A process for preparing lactobacillus-based recombinant vaccine candidate against multiple salmonella serovar in poultry

Abstract

The process comprises providing a recombinant vaccine construct, wherein said construct comprises genetically modified Lactobacillus plantarum NC8 as a live vector; modifying the genetic structure of said Lactobacillus plantarum NC8 to express conserved Salmonella antigens, including PagN, SopE2, and FliC, anchored by a ptrk 892 backbone with a constitutive promoter, phosphoglycerate mutase (PGM); incorporating Signal Lp_2145 and cAM12 Anchor sequences into said genetic construct to enhance surface expression of recombinant proteins on Lactobacillus plantarum NC8; administering said recombinant vaccine orally to poultry, leveraging the probiotic properties of Lactobacillus plantarum NC8 for effective colonization of the poultry gastrointestinal tract; inducing a prolonged and intensified immune response by ensuring sustained high-level expression of target antigens through the utilization of the robust constitutive promoter, phosphoglycerate mutase (PGM); and optimizing immunogenicity through the surface expression of recombinant proteins on Lactobacillus plantarum NC8, fostering a robust and precisely targeted immune response.

Description

[0001] A PROCESS FOR PREPARING LACTOBACILLUS-BASED RECOMBINANT VACCINE CANDIDATE AGAINST MULTIPLE SALMONELLA SEROVAR IN POULTRY

[0002] FIELD OF THE INVENTION

[0003] The present disclosure pertains to the field of veterinary medicine and biotechnology, specifically focusing on the development of vaccines for preventing Salmonella infections in poultry. More particularly, the invention relates to a novel process for preparing a Lactobacillus-based recombinant vaccine candidate targeting multiple Salmonella serovars in poultry. This invention encompasses methods and techniques involved in the synthesis, expression, and administration of the vaccine, aiming to provide an effective and innovative solution to mitigate the threat of Salmonella infections in poultry farming operations.

[0004] BACKGROUND OF THE INVENTION

[0005] Salmonella infections continue to be a serious hazard to the poultry business, affecting the health of the birds as well as the safety of consumers. This bacterial threat has a history of seriously infecting humans and poultry, which makes effective prevention measures imperative. Our novel vaccine addresses this urgent problem head-on, with an efficacy rate that exceeds 95% and the rare ability to stop vertical transmission. Our vaccine solves a significant need in the business by protecting not only the health of poultry but also the safety of poultry products, which are an essential source of protein for millions of people globally.

[0006] Developed using genetically modified Lactobacillus plantarum NC8, this innovative vaccine represents a breakthrough in poultry health. The objective of this report is to provide a thorough analysis of the synthesis, significance, clinical trials, and evaluation criteria supporting the patent application for this groundbreaking vaccine. The poultry industry faces significant challenges from Salmonella infections, resulting in economic losses and potential threats to human health through contaminated poultry products. Traditional vaccination strategies have limitations, necessitating innovative solutions. The use of Lactobacillus plantarum NC8 as the live vector offers unique advantages, leveraging its probiotic properties, safety, and effective colonization of the poultry gastrointestinal tract. The vaccine design, emphasizing oral administration and mucosal immunity, positions it as a comprehensive defense strategy against Salmonella. The inclusion of conserved Salmonella antigens in the genetic construct ensures broad applicability across various serovars.

[0007] The background of the invention revolves around the development of a groundbreakingrecombinant vaccine designed to address the significant threat posed by Salmonella infections inpoultry. This innovative vaccine utilizes genetically modified Lactobacillus plantarum NC8 as alive vector to express crucial Salmonella antigens, namely PagN, SopE2, and FliC. The geneticstructure of the vaccine is based on a plasmid with a ptrk 892 backbone, featuring a potentconstitutive promoter, phosphoglycerate mutase (pgm). To enhance immunogenicity, SignalLp_2145 and cAM12 Anchor sequences are incorporated, ensuring the effective surfaceexpression of recombinant proteins on Lactobacillus.

[0008] Poultry industry faces substantial challenges from Salmonella infections, leading to economiclosses and potential risks to human health through contaminated poultry products. Currentvaccination strategies have limitations, necessitating more effective and innovative solutions. Thechosen live vector, Lactobacillus plantarum NC8, offers probiotic properties, safety, and anexceptional ability to colonize the poultry gastrointestinal tract.The vaccine construct, anchored by ptrk 892, carries conserved Salmonella genes (PagN, SopE2,and FliC), ensuring its applicability across various serovars. The robust constitutive promoter, pgm, facilitates sustained and high-level expression of target antigens. Signal Lp_2145 andcAM12 Anchor sequences play a pivotal role in enhancing the vaccine's efficacy by driving thesurface expression of recombinant proteins on Lactobacillus. This strategic surface expressionoptimizes the interaction of antigens with the host immune system, fostering a robust andprecisely targeted immune response.

[0009] This innovative recombinant vaccine represents a significant advancement in addressing thechallenges posed by Salmonella infections in poultry. Its unique design leverages the propertiesof Lactobacillus plantarum NC8 and the strategic expression of key antigens, offering apromising solution to enhance poultry health, reduce economic losses, and mitigate potential risksto human health.

[0010] In view of the foregoing discussion, it is portrayed that there is a need to have aprocess for preparing Lactobacillus-based recombinant vaccine candidate against multiple Salmonella Serovar in poultry.

[0011] SUMMARY OF THE INVENTION

[0012] The present disclosure seeks to provide aprocess for preparing Lactobacillus-based recombinant vaccine candidate against multiple Salmonella Serovar in poultry.Leveraging the unique attributes of Lactobacillus plantarum NC8 as a live vector, the vaccine incorporates conserved Salmonella antigens — PagN,SopE2, and FliC — through a sophisticated genetic construct. This design ensures a broad-spectrumdefense against various Salmonella serovars, addressing the challenge of antigenic variability. Akey innovation lies in the use of the phosphoglycerate mutase (pgm) promoter for sustained andhigh-level expression of target antigens, intensifying the immune response. Additionally, SignalLp_2145 and cAM12 Anchor sequences are strategically employed for surface expression ofrecombinant proteins on Lactobacillus, optimizing immunogenicity. The emphasis on oraladministration, facilitated by Lactobacillus plantarum NC8, targets mucosal immunity and offersa more comprehensive defense against Salmonella infections in poultry. Beyond poultry health, the vaccine's potential to reduce zoonotic transmission adds a layer of versatility, positioning it asa valuable tool in broader public health efforts. This recombinant vaccine signifies a notableadvancement in poultry vaccination against Salmonella, offering a holistic and multifaceteddesign for enhanced efficacy and safety.

[0013] In an embodiment, a process for preparing Lactobacillus-based recombinant vaccine candidate against multiple Salmonella Serovar in poultry is disclosed. The process includes providing a recombinant vaccine construct, wherein the construct comprises genetically modified Lactobacillus plantarum NC8 as a live vector.

[0014] The process further includes modifying the genetic structure of IbeLactobacillus plantarum NC8 to express conserved Salmonella antigens, including PagN, SopE2, and FliC, anchored by a ptrk 892 backbone with a constitutive promoter, phosphoglycerate mutase (PGM).

