RECOMBINANT ATTENUATED SALMONELLA VACCINES (RASVS) AGAINST INFECTIONS BY AVIAN PATHOGENIC ESCHERICHIA COLI (APEC)

Notice of Pre-AIA or AIA Status The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . DETAILED ACTION Claim Status Claims 1-20 are currently pending. There are no cancellations, amends or new claims. Claims 1-20 will be examined on the merits. Priority Acknowledgement is made of applicant’s claim for priority based on PRO 63/568,108 application filed 03/21/2024. Information Disclosure Statement (IDS) Accordingly, the information disclosure statement is being considered by the examiner. Claim Rejections - 35 USC § 103 In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status. The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action: A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made. The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows: 1. Determining the scope and contents of the prior art. 2. Ascertaining the differences between the prior art and the claims at issue. 3. Resolving the level of ordinary skill in the pertinent art. 4. Considering objective evidence present in the application indicating obviousness or nonobviousness. Claims 1-6, 13-14, 16, 18 rejected under 35 U.S.C. 103 as being unpatentable over Han et al. (Published 2018; hereafter Han; PTO-892) in view of Kong et al (Published 2010; hereafter Kong; PTO-892) and evidenced by Lukomski et al. (Published 1995; hereafter Lukomski; PTO-892). Han teaches that “Avian pathogenic Escherichia coli (APEC), a leading cause of avian airsacculitis and colibacillosis, is responsible for significant economic loss in the poultry industry. APEC serogroups O1, O2, and O78 are predominantly associated with disease. Lipopolysaccharide (LPS) O-antigen has been shown to be a potent antigen for inducing specific protective immune responses”, which is pertinent to claims 1-5. Han teaches that “a bivalent vaccine derived from attenuated Salmonella expressing O-antigen polysaccharide provides protection against avian pathogenic Escherichia coli O1 and O2 infection”, which is pertinent to claims 1-5, 13, 16, 18. Han teaches that “immunization of chickens with the bivalent vaccine elicited production of serum IgG and mucosal sIgA antibodies against the LPS of both APEC O1 and O2. Antibodies induced by the bivalent vaccine promoted opsonization, provoked complement-mediated bactericidal activity, and elicited protection against lethal challenge with both virulent APEC O1 and O2 strains”, which is pertinent to claims 1-5, 13, 16. Han teaches “The epidemiological serogroup distribution of APEC is not limited to E. coli O1, O2, and O78, but also encompasses a diverse range of less prevalent serogroups including O4, O6, O7, O8, O15, O18, O35, O53, O55, O65”, which is pertinent to claims 1-4. Han teaches that virulence determinants that support avian colibacillosis, such as adhesins (F1, P-fimbriae), iron acquisition systems (aerobactin and yersiniabactin), antibactericidal factors (lipopolysaccharide, K1-capsule, outer membrane protein, protein for increased serum survival), are viable targets for vaccine design, which is pertinent to claims 6, 18. Han teaches that “an ideal APEC vaccine candidate is not limited to a specific serovar; rather, must confer a broad spectrum of efficacy. Multivalent vaccine strategies employing oil-emulsified bacterin as well as fimbriae subunit-based vaccine have provided protection against APEC O1, O2, and O78 serogroups, which is pertinent to claim 1-6, 13, 16. Han teaches that O-antigen polysaccharide as the basis for vaccine construct against APEC infection in chickens because (1) LPS O-antigen is an important protective immunogen in all of the APECs, which is utilized in a vaccine candidate against E. coli O78; (2) the prevalent serogroups are O1, O2 and O78 worldwide, and O-antigen-based vaccine has the potential to induce cross-protection against heterologous strains in the same serogroup; thus, a multivalent vaccine comprised of O1, O2, and O78 O-antigens would provide protection against the most prevalent serogroups worldwide; (3) establishing the multivalent proof of principle, this platform is extremely versatile and can be readily modified to include additional O-antigen serogroups to tailor vaccine cocktails for local or emergent demand and regional diversity, which is pertinent to claims 1-2, 6, 13, 16, 18. Han teaches that the gene cluster specifying the production of APEC O1 O-antigen polysaccharide on the lipid A core was introduced into attenuated S. Typhimurium strain S739 (Dasd-66 Δcrp-24 Δcya-25), which express dual O antigen polysaccharides of homologous Salmonella O-antigen and APEC O1 