmonella bacterium of any one of the previous claims, wherein the bacterium is a Salmonella enterica bacterium.

21. The recombinant Salmonella bacterium of any one of claims the previous claims, wherein the bacterium is a Salmonella enterica subsp. enterica serovar Paratyphi A bacterium, a Salmonella enterica subsp. enterica serovar Enteritidis bacterium, a Salmonella enterica subsp. enterica serovar Typhi bacterium, a Salmonella enterica subsp. enterica serovar Typhimurium bacterium, Salmonella enterica subsp. enterica serovar Dublin, Salmonella Pullorum, Salmonella Gallinarum, or Salmonella enterica subsp. enterica serovar Choleraesuis.

22. A pharmaceutical composition comprising the recombinant Salmonella bacterium of any one of the previous claims, and a pharmaceutically acceptable carrier.

23. A vaccine comprising the recombinant Salmonella bacterium of any one of claims 1- 19 or the pharmaceutical composition of claim 22.

24. A method for eliciting an immune response against APEC in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 22 or the vaccine of claim 23.

25. A method for vaccinating a subject against APEC, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 22 or the vaccine of claim 23.

26. The method of claim 24 or 25, wherein the subject is a chicken, turkey, goose, or duck.

27. The method of any one of claims 24-26, wherein the pharmaceutical composition or vaccine is administered by spray or oral immunization.

28. A method for producing the recombinant Salmonella bacterium of any one of claims 1-21, the method comprising culturing the bacterium in media. 120 ME151672980v.1

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