[0015] The process further includes incorporating Signal Lp_2145 and cAM12 Anchor sequences into the genetic construct to enhance surface expression of recombinant proteins on Lactobacillus plantarum NC8.

[0016] The process further includes administering the recombinant vaccine orally to poultry, leveraging the probiotic properties of Lactobacillus plantarum NC8 for effective colonization of the poultry gastrointestinal tract.

[0017] The process further includes inducing a prolonged and intensified immune response by ensuring sustained high-level expression of target antigens through the utilization of the robust constitutive promoter, phosphoglycerate mutase (PGM).

[0018] The process further includes optimizing immunogenicity through the surface expression of recombinant proteins on Lactobacillus plantarum NC8, fostering a robust and precisely targeted immune response.

[0019] An object of the present disclosure is to enhance the efficacy of a recombinant vaccine against Salmonella in poultry. This is achieved through a unique approach utilizing Lactobacillus plantarum NC8 as the live vector, capitalizing on its probiotic attributes and proven safety in food and feed applications.

[0020] Another object of the present disclosure is to provide a broad-spectrum defense against various Salmonella serovars. This addresses the challenge of antigenic variability commonly encountered with traditional vaccine platforms.

[0021] Another object of the present disclosure is to adopt the phosphoglycerate mutase (pgm) promoter is intended to ensure sustained and high-level expression of target antigens. This feature aims to prolong and intensify the immune response, distinguishing the vaccine from conventional methods and enhancing its effectiveness.

[0022] Another object of the present disclosure is to incorporate Signal Lp_2145 and cAM12 Anchor sequences into the genetic construct represents an innovative strategy for the surface expression of recombinant proteins on Lactobacillus. This design is intended to optimize immunogenicity and overall vaccine effectiveness by facilitating enhanced interaction with the host immune system.

[0023] Another object of the present disclosure is to emphasis on oral administration facilitated by Lactobacillus plantarum NC8 underscores a focus on mucosal immunity. This approach potentially provides a more comprehensive defense against Salmonella infections, particularly within the gastrointestinal tract, enhancing the vaccine's efficacy.

[0024] Another object of the present disclosure is to beyond poultry health, the invention's potential to reduce zoonotic transmission of Salmonella from poultry to humans adds versatility and positions it as a valuable tool in broader public health efforts. Addressing concerns related to food safety and zoonotic diseases strengthens its significance.

[0025] Yet another object of the present invention is to deliver an expeditious and cost-effective use of Lactobacillus plantarum NC8 as a live vector that offers probiotic properties and safety, distinguishing it from traditional vaccine platforms. Surface expression of antigens enhances immunogenicity, resulting in a robust immune response. Oral administration simplifies vaccine delivery in the poultry farming industry for Salmonella infection prevention. Contribution to zoonotic disease prevention by reducing Salmonella transmission from poultry to humans highlights its broader impact.

[0026] To further clarify the advantages and features of the present disclosure, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which are illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail in the accompanying drawings.

[0027] BRIEF DESCRIPTION OF FIGURES

[0028] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read concerning the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

[0029] Figure 1 illustrates a flow chart of a process for preparing Lactobacillus-based recombinant vaccine candidate against multiple Salmonella Serovar in poultry in accordance with an embodiment of the present disclosure;

[0030] Figure 2 illustrates a vector design and an expression cassette;

[0031] Figure 3 illustrates a process flow of the disclosed invention;

[0032] Figure 4 illustrates expression vector, plasmid digestion, expression of recombinant protein in Lactobecillusplanterum after transformation;

[0033] Figure 5 illustrates (a, b) copper-colored liver with Hepatomegaly, (c) Fibrinouspericarditis;

[0034] Figure 6 illustrates swab taken from liver of challenged bird of control group grown on (a) XLD, (b) SS agar, and (c) confirmation of Salmonella by PCR;

[0035] Figure 7 illustrates a graph 1) indirect ELISA reactivity against Salmonella antigens represented by an OD measured at 450 nm, (a, b) Serum IgG and (c, d) Gut mucosal IgA binding endpoint titers after immunisation. Data shown represent mean OD450 nm values, and a graph 2) indirect ELISA reactivity against Salmonella antigens represented by an OD measured at 450nm (a) Serum IgG and (b) Gut Mucosal IgA binding endpoint titers, 70 days after immunisation; and

[0036] Figure 8 illustrates a histopathologic changes and immunohistochemical staining S. gallinarum-challanged chicks, 5 days postchallenge.

[0037] Further, skilled artisans will appreciate those elements in the drawings are illustrated for simplicity and may not have necessarily been drawn to scale. For example, the flow charts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present disclosure. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawings may show only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having the benefit of the description herein.

[0038] DETAILED DESCRIPTION:

[0039] To promote an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

[0040] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

[0041] Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0042] The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises...a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.

[0043] Unless otherwise defined, 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 invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.

[0044] Embodiments of the present disclosure will be described below in detail concerning the accompanying drawings.

[0045] Referring to Figure 1, a flow chart of a process for preparing Lactobacillus-based recombinant vaccine candidate against multiple Salmonella Serovar in poultryis illustrated in accordance with an embodiment of the present disclosure. At step 102, method 100 includes providing a recombinant vaccine construct, wherein the construct comprises genetically modified Lactobacillus plantarum NC8 as a live vector.

[0046] At step 104, method 100 includes modifying the genetic structure of IbeLactobacillus plantarum NC8 to express conserved Salmonella antigens, including PagN, SopE2, and FliC, anchored by a ptrk 892 backbone with a constitutive promoter, phosphoglycerate mutase (PGM). At step 106, method 100 includes incorporating Signal Lp_2145 and cAM12 Anchor sequences into the genetic construct to enhance surface expression of recombinant proteins on Lactobacillus plantarum NC8.

[0047] At step 108, method 100 includes administering the recombinant vaccine orally to poultry, leveraging the probiotic properties of Lactobacillus plantarum NC8 for effective colonization of the poultry gastrointestinal tract.

[0048] At step 110, method 100 includes inducing a prolonged and intensified immune response by ensuring sustained high-level expression of target antigens through the utilization of the robust constitutive promoter, phosphoglycerate mutase (PGM).

[0049] At step 112, method 100 includes optimizing immunogenicity through the surface expression of recombinant proteins on Lactobacillus plantarum NC8, fostering a robust and precisely targeted immune response.

[0050] In another embodiment, the method 100 further comprisespreventing vertical transmission of Salmonella in poultry using a recombinant vaccine, comprisingadministering the vaccine to poultry to induce immunity against Salmonella. Then, ensuring vaccinated flocks produce Salmonella- ee, eggs, thereby preventing vertical transmission of the pathogen. Then, conducting trials to validate the efficacy of the vaccine in preventing vertical transmission. Thereafter, monitoring vaccinated laying hens for the absence of Salmonella contamination in their eggs, compared to unvaccinated groups.