O-antigen, and strain S740 (Δasd-66 Δcrp-24 Δcya-25 ΔrfbP45), which only expresses APEC O1 O-antigen polysaccharide, which is pertinent to claims 1-6, 13-14. Han teaches “in the case of immunization with MGN1180 (Δcya-28::rfbO78 Δcrp-28 Δrfc), in which single heterologous APEC O78 O-antigen was expressed on the surface, and MGN996 (Δcya-28::rfbO78 Δcrp-28), in which dual O-antigen polysaccharides were expressed”, which is pertinent to claim 2. However, Han does not teach that recombinant Salmonella bacterium does not express LPS O4 antigen as claim 1. Han does not teach the bacterium comprises APEC's rfbo78 gene cluster as claim 2. Han does not teach a deletion mutation in the Salmonella rfbo4 gene cluster as claim 3. Han does not teach the APEC's rfb078 gene cluster replaces the entire Salmonella rfbo4 gene cluster in the genome of the Salmonella bacterium as claim 4. Han does not teach the rfbo4 gene cluster comprises a nucleic acid sequence having at least 85% identity to the sequence of SEQ ID NO: 4 or wherein the rfbo4 gene cluster comprises the nucleic acid sequence of SEQ ID NO: 4 as claim 5. Kong teaches that regulated delayed expression of rfc enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines, which is pertinent to claim 1-5, 13, 16. Kong teaches that the Salmonella rfc gene encodes the O-antigen polymerase, which is pertinent to claim 1-5. Kong teaches that four LPS genes that have been shown previously to be useful deletion targets for Salmonella vaccines are galE, pmi, rfaH and rfc. Two of these genes, galE and pmi, are involved in synthesizing sugars required for core and/or O-antigen synthesis. GalE is a UDP-galactose epimerase that inter-converts UDP-glucose and UPD-galactose, an essential part of core sugar and O-antigen. This mutant synthesized core-defective LPS in the absence of galactose but made normal LPS when galactose was available in the growth media. S. Typhimurium galE strains are avirulent and immunogenic. However, this same mutation, transferred to S. Typhi, was not attenuated and was poorly immunogenic in humans, which is pertinent to claims 1-6. Kong teaches that Rfc (Wzy) is the O-antigen polymerase that, in conjunction with Wzx (transporter), Wzz (length determinant) and WbaP (O-antigen synthesis initiation), synthesizes, assembles, and transports the O-antigen to the periplasm, where WaaL (ligase) ligates O-antigen to lipid A to form complete LPS. rfc mutants exhibit a characteristic “semi-rough” phenotype, making lipid A and core capped with a single O-antigen subunit. An Salmonella Typhimurium rfc mutant administered orally to mice was avirulent and protective against oral challenge with wild-type S. Typhimurium, which is pertinent to claims 1-6, 13-14, 16. See for example table 1. Kong teaches that strains with mutations in rfc do not transiently express full length O-antigen and therefore may be impaired in their ability to invade and colonize host tissues effectively, and that a tightly regulated araC PBAD activator-promoter has been used extensively to regulate gene expression. The rfaH promoter was replaced with araC PBAD and the resulting strain exhibited arabinose-dependent O-antigen synthesis. The promoter substitution mutation was then introduced into attenuated S. Typhimurium strain χ9241 (ΔpabA ΔpabB ΔasdA). The resulting strain was found to be superior to a χ9241 ΔrfaH strain for delivery of the pneumococcal protein PspA, eliciting significantly greater protection against challenge with virulent Streptoccocus pneumoniae, which is pertinent claim 1-4, 20. Kong teaches that the rfc promoter was replaced with an araC PBAD promoter to create arabinose inducible production of Rfc and thus regulate rfc expression to mimic transient expression of smooth LPS, which is pertinent to claim 1-2. Kong teaches a series of rfc promoter mutations in vitro and introduced one of them into vaccine strain χ9241, and that the resulting strain was evaluated for its ability to deliver the heterologous protein PspA to mice, which is pertinent to claims 1-5. Kong teaches the chromosomal structure of the araC PBAD rfc mutant strains, deletion-insertion mutations resulting in arabinose-regulated rfc expression, which is pertinent to claims 1-5. See for example Figure 1; Table 1. PNG media_image1.png 1018 1059 media_image1.png Greyscale PNG media_image2.png 722 1104 media_image2.png Greyscale Kong teaches that Rfc (also known as Wzy) is the Salmonella O-antigen polymerase but does not make a reference to “Salmonella LPS O4 antigen” as claim 1. However, the O4 polysaccharide gene cluster of Escherichia coli is similar to the Salmonella as evidenced by Lukomski, which is pertinent to