[0051] In another embodiment, the method 100 further comprises synthesizing a plasmid containing Salmonella antigen sequences, comprisingdesigning and codon optimizing nucleotide sequences encoding PagN, SopE2, and FliC antigens for expression in Lactobacillus plantarum, with strategic addition of Furin cleavage sites between each antigen. Then, synthesizing the codon-optimized expression cassette containing PagN, SopE2, and FliC antigens with Furin cleavage sites. Then, selecting the plasmid vector Ptrk892 as the backbone for the expression construct. Then, digesting Ptrk892 with BstXI and Notl enzymes to create compatible ends for ligation. Then, ligating the synthesized expression cassette into the digested Ptrk892 plasmid vector to integrate the cassette. Then, adding a His tag during ligation to facilitate validation of recombinant protein expression. Then, incorporating Signal Lp_2145 and cAM12 Anchor sequences into the genetic construct to enable surface expression of recombinant proteins on Lactobacillus plantarum NC8, enhancing antigen presentation and immune response induction. Then, verifying the constructed plasmid through sequencing to confirm correct insertion of the expression cassette and presence of the His tag. Thereafter, validating expression of the recombinant proteins through assays such as Western blotting and immunofluorescence, utilizing the His tag for detection.

[0052] In another embodiment, the design and codon optimization of nucleotide sequences are performed using bioinformatics tools to maximize expression efficiency in Lactobacillus plantarum, wherein the synthesized expression cassette is obtained through gene synthesis techniques utilizing commercially available services, wherein Ptrk892 plasmid vector is chosen based on its compatibility with Lactobacillus plantarum and suitability for gene expression applications, wherein the addition of a His tag during ligation enables purification and detection of the expressed recombinant proteins for downstream applications, wherein Signal Lp_2145 and cAM12 Anchor sequences are strategically incorporated into the genetic construct to promote efficient surface expression of recombinant proteins on Lactobacillus plantarum NC8.

[0053] In another embodiment, the method 100 further comprises transforming and expressing a construct in Lactobacillus plantarum, comprisingpreparing Lactobacillus plantarum cells and rendering them competent for transformation. Then, mixing purified plasmid DNA containing the construct with competent Lactobacillus plantarum cells and subjecting them to transformation techniques such as electroporation or chemical transformation. Then, allowing the transformed cells to recover in a suitable growth medium. Then, plating the transformed cells on selective agar plates containing appropriate antibiotics to select cells harboring the expression construct. Thereafter, allowing the transformed Lactobacillus plantarum cells to express the antigen sequences under suitable growth conditions.

[0054] In another embodiment, the transformation is achieved by subjecting the mixture of purified plasmid DNA and competent Lactobacillus plantarum cells to an electric field in the case of electroporation or utilizing chemical agents to facilitate DNA uptake in the case of chemical transformation, wherein recovery of transformed cells involves incubating them in a growth medium that supports cell growth and allows for the expression of resistance genes carried by the expression construct, wherein selection of transformed cells is performed by plating them on agar plates supplemented with antibiotics that selectively kill cells lacking the expression construct while allowing transformed cells to survive and grow, wherein expression of the antigen sequences by transformed Lactobacillus plantarum cells is achieved by providing suitable growth conditions such as temperature, pH, and nutrient availability conducive to protein synthesis.

[0055] In another embodiment, the method 100 further comprises detecting expressed antigens by Western blotting, comprisingextracting proteins from transformed Lactobacillus plantarum cells expressing the Salmonella antigens. Then, separating the extracted proteins using SDS-PAGE gel electrophoresis. Then, transferring the separated proteins from the gel to a nitrocellulose or PVDF membrane. Then, blocking non-specific binding sites on the membrane using a blocking agent such as BSA or milk. Then, incubating the membrane with primary antibodies specific to the Salmonella antigens. Then, washing the membrane to remove unbound primary antibodies. Then, incubating the membrane with secondary antibodies conjugated with enzymes such as HRP. Thereafter, detecting the presence of antigen-antibody complexes using suitable detection methods like chemiluminescence.

[0056] In another embodiment, the protein extraction is performed using methods such as sonication, mechanical disruption, or enzymatic digestion to release proteins from the transformed Lactobacillus plantarum cells, wherein SDS-PAGE gel electrophoresis is conducted to separate the extracted proteins based on their molecular weights.

[0057] In another embodiment, the transfer of separated proteins to a nitrocellulose or PVDF membrane is achieved through techniques such as electroblotting or capillary transfer, wherein blocking of non-specific binding sites on the membrane is carried out by incubating the membrane with blocking agents such as BSA or milk to prevent false positive signals, wherein primary antibody incubation involves incubating the membrane with specific primary antibodies raised against the Salmonella antigens of interest.

[0058] In another embodiment, the washing of the membrane is performed to remove unbound primary antibodies and reduce background noise, wherein secondary antibody incubation entails incubating the membrane with secondary antibodies conjugated with enzymes such as HRP to amplify the signal, wherein detection of antigen-antibody complexes is achieved using suitable detection methods such as chemiluminescence, fluorescence, or colorimetric assays.

[0059] The synthesis of a plasmid containing Salmonella antigen sequences involves a multi-step process aimed at optimizing expression and ensuring efficacy: Firstly, nucleotide sequences encoding PagN, SopE2, and FliC antigens are meticulously designed and codon-optimized for expression within Lactobacillus plantarum. To enhance processing efficiency, Furin cleavage sites are strategically introduced between each antigen. Next, the codon-optimized expression cassette, encompassing the aforementioned antigens along with Furin cleavage sites, is synthesized by GenScript Private Ltd. For vector selection and preparation, the plasmid vector Ptrk892, sourced from Addgene, is chosen as the backbone for the expression construct. Ptrk892 undergoes digestion with BstXI and Notl enzymes to generate compatible ends for subsequent ligation. Following this, the synthesized expression cassette is ligated into the Ptrk892 plasmid vector, previously digested with BstXI and Notl enzymes. This ligation step ensures the seamless integration of the cassette into the vector. During ligation, a His tag is appended to the construct, facilitating the validation of recombinant protein expression. The presence of the His tag enables both purification and detection of the expressed proteins. Additionally, innovative surface expression elements, including Signal Lp_2145 and cAM12 Anchor sequences, are incorporated into the genetic construct. These elements are specifically designed to promote surface expression of the recombinant proteins on Lactobacillus plantarum NC8, thereby enhancing antigen presentation and inducing a robust immune response. Finally, the constructed plasmid undergoes rigorous verification and validation processes. Sequencing is employed to confirm the correct insertion of the expression cassette and the presence of the His tag. Furthermore, expression of the recombinant proteins is validated through various assays, such as Western blotting and immunofluorescence, leveraging the His tag for detection. This comprehensive approach ensures the reliability and efficacy of the synthesized plasmid for subsequent applications in vaccine development against Salmonella.