claims 1-5. Lukomski teaches “the identification of the O antigen polymerase (rfc) gene in Escherichia coli O4 by insertional mutagenesis using a nonpolar chloramphenicol resistance gene”, which is pertinent to claims 1-5. Lukomski teaches that “Computer analysis of the O4 polysaccharide gene cluster of Escherichia coli revealed the presence of two open reading frames (ORFs) encoding strongly hydrophobic polypeptides. O antigen polymerase, which is encoded by the rfc gene, is a potential membrane protein and therefore should be hydrophobic. To identify the rfc gene, these two ORFs were subjected to insertional mutagenesis. A chloramphenicol resistance cassette was designed which, when properly inserted, does not cause a polar effect in downstream genes. Each of two ORFs, cloned into a plasmid vector, was inactivated with this cassette. Two types of mutants bearing chromosomal insertions of the cassettes in each ORF were constructed by homologous recombination. These mutants were characterized by PCR, Southern blotting, and transverse-alternating-field electrophoresis. Only one class of mutants exhibited the expected O polymerase-deficient phenotype; they produced O4-specific, semirough lipopolysaccharide. Therefore, this ORF was identified as the rfc gene. The chromosomal rfc mutation was complemented in trans by the rfc gene expressed from a plasmid vector, which is pertinent to claims 1-5. Lukomski teaches “The O4 units consist of pentasaccharides containing N-acetylglucosamine, N-acetyl fucosamine, L-rhamnose, and glucose (1:1:1:2 molar ratio). The genes responsible for the biosynthesis of the O4 polysaccharide of a human uropathogenic E. coli have been cloned and expressed in E. coli K-12. Qualitative sugar analyses of LPSs in recombinant strains, as well as boundaries of the complete O4 region, have been established. On the basis of available sequence data, a methodology was developed to identify the gene encoding the O4 polymerase. Experiments on rfc identification, cloning, and expression. Structural and functional similarities between rfc genes and Rfc proteins of E. coli, Salmonella typhimurium, and Shigella flexneri”, which is pertinent to claim 1-4, 14, 16. SEQ ID NO: 4 was searched, and it was found that SEQ ID NO:4 is 99.83 % identical to a sequence in the genome of E. coli APEC O78 (accession CP004009.1), which is pertinent to claims 4-5. See Figure below. PNG media_image3.png 823 1270 media_image3.png Greyscale Therefore, it would have been obvious to one of ordinary skill in the art to modify the teachings of Han, Kong and Lukomski, thereby arriving at the invention of claims 1-6, 13-14, 16, 18, because Han teaches a bivalent vaccine derived from attenuated Salmonella expressing O-antigen polysaccharide that provides protection against avian pathogenic Escherichia coli O1 and O2 infection, and that O-antigen polysaccharide is the basis for vaccine construct against APEC infection in chickens, and a vaccine candidate against E. coli O78, and Kong teaches that regulated delayed expression of rfc gene, which encodes O-polymerase involved in the production of LPS O4 as taught by Lukomski, enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines. Thus, it would have been beneficial and desirable to improve the attenuated Salmonella strain of Han by expressing O-antigen polysaccharide to provide protection against avian pathogenic Escherichia coli O78 infection, and enhancing the immunogenicity and protective efficacy of the live Salmonella vaccine by altering the expression of the rfc gene as a way to control the production of O4 LPS as taught by Wong, it would been obvious to substitute these known equivalents; MPEP 2144.06. See MPEP 2144(II): “The strongest rationale for combining references is a recognition, expressly or impliedly in the prior art … that some advantage or expected beneficial result would have been produced by their combination. Claims 7-9 rejected under 35 U.S.C. 103 as being unpatentable over Han et al. (Published 2018; hereafter Han; PTO-892) in view of Kong et al (Published 2010; hereafter Kong; PTO-892) as in claims 1-6, 13-14, 16, 18, and evidenced by Lukomski et al. (Published 1995; hereafter Lukomski; PTO-892) and further view of Mellata (Published 2010; hereafter Mellata; PTO-892). Han, Kong and Lukomski teach all the limitations of claims 1-6, 13-14, 16, 18 as fully disclosed and incorporated herein. However, Han, Kong and Lukomski do not teach Salmonella bacterium comprising the APEC plasmid is plasmid pAPEC-1, pAPEC-2, pAPEC-3, or a combination thereof, derived from APEC strain X7122 as claim 7. Han, Kong and Lukomski do not teach recombinant Salmonella bacterium comprising all three plasmids pAPEC-1, pAPEC-2 and pAPEC as claim 8. Han, Kong and Lukomski do not teach SEQ ID NO: 1-3 as claim 9. Mellata teaches “the contribution to virulence of three large plasmids of avian pathogenic Escherichia coli X7122 (O78:K80:H9) and three plasmids, pAPEC-1 (103 kb), pAPEC-2 (90 kb), and pAPEC-3 (60 kb), from APEC strain χ7122”, which is pertinent to claims 7-9. The SEQ ID NOs: 1-3 were searched. It was found that SEQ ID NO: 1 is 100% identical to pAPEC-1 from E. coli strain χ7122, also known as E. coli APEC O78K80H9 (GenBank; accession number: CP000836.1), which is pertinent to claim 9. See Figure below. SEQ ID NO:2 is 100% identical to a plasmid sequence from E. coli strain χ7122, also known as E. coli APEC O78K80H9 (GenBank; accession number: FR851303.1), which is pertinent to claim 9. See Figure below. The SEQ ID NO: 3 is 100% identical to a plasmid sequence from E. coli strain χ7122, also known as E. coli APEC O78K80H9 (GenBank; accession number: FR851304.1), which is pertinent to claim 7-9. See Figure below. PNG media_image4.png 755 1191 media_image4.png Greyscale Mellata teaches that “in vivo assays showed that curing the three plasmids resulted in severe attenuation of virulence. The presence of different plasmids and combinations of plasmids resulted in strains with different pathotypes and levels of virulence, reflecting the diversity of APEC strains associated with colibacillosis in chickens”, which is pertinent to claim 8. Mellata teaches “results associated the decrease in growth of some strains in some media with the virulence of APEC, and the mechanism was associated with some combinations of plasmids that included pAPEC-1”, which is pertinent to claim 8. Mellata teaches “detection of adhesin genes (fimH, tsh, csgA, ecpA, pilS and stgA), iron acquisition genes (iroN, iucC, sitA, eitA, feoB, fepA and mntH), and genes encoding other virulence factors (traT, etsA, cvaC, ompT, hylF, and iss) in the different strains with either the wild-type χ7122 or E. coli K-12 χ6092 background was performed by PCR amplification using primers specified in Table 2”, which is pertinent to claims 6, 10. See for example, Figure 2. Mellata teaches growth curves for different strains in different media. The amounts of growth of wild-type strain x7122 and its plasmid-containing derivatives in different media (LB medium, MM9 medium, RPMI-1640, and GFTS-2 medium), which is pertinent to claim 8. PNG media_image5.png 930 1160 media_image5.png Greyscale PNG media_image6.png 916 1181 media_image6.png Greyscale PNG media_image7.png 878 1175 media_image7.png Greyscale Therefore, it would have been obvious to one of ordinary skill in the art to modify the teachings of Han, Kong and Lukomski, and Mellata thereby arriving at the invention of claims 7-9, because Han teaches a bivalent vaccine derived from attenuated Salmonella expressing O-antigen polysaccharide that provides protection against avian pathogenic Escherichia coli O1 and O2 infection, and that O-antigen polysaccharide is the basis for vaccine construct against APEC infection in chickens, and a vaccine candidate against E. coli O78, and Kong teaches that regulated delayed expression of rfc gene, which encodes O-polymerase involved in the production of LPS O4 as taught by Lukomski, enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines. Since Mellata teaches E. coli strain χ7122, also known as E. coli APEC O78K80H9 and its native pAPEC 1-3 plasmids, levels of virulence and attenuation of these plasmids in culture media and the implication of these results in chicken, it would have been beneficial and desirable to improve the attenuated Salmonella strain of Han by expressing O-antigen polysaccharide to provide protection against avian pathogenic Escherichia coli O78 infection, and enhancing the immunogenicity and protective efficacy of the live Salmonella vaccine by altering the expression of the rfc gene as a way to control the production of O4 LPS as taught by Wong, and the E. coli strain/plasmids and cultivation conditions taught by Mellata, it would been obvious to substitute these known equivalents; MPEP 2144.06. See MPEP 2144(II): “The strongest rationale for combining references is a recognition, expressly or impliedly in the prior art … that some advantage or expected beneficial result would have been produced by their combination. Claims 10-12, 15, 17, 19-20 rejected under 35 U.S.C. 103 as being unpatentable over Han et al. (Published 2018; hereafter Han; PTO-892) in view of Kong et al (Published 2010; hereafter Kong; PTO-892) as in claims 1-6, 13-14, 16, 18, and evidenced by Lukomski et al. (Published 1995; hereafter Lukomski; PTO-892) and