[0060] The transformation and expression of the construct in Lactobacillus plantarum involves a series of systematic steps: Firstly, host cells of Lactobacillus plantarum are prepared and rendered competent for transformation, ensuring their receptiveness to the introduced genetic material. Subsequently, the purified plasmid DNA containing the desired construct is mixed with the competent Lactobacillus plantarum cells. This mixture undergoes transformation using techniques such as electroporation or chemical transformation, facilitating the uptake of the foreign DNA by the bacterial cells. Following transformation, the transformed cells are allowed to recover in a suitable growth medium, providing them with the necessary nutrients and conditions to recuperate and adapt to the genetic changes. Next, to select for cells harboring the expression construct, the transformed cells are plated onto selective agar plates containing appropriate antibiotics. This selective pressure ensures the survival and proliferation of cells carrying the desired genetic material. Finally, the transformed Lactobacillus plantarum cells are provided with suitable growth conditions to express the antigen sequences encoded by the construct. This expression step allows for the production of the target antigens within the bacterial host. Throughout this process, various techniques such as competent cell preparation, transformation (via electroporation or chemical methods), recovery, selection using antibiotics, and expression under suitable growth conditions are employed to facilitate successful transformation and subsequent expression of the desired antigens.

[0061] Detection of expressed antigens by Western blotting involves a series of steps: Firstly, proteins are extracted from the transformed Lactobacillus plantarum cells expressing the Salmonella antigens. These proteins are then separated using SDS-PAGE gel electrophoresis. Subsequently, the separated proteins are transferred from the gel to a nitrocellulose or PVDF membrane. To prevent non-specific binding, the membrane is blocked using a blocking agent such as BSA or milk. The membrane is then incubated with primary antibodies specific to the Salmonella antigens, followed by washing to remove unbound antibodies. Afterward, the membrane is incubated with secondary antibodies conjugated with enzymes such as HRP. Finally, the presence of antigen-antibody complexes is detected using suitable detection methods like chemiluminescence. Various techniques including protein extraction, SDS-PAGE, western blotting, antibody incubation, and detection are utilized throughout the process.

[0062] Bird immunization involves administering vaccines to birds to induce protective immunity against specific pathogens. This comprehensive process includes several key steps: Firstly, the preparation of the vaccine, which entails combining antigens of interest with adjuvants to enhance the immune response. Next, the selection of the appropriate route of administration, which can include subcutaneous, intramuscular, or oral methods. Dose determination is crucial and involves assessing factors such as bird species, age, and immune status to determine the optimal vaccine dosage. Establishing a vaccination schedule is essential, including determining the timing of primary and booster vaccinations to ensure sustained immunity. Throughout the process, monitoring of birds postvaccination is conducted to identify any adverse reactions and assess the immune response elicited by the vaccine. ELISA (Enzyme-Linked Immunosorbent Assay) is a widely employed technique for quantifying specific antibodies in biological samples. To estimate IgG and IgA antibody titers in immunized birds, ELISA involves several steps: Firstly, ELISA plates are coated with specific Salmonella antigens. Then, serum or mucosal samples are collected from immunized birds, followed by incubation of these samples on the antigen-coated plates to allow binding of IgG or IgA antibodies. Subsequently, the plates are washed to remove unbound components, and enzyme-linked secondary antibodies specific to bird IgG or IgA are added. After the addition of a substrate solution for colordevelopment, the absorbance of the colored reaction product is measured at appropriate wavelengths. Finally, data analysis is performed to quantify the levels of IgG and IgA antibodies in the samples.

[0063] To challenge immunized birds with a virulent Salmonella Gallinarum strain, several steps are involved: Firstly, the virulent strain is cultured under appropriate conditions to obtain a high-density culture. Subsequently, the appropriate challenge dose is determined considering factors like bird species, age, and strain virulence. The virulent Salmonella strain is then administered to immunized birds via suitable routes such as oral gavage or intramuscular injection. Post-challenge, the immunized birds are monitored for clinical signs of Salmonella infection, and their survival rates are assessed. Post-mortem examinations are conducted on deceased birds to evaluate pathological changes and bacterial colonization. These processes complement the synthesis of the plasmid, transformation, and expression in Lactobacillus plantarum, detection of expressed antigens, bird immunization, ELISA for antibody titration, and challenge with the virulent Salmonella Gallinarum strain.

[0064] Figure 2 illustrates a vector design and an expression cassette.

[0065] This groundbreaking recombinant vaccine introduces a novel design that utilizes genetically modified Lactobacillus plantarum NC8 to express key Salmonella antigens — PagN, SopE2, and FliC. The genetic structure incorporates a plasmid with a ptrk 892 backbone, incorporating a strong constitutive promoter, phosphoglycerate mutase (pgm). To enhance immunogenicity, the construct integrates Signal Lp_2145 and cAM12 Anchor sequences, ensuring the effective surface expression of recombinant proteins on Lactobacillus.

[0066] Poultry faces a substantial threat from Salmonella infections, resulting in economic losses and potential hazards to human health through contaminated poultry products. Acknowledging the limitations of current vaccination strategies, the need for more effective and innovative solutions is evident. The chosen live vector, Lactobacillus plantarum NC8, has probiotic properties, safety, and a remarkable ability to effectively colonize the poultry gastrointestinal tract.

[0067] The vaccine construct, anchored by ptrk 892, carries the conserved Salmonella genes PagN, SopE2, and FliC, ensuring broad applicability across various serovars. Sustained and high-level expression of target antigens is achieved through the implementation of the robust constitutive promoter, phosphoglycerate mutase (pgm). Further enhancing the vaccine's efficacy, Signal Lp_2145 and cAM12 Anchor sequences strategically drive the expression of recombinant proteins on the surface of Lactobacillus. This surface expression optimizes the interaction of antigens with the host immune system, fostering a robust and precisely targeted immune response.

[0068] This recombinant vaccine against Salmonella in poultry presented here exhibits a novel and multifaceted design that holds significant promise for advancing the field of poultry vaccination. At the core of its innovation lies the strategic use of Lactobacillus plantarum NC8 as the live vector, harnessing its probiotic attributes and proven safety in food and feed applications. This distinctive approach diverges from traditional vaccine platforms, marking a paradigm shift in the quest for more effective and versatile solutions. The inclusion of conserved Salmonella antigens — PagN, SopE2, and FliC — in the genetic construct is a key feature that imparts broad- spectrum defense against multiple pathogenic Salmonella serovar. This addresses a longstanding challenge posed by the antigenic variability of Salmonella, providing a comprehensive solution that goes beyond the limitations of existing vaccines. Moreover, the adoption of the phosphoglycerate mutase (pgm) promoter ensures sustained and high-level expression of target antigens, contributing to a prolonged and intensified immune response. The incorporation of Signal Lp_2145 and cAM12 Anchor sequences into the genetic construct represents an innovative strategy for surface expression of recombinant proteins on Lactobacillus. This optimization of immunogenicity is a critical aspect of the vaccine's design, further distinguishing it from conventional approaches. The emphasis on oral administration, facilitated by Lactobacillus plantarum NC8, underscores a deliberate focus on mucosal immunity, potentially offering a more comprehensive defense against Salmonella infections. Beyond its impact on poultry health, the recombinant vaccine exhibits potential in reducing zoonotic transmission of Salmonella from poultry to humans. This layer of versatility adds a public health dimension to its significance, positioning it as a valuable tool in broader disease prevention efforts. The ease of oral administration, coupled with the safety profile of Lactobacillus plantarum NC8, contributes to its practicality in real- world poultry farming scenarios. The vaccine's potential becomes even more apparent when considering the limitations of existing Salmonella vaccines. The prevailing focus on S. Typhimurium and S. Enteritidis leaves a critical gap in protection against other relevant serovars, particularly those with implications for human infections. The recombinant vaccine, with its unique genetic construct and emphasis on conserved antigens, serves as a pioneering solution to this limitation.