further view of Baek et al. (Published 2009; hereafter Baek; PTO-892) and Curtiss et al. (US 10988729 B2; hereafter Curtiss; PTO-892). Han, Kong and Lukomski teach all the limitations of claims 1-6, 13-14, 16, 18 as fully disclosed and incorporated herein. However, Han, Kong and Lukomski do not teach do not teach a deletion mutation in the Irp gene (Alrp); an asd gene (asd+); and/or a pmi gene (pmi+) as claim 10. Han, Kong and Lukomski do not teach the bacterium further comprises, or is derived from a parent Salmonella bacterium strain comprising: a araC ParaBAD-regulated murA gene (APmurA::TT araC ParaBADmurA deletion-insertion mutation);an insertion of a c2 gene operably linked to an arabinose regulatable promoter araC ParaBAD (araC ParaBAD c2);a deletion mutation in the gmd and a deletion in the fcl genes (A(gmd-fcl));anda deletion-insertion mutation that inactivates the expression of a relA gene and inserts a lacl gene (ArelA::araC ParaBAD lac/ TT deletion-insertion mutation) as claim 11. Han, Kong and Lukomski do not teach araC ParaBAD-regulated murA gene (APmUrA::TT araC ParaBADmurA deletion-insertion mutation);an insertion of a c2 gene operably linked to an arabinose regulatable promoter araC ParaBAD (araCParaBAD c2);a deletion mutation of the gene cluster wza-wcaM (A(wza-wcaM));a deletion-insertion mutation that inactivates the expression of a relA gene and inserts a lacl gene (ArelA::araC ParaBAD lac/ TT deletion-insertion mutation) a deletion mutation in the recF gene (ArecF);a deletion mutation in the sifA gene (AsifA);a deletion mutation in the waaL gene (AwaaL46); anda deletion in a pagL gene and an insertion of a rhaRS PrhaBAD-regulated waaL gene (ApagL::TT rhaRS PrhaBAD waaL) as claim 12. Han, Kong and Lukomski do not teach pharmaceutical composition comprising the recombinant Salmonella bacterium of claim 1, and a pharmaceutically acceptable carrier, which is pertinent to claim 15. Han, Kong and Lukomski do not teach the pharmaceutical composition is administered by spray or oral immunization as claim 19. Han, Kong and Lukomski do not teach the method comprising culturing the bacterium in media as claim 20. Baek teaches that “Leucine-responsive regulatory protein (Lrp) is a global gene regulator that influences expression of a large number of genes including virulence-related genes in Escherichia coli and Salmonella”, which is pertinent to claim 10. Baek teaches that “the hyperinvasive phenotype of the Δlrp mutant may be useful to include in a live Salmonella vaccine, which is pertinent to claim 10. Baek teaches that “live attenuated Salmonella strains make good vaccines because they invade and stimulate host lymphoid tissues, inducing a robust immune response. However, the process of making a strain safe for human or animal use may reduce the strain's capacity to invade immune cells, thereby reducing its effectiveness at inducing a strong immune response. Inclusion of Δlrp in an attenuated vaccine strain may result in a safe vaccine better able to invade and colonize host tissues, thereby stimulating the desired immune response, which is pertinent to claim 10. Baek teaches the plasmid, pYA3337 which is asd-based cloning vector (pSC101 ori) with Ptrc promoter, Asd+, which is pertinent to claim 10. See for example, Table 1. Curtiss teaches compositions and methods for making and using recombinant bacteria that are capable of regulated attenuation and/or regulated expression of one or more antigens of interest, which is pertinent to claims 1-20. See for example, Abstract. Curtiss teaches “a recombinant derivative of a pathogenic bacterium, wherein the bacterium is a Salmonella enterica subsp. Enterica bacterium, and wherein the bacterium comprises an araC P.sub.araBAD-regulated murA gene; a deletion-insertion mutation that inactivates the expression of asdA gene and inserts a c2 gene; a deletion in a pmi gene; a deletion in a pagL gene; a rhaRS P.sub.rhaBAD-regulated waaL gene; a deletion in a wza-wcaM gene; a deletion-insertion mutation that inactivates the expression of a RelA gene and inserts a lacI gene; a deletion in a recF gene; and a deletion in a sifA gene”, which is pertinent to claims 11-13. See for example, claim 1. Curtiss teaches “the bacterium of claim 1, wherein the araC P.sub.araBAD-regulated murA gene is ΔP.sub.murA25::TT araC P.sub.BAD murA; the deletion-insertion mutation that inactivates the expression of the asdA gene and inserts the c2 gene is ΔasdA27::TT araC P.sub.BAD c2; the deletion in the pmi gene is Δpmi-2426; the deletion in the pagL gene and the rhaRS P.sub.rhaBAD-regulated waaL gene are ΔwaaL46 ΔpagL64::TT rhaRS P.sub.rhaBAD waaL; the deletion in the wza-wcaM gene