[0069] In large-scale vaccination trials, the observed homologous immunity, and the reduction in detection rates of S. Typhimurium and S. Enteritidis highlight the potential impact of the recombinant vaccine. By addressing the challenge of cross-immunity between different serovars, it offers a more comprehensive and broadly effective solution. The utilization of Lactobacillus plantarum NC8 as the live vector provides advantages in terms of safety, effective colonization of the poultry gastrointestinal tract, and leveraging probiotic properties. The recombinant vaccine presented here represents a pivotal advancement in the field of poultry vaccination against Salmonella. Its innovative design, incorporating unique genetic elements and leveraging the properties of Lactobacillus plantarum NC8, positions it as a promising candidate for the effective and safe prevention of Salmonella infections in poultry. Beyond poultry health, its potential to contribute to public health by minimizing the risk of zoonotic transmission underscores its significance in the broader context of disease prevention and control.

[0070] The invention described herein represents a revolutionary development in the field of poultry vaccine technology, specifically designed to combat Salmonella infections in poultry. Salmonella is a common pathogen of great concern in the poultry industry due to its adverse effects on poultry health and potential risks to consumers of poultry products. Salmonella can cause serious illness in poultry and humans, requiring effective preventative measures to protect public health and maintain the integrity of poultry production.

[0071] • Vaccine Development: Current innovation is a meticulously designed recombinant vaccine aimed at preventing Salmonella infections in poultry. The development of this vaccine is driven by the urgent need for a safe and effective solution to combat Salmonella infection and its impact on poultry.

[0072] • Outstanding Efficacy: This pioneering vaccine has demonstrated outstanding effectiveness, consistently achieving success rates higher than 95% in rigorous clinical trials. This exceptionally high level of efficacy highlights the potential of vaccines to protect poultry health and, therefore, ensure the safety of poultry products for consumers.

[0073] • Preventing Vertical Transmission: Vaccines have been shown to be effective in preventing vertical transmission of Salmonella in poultry. Eggs from vaccinated flocks remained free of Salmonella contamination, highlighting the ability of vaccines to protect current and future generations of poultry.

[0074] • Clinical Trials: Extensive clinical trials are conducted on large-scale poultry farms involving broilers and layers to test the effectiveness of the vaccine. The vaccination protocol involves vaccinating day-old chicks, followed by a booster dose on the seventh day. The birds are then challenged with various Salmonella serovars to evaluate vaccine effectiveness.

[0075] • Benefits of Vaccination: The vaccinated chicks showed no signs of systemic infection or carried Salmonella. This is in stark contrast to the unvaccinated control groups, who had systemic infections of the liver, spleen, and cecum.

[0076] • Scalability and Effectiveness: Mass testing with 30,000 chicks on various farms confirmed the vaccine's effectiveness. Randomly selected vaccinated chicks demonstrated robust cell- and humoral-mediated responses against Salmonella, demonstrating the scalability and effectiveness of the vaccine in other poultry facilities together.

[0077] • Protection against Lethal Doses: Vaccine shows impressive protection in excess of 95% against lethal doses of Salmonella serovars, including Salmonella gallinarum, S. pullorum, S. typhimurium, and S. enteritidis. This level of protection demonstrates the outstanding effectiveness of the vaccine.

[0078] • Salmonella Challenge Trial in Laying Hens: In a pivotal trial, 150 vaccinated laying hens are challenged with various strains of Salmonella. These vaccinated hens demonstrated resilience against these strains and consistently produced Salmonella-free eggs. On the other hand, eggs from unvaccinated laying hens contain Salmonella bacteria.

[0079] • Significance and Potential Impact: This invention represents an important step in the development of vaccines for poultry. Recombinant Lactobacillus vaccines, with their high efficacy and unprecedented protection against Salmonella, have significant implications for poultry health and food safety. It offers a promising solution to a long-standing industry concern, the threat of Salmonella contamination in poultry.

[0080] • Market Introduction: Inventors are excited about the prospects of commercializing this revolutionary vaccine, which has the potential to revolutionize poultry production, improving food safety and improve public health by reducing the threat of persistent Salmonella infections in poultry. This innovation represents significant progress for poultry and consumer welfare, as well as the long-term sustainability of the poultry industry.

[0081] Advantages of the Invention

[0082] This invention represents a revolutionary development in the field of poultry vaccine technology. This advancement in the form of a recombinant vaccine offers a unique solution to a long-standing problem, making it a strong candidate for a patent.

[0083] • Public Health Meaning: Salmonella infection in poultry poses a threat not only to poultry health but also to public safety due to consumption of contaminated poultry products. This invention directly addresses this public health problem, which makes it very important.

[0084] • Highly Effective: The vaccine has demonstrated a success rate of over 95% in clinical trials. Such a high level of efficacy is a strong point in favor of a patent, as it shows the practical utility of the invention.

[0085] • Preventing Vertical Transmission: The ability of the vaccine to prevent vertical transmission of Salmonella in poultry, ensuring that eggs produced by vaccinated flocks are free of contamination, highlights its long-term benefits for the poultry industry.

[0086] • Rigorous Clinical Trials: The rigorous clinical trials conducted on a large scale involving broilers and layers provide strong evidence of the vaccine's effectiveness and practicality.

[0087] • Scalability: The scalability of the vaccine, as demonstrated by successful testing on 30,000 chicks in various farms, underlines its potential to be widely adopted in poultry production settings.

[0088] • Protection against Multiple Serovar: The vaccine's protection against various Salmonella serovar, including lethal doses, showcases its versatility and potential for broad application.

[0089] • Improving Food Safety: The ability of vaccines to produce salmonella-free eggs from vaccinated laying hens highlights the role of vaccines in improving food safety, which can be of primary importance to consumers.

[0090] • Industry Impact: This invention represents a significant advance in poultry vaccine development, making it attractive from a market perspective. It offers a unique solution to a longstanding industry concern, with the potential to change the way poultry is raised.

[0091] • Commercial Potential: The marketing prospects of this improved vaccine are promising. It has the potential to revolutionize poultry production, improve food safety and improve public health by addressing the persistent threat of Salmonella infection in poultry, bringing commercial value to it. This vaccine has groundbreaking nature; high efficacy, scalability, and its potential to address a critical issue in the poultry industry and public health.

[0092] Application Area of the Invention Our product has a significant application in the field of the poultry industry. The poultry industry has long grappled with the pervasive threat of Salmonella infection, posing significant challenges to both the health of poultry and the safety of poultry products for consumers. Salmonella not only jeopardizes the well-being of poultry but also represents a critical concern regarding public health due to its potential to cause severe illnesses in both poultry and humans. Given these circumstances, the development of effective preventive measures has become an urgent priority.

[0093] Our vaccine addresses this pressing need by offering an innovative and effective solution for countering Salmonella infections in poultry. With an exceptional efficacy rate exceeding 95%, our vaccine not only safeguards the health and well-being of poultry but also ensures the safety of poultry products, which constitute a vital source of protein for millions of people worldwide. The extensive trials user conducted on large-scale poultry farms, focusing on broiler chicks and layers, have yielded highly promising results, underscoring the remarkable efficacy of our vaccine. Notably, the eggs produced by the vaccinated layers are found to be entirely free from Salmonella infection, signifying the prevention of vertical transmission of the pathogen. These field trials are carried out successfully and have provided us with invaluable insights.

[0094] Key findings from our research include:

[0095] • Absence of Systemic Infection and Carrier State:

[0096] Chicks vaccinated with our Lactobacillus-based recombinant vaccine showed no symptoms of systemic infection and are not carriers of Salmonella. In contrast, the unvaccinated control groups exhibited systemic infection of Salmonella in the liver, spleen, and caecum.

[0097] • Mass Trials on 30,000 Chicks Across Various Farms:

[0098] Mass trials involving 30,000 chicks from different farms revealed a robust humoral and cell- mediated response against Salmonella in randomly selected vaccinated chicks, underscoring the vaccine's scalability and effectiveness in diverse poultry farming environments.

[0099] • 95% Protection Against Lethal Doses of Salmonella:

[0100] Our vaccine demonstrated an impressive >95% protection rate against lethal doses of Salmonella serovars in the tested chicks, highlighting its robust protective capabilities.

[0101] • Salmonella Challenge Trials on 150 Layers:

[0102] Following vaccination, 150 layers are challenged with Salmonella gallinarum, S. pullorum, S. Typhimurium, and S. enteritidis. The vaccinated layers not only exhibited resilience against these strains but also produced Salmonella-free eggs. In contrast, non-vaccinated layers' eggs showed the presence of Salmonella.

[0103] These results mark a significant milestone in poultry vaccine development, as our Lactobacillus-based recombinant vaccine is the first to achieve such high levels of efficiency and protection against Salmonella. The implications for poultry health and food safety are substantial, and user are excited about the potential impact of this breakthrough in the field.

[0104] Industrial Application and Commercial Viability:

[0105] This Lactobacillus-based recombinant vaccine for poultry has significant industrial applications. Lactobacillus plantarum, a well-characterized and safe probiotic widely used in food and feed, serves as the live vector, ensuring commercial viability and ease of adoption in the poultry farming industry. The surface expression of antigens within this vector enhances their immunogenicity, resulting in a robust immune response. The oral administration aspect simplifies vaccine delivery, making it practical for large-scale poultry production. The vaccine's potential to reduce zoonotic transmission of Salmonella from poultry to humans adds a layer of versatility, positioning it as a valuable tool in broader public health efforts. The cost-benefit analysis indicates substantial benefits, including a remarkable 95% protection rate against various Salmonella strains, which enhances poultry health, reduces economic losses, and contributes to food safety. Ensuring a consistent availability of a stable and accessible supply chain for raw materials and components is crucial for the widespread adoption of this innovative poultry vaccine. The stability and accessibility of the supply chain for raw materials, especially those required for the genetic modification of Lactobacillus plantarum NC8, need to be carefully considered. A sustainable and commercially viable supply chain will support the large-scale production and distribution of the vaccine, making it more feasible for widespread implementation in the poultry industry. 1. Patent Act Compliance:

[0106] The described invention, with its novel approach, significant efficacy, and industrial application, aligns with the provisions of Section 3 and 4 of the Patent Act 1970. These sections typically address the patentability criteria, including novelty, inventive step, and industrial applicability. The invention's compliance with these criteria positions it as eligible for patent protection, emphasizing its potential contribution to the field of poultry vaccination. Considering the global nature of poultry production and the potential international impact of the vaccine, it's crucial to evaluate the intellectual property landscape beyond national borders. A thorough analysis of existing patents and intellectual property considerations in relevant jurisdictions should be conducted to ensure that the patent filing strategy aligns with international standards and maximizes protection globally.

[0107] 2. Ethical and Environmental Considerations:

[0108] The invention, as described, appears to contribute positively to public health and food safety by addressing Salmonella infections in poultry. There are no indications of anything in the patent that is contrary to natural law, public order, morality, or creates serious prejudice to human, animal, plant life, or the environment. A thorough ethical and environmental assessment should be included in the patent filing documentation to address any potential concerns and demonstrate the responsible development and deployment of the vaccine.

[0109] 3. Future Technological Impact:

[0110] With its remarkable protection rate, versatility against various Salmonella strains, and potential to revolutionize poultry production, the vaccine under discussion has the potential to lead to a gradual replacement of current techniques in the manufacturing of chicken vaccines by new ones. Additionally, the revolutionary effect on public health and food safety suggests the possibility of more advancements and the creation of new technologies in the larger field of preventing disease. This future technological impact strengthens the case for patent protection, as the invention may pave the way for further innovations in the prevention of poultry diseases.

[0111] 4. Strategic Partnerships for Manufacturing and Distribution:

[0112] Considering the potential widespread adoption of the vaccine, establishing strategic partnerships for manufacturing and distribution will be crucial. Identifying potential partners with expertise in vaccine production, distribution networks, and regulatory compliance will strengthen the overall commercialization strategy. Collaborative efforts can expedite the availability of the vaccine to the market. A robust market entry and commercialization strategy should be outlined, considering factors such as target markets, pricing models, and marketing approaches. Clearly defining the value proposition of the vaccine, addressing market needs, and establishing a comprehensive go-to-market plan will contribute to the successful introduction of the vaccine into the poultry industry. Assessing the long-term sustainability and impact of the vaccine on the poultry industry, public health, and food safety is integral. This includes considerations of environmental impact, continuous efficacy, and potential for global impact position it as a strong candidate for patent protection. The inclusion of comprehensive documentation addressing the outlined considerations will further strengthen the patent application and support the successful development and commercialization of this groundbreaking vaccine against Salmonella in poultry. An in-depth analysis of the competitive landscape in the field of poultry vaccines, particularly those targeting Salmonella infections, is essential. Identifying existing patents, potential competitors, and their technological approaches will provide valuable insights. This analysis will not only inform the patent filing strategy but also help in positioning the invention within the context of existing solutions, highlighting its unique features and advantages.

[0113] This detailed and comprehensive analysis covers critical aspects to support a robust patent filing strategy, emphasizing the innovation's unique features, potential impact, and long-term sustainability.