is Δ(wza-wcaM)-8; the deletion-insertion mutation that inactivates the expression of the RelA gene and inserts the lad gene is ΔrelA197::araC P.sub.BAD lacI TT; the deletion in the recF gene is ΔrecF126; and the deletion in the sifA gene is ΔsifA26., which is pertinent to claims 11-13. See for example, claim 2. Curtiss teaches “the bacterium further comprises a gene encoding an antigen of interest operably linked to a repressor-regulatable promoter”, which is pertinent to claims 11-13. See for example, claim 3. Curtiss teaches the phenotype is regulated-delayed attenuation, and the gene conferring the phenotype is fur. The phenotype is regulated-delayed expression of an antigen of interest, and the gene conferring the phenotype encodes an antigen of interest. The phenotype is the regulated-delayed lysis in vivo, wherein the lysis is enabled to occur in a cytosol due to mutation in a sifA gene. The phenotype is regulated synthesis of O-antigen, and the gene conferring the phenotype is selected from the group consisting of waaG, rfaH, waaJ, wbaP, wzy (also known as rfc), waaP, waaO, waaF, waaP, waaC, waaA, waaL and wbaP, which is pertinent to claims 1-5, 11-13. See for example, column 5; lines 12- Curtiss teaches “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”, which is pertinent to claims 1-12. See for example, column 28; lines 34-37. Curtis teaches “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 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 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, which is pertinent to claim 11. See column 20; lines 61-67; column 21; lines 1-7. Curtiss teaches “a triple sugar regulated Recombinant Attenuated Salmonella Vaccine (RASV) strains. These strains deliver multiple conserved protective Salmonella surface/secreted antigens with their natural conformations to induce protective immunity against multiple virulent Salmonella serovars. As an example, the RASVs may have a rhamnose-regulated O-antigen synthesis, combined with a mannose-regulated O-antigen side chain synthesis to expose conserved inner core, and an arabinose-regulated production of Generalized Modules for Membrane Antigens (GMMA), or outer membrane vesicles, in vivo for enhancing production of conserved outer membrane proteins (OMPs). RASVs may be constructed in two Salmonella serovars, group B S. Typhimurium and group D S. Enteritidis, to express conserved immunogen genes and to maximize anti-Salmonella humoral, cellular and mucosal immune responses. The disclosed RASVs have rational design features different from other RASVs that enhance success. Specifically, the disclosed RASVs provide safe and highly effective Salmonella vaccines with low cost and can be used to develop S. Typhi or S. Paratyphi A RASVs for human use”, which is pertinent to claims 1-20. See for example, Column 4; lines 1-23. Curtiss teaches the method comprises administering to the host an effective amount of a pharmaceutical composition comprising a recombinant bacterium, which is pertinent to claim 15. See for example, claim 1. Curtiss teaches “a pharmaceutically acceptable carrier”, which is pertinent to claims 15, 17. See for example claims 11, 15. Curtiss teaches the pharmaceutical composition is administered to the subject by spray or oral immunization, which is pertinent to claim 19. See for example, claim 15. Curtiss teaches “an innovative RASV platform to overproduce protective Salmonella antigens in vivo. This system is a unique triple sugar regulated system, double shutoff of O-antigen synthesis by rhamnose and mannose and overproduction of GMMA or outer membrane vesicles by arabinose. It will also incorporate the RDA and RDPS systems. These systems will not increase the virulence (by the introduction of these self-antigens) because most of the antigen genes are not highly expressed in vivo (106). The overexpression of antigen genes will also attenuate the strain, as shown by overexpression of the flagellin gene. The virulence of strains with or without chromosomal mutations for these antigen genes when carrying antigen gene expression plasmids can also be evaluated, as discussed further below. In case the expression plasmid increases the virulence high enough to cause disease, the strain or the plasmid can be modified to guarantee the attenuation attributes. Levels of gene expression can be modified up or down, as necessary, by switching the sugar regulated promoters, altering promoter and Shine-Dalgarno nucleotide sequence and the spacing between these elements and the start codon of the regulated gene”, which is pertinent to claim 20. See for example, column 41; lines 64-67; column 42; lines 1-19. Curtiss teaches that “The strain requires 0.001% galactose in growth media to form complex LPS O-antigen in either Nutrient broth or LB broth”, which is pertinent to claim 20. See for example, column 53; lines 13-24. See also column 54; lines 16-35. Therefore, it would have been obvious to one of ordinary skill in the art to combine the teachings of Han, Kong and Lukomski and Curtiss and Baek, thereby arriving at the invention of claims 10-12, 15, 17, 19-20 because Han teaches a bivalent vaccine derived from attenuated Salmonella expressing O-antigen polysaccharide provides protection against avian pathogenic Escherichia coli O1 and O2 infection, and the need for a vaccine that can confer protection against E. coli O78 infections, and Curtiss teaches an innovative regulated recombinant attenuated Salmonella vaccine platform (RASV) to overproduce protective Salmonella antigens in vivo. In addition, Kong does teach that regulated delayed expression of rfc gene, which encodes O-polymerase involved in the production of LPS O4 as taught by Lukomski, enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines and Baek teaches that the Inclusion of Δlrp in an attenuated vaccine strain may result in a safe vaccine better able to invade and colonize host tissues, thereby stimulating the desired immune response. Thus, it would be beneficial and very desirable to combine the teachings of Han, Kong and Lukomski, and Curtiss and Baek, since the attenuated Salmonella live vaccines have shown to be successful to provide protection against bacterial infections, and the RASV platform of Curtiss can be modified to accommodate the production of one or more antigens of interest, such as LPS O4 regulated delayed expression of rfc gene from E. coli for enhancing the immunogenicity and protective efficacy of a heterologous antigen delivery by a live attenuated Salmonella enterica vaccines against E. coli O78 infections and Baek teaches that the inclusion of Δlrp in an attenuated vaccine strain may result in a safe vaccine better able to invade and colonize host tissues, thereby stimulating the desired immune response, it would been obvious to substitute/combine these known equivalents; MPEP 2144.06. See MPEP 2144(II): “The strongest rationale for combining references is a recognition, expressly or impliedly in the prior art … that some advantage or expected beneficial result would have been produced by their combination. Reasonable expectation of success would be expected because Curtiss et al. (US 10988729 B2; hereafter Curtiss; PTO-892) specifically suggests compositions and methods for making and using a genetically modified Salmonella capable of regulated attenuation and/or regulated expression of one or more antigens of interest and does not specify any antigen that should not be used. Additionally, KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007), discloses that combining prior art elements according to known methods to yield predictable results, is obvious unless its application is beyond that person's skill. KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007) also discloses that the combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results. In the instant case, all elements (i.e., the recombinant attenuated Salmonella vaccine (RASV) platform with regulated delayed attenuation and regulated delayed lysis in vivo attributes that synthesizes protective antigens to enable vaccination of avians, such as chickens and vaccinal antigens/immunogenic proteins from E. coli APEC species, such as E. coli O78 strain, the regulated delayed expression of rfc gene, which encodes O-polymerase involved in the production of LPS O4 to enhance the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines) were known in the art. In addition, combining these elements yields a method/composition wherein each element merely performs the same function as it does separately; thus, the results of the combination would be recognized as predictable to one of ordinary skill in the art. Therefore, the claimed invention is prima facie obvious in view of the teachings of the prior art, absent any convincing evidence to the contrary. Conclusion No claims are allowed. Any inquiry concerning this communication or earlier communications from the examiner should be directed to PRICILA HAUK TEODORO whose telephone number is (571)272-2784. The examiner can normally be reached 6:15-3:00. Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice. 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If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /PRICILA NMN HAUK TEODORO/Examiner, Art Unit 1645 /HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684