[0114] Figure 3 illustrates a process flow of the disclosed invention. The presented invention introduces a groundbreaking approach to developing a recombinantvaccine against Salmonella in poultry. Leveraging the unique attributes of Lactobacillusplantarum NC8 as a live vector, the vaccine incorporates conserved Salmonella antigens — PagN,SopE2, and FliC — through a sophisticated genetic construct. This design ensures a broad- spectrumdefense against various Salmonella serovars, addressing the challenge of antigenic variability. Akey innovation lies in the use of the phosphoglycerate mutase (pgm) promoter for sustained andhigh-level expression of target antigens, intensifying the immune response. Additionally, SignalLp_2145 and cAM12 Anchor sequences are strategically employed for surface expression ofrecombinant proteins on Lactobacillus, optimizing immunogenicity. The emphasis on oraladministration, facilitated by Lactobacillus plantarum NC8, targets mucosal immunity and offersa more comprehensive defense against Salmonella infections in poultry. Beyond poultry health, the vaccine's potential to reduce zoonotic transmission adds a layer of versatility, positioning it asa valuable tool in broader public health efforts. This recombinant vaccine signifies a notableadvancement in poultry vaccination against Salmonella, offering a holistic and multifaceted design for enhanced efficacy and safety.

[0115] Figure 4 illustrates expression vector, plasmid digestion, expression of recombinant protein in Lactobecillusplanterum after transformation.

[0116] Live Vector Selection:

[0117] The invention employs Lactobacillus plantarum NC8 as the vaccine. This choice is based on the strain's proven probiotic properties, safety in food and feed applications, and its remarkable ability to colonize the poultry gastrointestinal tract. The use of Lactobacillus as a live vector sets this vaccine apart from traditional platforms.

[0118] Genetic Construct Design:

[0119] The genetic construct is designed to carry conserved Salmonella genes-PagN, SopE2, and FliC. This ensures a broad-spectrum defense against various Salmonella serovars, addressing the challenge of antigenic variability. The construct incorporates a plasmid with a ptrk 892 backbone, featuring a robust constitutive promoter, phosphoglycerate mutase (pgm). This promotes sustained and high-level expression of target antigens.

[0120] Enhancing Immunogenicity:

[0121] To enhance immunogenicity, the genetic construct includes Signal Lp_2145 and cAM12 Anchor sequences. These sequences play a crucial role in the surface expression of recombinant proteins on Lactobacillus, optimizing the interaction of antigens with the host immune system. This strategic surface expression fosters a robust and precisely targeted immune response.

[0122] Oral Administration and Mucosal Immunity:

[0123] The emphasis on oral administration facilitated by Lactobacillus plantarum NC8 is a key aspect of the vaccine's working. This approach underscores a focus on mucosal immunity, potentially providing a more comprehensive defense against Salmonella infections, particularly in the gastrointestinal tract of poultry.

[0124] Figure 5 illustrates (a, b) copper-colored liver with Hepatomegaly, (c) Fibrinouspericarditis.

[0125] Figure 6 illustrates swab taken from liver of challenged bird of control group grown on (a) XLD, (b) SS agar, and (c) confirmation of Salmonella by PCR. Figure 7 illustrates a graph 1) indirect ELISA reactivity against Salmonella antigens represented by an OD measured at 450 nm, (a, b) Serum IgG and (c, d) Gut mucosal IgA binding endpoint titers after immunisation. Data shown represent mean OD450 nm values, and a graph 2) indirect ELISA reactivity against Salmonella antigens represented by an OD measured at 450nm (a) Serum IgG and (b) Gut Mucosal IgA binding endpoint titers, 70 days after immunisation.

[0126] Figure 8 illustrates a histopathologic changes and immunohistochemical staining S. gallinarum-challanged chicks, 5 days postchallenge.

[0127] Zoonotic Disease Prevention:

[0128] The invention's versatility extends beyond poultry health by contributing to zoonotic disease prevention. By reducing Salmonella transmission from poultry to humans, the recombinant vaccine adds an extra layer of value in addressing public health concerns related to food safety.

[0129] The working of this innovative recombinant vaccine involves a carefully orchestrated combination of live vector selection, genetic construct design, and strategic emphasis on oral administration. The incorporation of unique features, such as Signal Lp_2145 and cAM12 Anchor sequences, contributes to the optimization of immunogenicity. Overall, the invention presents a comprehensive and effective solution for combating Salmonella infections in poultry while offering broader implications for public health.

[0130] The presented recombinant vaccine against Salmonella in poultry using Lactobacillus plantarum NC8 holds promising industrial applications across various sectors, contributing to advancements in animal health, food safety, and public health. Some of the key industrial applications include:

[0131] Poultry Farming Industry:

[0132] The primary industrial application lies within the poultry farming sector. The vaccine offers a highly effective and innovative solution for preventing and controlling Salmonella infections in poultry. Its unique design, including the use of Lactobacillus plantarum NC8 as a live vector and the incorporation of conserved Salmonella antigens, addresses the limitations of traditional vaccination strategies. This can result in improved poultry health, increased productivity, and reduced economic losses for poultry farmers.

[0133] Animal Health and Veterinary Medicine:

[0134] The recombinant vaccine contributes significantly to animal health and veterinary medicine. By providing a broad-spectrum defense against various Salmonella serovars, it becomes a valuable tool for veterinarians and animal health professionals in controlling infectious diseases in poultry populations. The focus on mucosal immunity and the oral administration method also align with practical and effective vaccination practices in the veterinary field.

[0135] Food Safety and Zoonotic Disease Prevention:

[0136] The vaccine's potential to reduce zoonotic transmission of Salmonella from poultry to humans has direct applications in enhancing food safety. Poultry products are a common source of Salmonella contamination, and a vaccine that mitigates transmission contributes to safer poultry products reaching consumers. This aspect is particularly relevant to the food processing and distribution industries.

[0137] Biotechnology and Pharmaceutical Manufacturing:

[0138] The production and commercialization of the recombinant vaccine involve biotechnological processes. The manufacturing of genetically modified Lactobacillus plantarum NC8, the formulation of the vaccine, and the large-scale production of doses represent applications in the biotechnology and pharmaceutical manufacturing sectors. This may involve fermentation processes, downstream processing, and quality control measures.

[0139] Research and Development in Vaccinology:

[0140] The invention serves as a model for advancements in vaccinology. Researchers and companies involved in vaccine development can draw insights from the unique features of this recombinant vaccine, such as the use of specific bacterial vectors, innovative genetic constructs, and strategies for optimizing antigen expression. This may inspire further research and the development of novel vaccines for other pathogens.

[0141] Public Health Initiatives:

[0142] The broader implications of the vaccine in reducing zoonotic transmission make it relevant to public health initiatives. Health authorities and policymakers can consider the use of such vaccines as part of comprehensive strategies to address foodborne illnesses and enhance overall public health. This could lead to collaborations between the veterinary and public health sectors.

[0143] The industrial applications of the recombinant vaccine extend across poultry farming, animal health, food safety, biotechnology, and public health. Its multifaceted design and potential to both poultry and zoonotic health concerns make it a valuable innovation with farreaching impacts in various industrial contexts.

[0144] The innovation in this recombinant vaccine against Salmonella in poultry is marked by several unique features, setting it apart from traditional vaccine platforms. The use of Lactobacillus plantarum NC8 as the live vector is distinctive, capitalizing on its probiotic attributes, safety profile, and ability to colonize the poultry gastrointestinal tract effectively. The inclusion of conserved Salmonella antigens — PagN, SopE2, and FliC — in the genetic construct ensures a broad-spectrum defense against various Salmonella serovars, addressing the challenge of antigenic variability. The adoption of the phosphoglycerate mutase (pgm) promoter ensures sustained and high-level expression of target antigens, prolonging and intensifying the immune response. Furthermore, the incorporation of Signal Lp_2145 and cAM12 Anchor sequences into the genetic construct represents an innovative strategy for surface expression of recombinant proteins on Lactobacillus, optimizing immunogenicity. This multifaceted approach contributes to the novelty and potential of the vaccine.The patent evaluation criteria reveal the novelty, industrial application, and significant benefits of the vaccine. The invention's response to potential threats, extensive patent searches, and adherence to patentability criteria position it favorably for patent protection. The comprehensive invention disclosure provides a detailed background, emphasizing the urgency of addressing Salmonella infections in poultry. The disclosed information covers synthesis, clinical trials, scalability, protection against various serovars, and the vaccine's potential impact on food safety and public health.

[0145] The invention described herein represents the development in the field of poultry vaccine technology, specifically designed to combat Salmonella infections in poultry. Salmonella is a common pathogen of great concern in the poultry industry due to its adverse effects on poultry health and potential risks to consumers of poultry products. Salmonella can cause serious illness in poultry and humans, requiring effective preventative measures to protect public health and maintain the integrity of poultry production.

[0146] This comprehensive patent report elucidates the development, optimization, and successful application of a novel recombinant vaccine against Salmonella in poultry. Leveraging the plasmid ptrk892 from Addgene as the backbone of the expression vector, the vaccine incorporates conserved Salmonella genes (PagN, SopE2, and FliC) cloned between Bstxl and Notl restriction sites. Additional elements such as Signal Lp_2145 and cAM12 Anchor are included for efficient surface expression of the recombinant proteins on Lactobacillus plantarum NC8. The vaccine, delivered orally, demonstrates remarkable efficacy in protecting broiler chicks and layer hens against lethal Salmonella challenges.

[0147] Plasmid Construction: The construction of the recombinant plasmid involves a meticulous process to ensure the stability and efficacy of the expression vector. The ptrk892 plasmid, acquired from Addgene, serves as the robust backbone for the expression cassette.

[0148] • Gene Cloning: The genes PagN, SopE2, and FliC, known for their conservation across various Salmonella serovars, are amplified and cloned into the ptrk892 plasmid. This process involves the utilization of high-fidelity polymerases to maintain the integrity of the genetic material.

[0149] • Expression Cassette Design: The expression cassette is strategically designed to maximize protein expression and stability. The strong constitutive promoter phosphoglycerate mutase (pgm) is chosen to drive the expression of the PagN, SopE2, and FliC genes consistently.

[0150] • Surface Expression Enhancers: To enhance surface expression on Lactobacillus plantarum NC8, Signal Lp_2145 and cAM12 Anchor are incorporated into the expression cassette. These elements play a crucial role in directing the recombinant proteins to the bacterial surface, increasing their accessibility to the host immune system.

[0151] • Selection Markers: Erythromycin resistance genes are strategically positioned within the plasmid to provide selective pressure during the transformation process. This ensures the survival and propagation of transformed Lactobacillus plantarum NC8 cells harboring the recombinant plasmid.

[0152] • Restriction Site Ligation: The entire expression cassette, comprising PagN, SopE2, FliC genes, Signal (Lp_2145), cAM12 Anchor, and the HIS tag for validation, is ligated between Bstxl and Notl restriction sites in the ptrk892 plasmid. This precise ligation ensures the correct orientation and integrity of the inserted genes.

[0153] • Plasmid Validation: To confirm the successful construction of the recombinant plasmid, agarose gel electrophoresis is performed after digestion with Bstxl and Notl restriction enzymes. The presence of the expected band size is indicative of a successful insertion.

[0154] • Sequencing Verification: Sanger sequencing is employed to verify the nucleotide sequence of the constructed plasmid. This step ensures the absence of mutations or unintended modifications, validating the accuracy of the recombinant plasmid.

[0155] Plasmid Transformation

[0156] Lactobacillus plantarum NC8 cells are made competent through a meticulously optimized protocol involving the use of divalent cations and electrocompetent buffers. Briefly, overnight cultures of L. plantarum NC8 are subcultured in fresh media and grown to mid-log phase. Cells are harvested, washed, and resuspended in an ice-cold electroporation buffer containing divalent cations, rendering them competent for transformation.

[0157] The recombinant plasmid, harboring the expression cassette with PagN, SopE2, and FliC genes, along with Signal Lp_2145 and cAM12 Anchor, is introduced into the competent L. plantarum NC8 cells using an electroporation apparatus. A precisely calibrated electric pulse promotes the uptake of the plasmid by the bacterial cells, ensuring efficient transformation.

[0158] Post-electroporation, cells are immediately rescued in a recovery medium to allow the expression of antibiotic resistance and recovery of membrane integrity. The transformed cells are then plated on MRS agar supplemented with erythromycin for selection, and the plates are incubated at 37 degrees Celsius under anaerobic conditions for 24 hours.

[0159] Successful transformants are identified by the appearance of colonies on selective agar, and individual colonies are picked for further analysis. To confirm the stable integration of the recombinant plasmid, several colonies are subjected to plasmid extraction, and the presence of the expression cassette is verified through restriction enzyme digestion and subsequent agarose gel electrophoresis.

[0160] This rigorous transformation process ensures the stable and reliable incorporation of the recombinant plasmid into L. plantarum NC8, laying the foundation for the subsequent successful expression and delivery of the Salmonella antigens. The careful optimization of this transformation protocol contributes to the reproducibility and scalability of the vaccine production process.

[0161] Confirmation of Recombinant Protein Expression Following the successful transformation of Lactobacillus plantarum NC8 with the recombinant plasmid, rigorous steps are taken to confirm the accurate expression of the recombinant proteins.

[0162] • Plasmid Extraction: A carefully optimized plasmid extraction protocol is employed to isolate the recombinant plasmid from the transformed Lactobacillus culture. This ensured the retrieval of high-quality DNA for subsequent analyses.

[0163] • Restriction Enzyme Digestion: To confirm the integrity of the inserted expression cassette, the extracted plasmid is subjected to enzymatic digestion using Bstxl and Notl restriction enzymes. This step aimed to verify the presence of the expected DNA fragments corresponding to the expression cassette, which are visualized through gel electrophoresis.

[0164] • Sanger Sequencing: To validate the sequence fidelity of the inserted genes and ensure that no unintended mutations are introduced during the cloning process, the excised band from the gel is sent for Sanger sequencing. This additional verification step confirmed the accurate integration of PagN, SopE2, and FliC genes into the plasmid.

[0165] • Western Blotting: Western blotting techniques are employed to detect and confirm the expression of the recombinant proteins in Lactobacillus. The extracted proteins are separated through polyacrylamide gel electrophoresis and subsequently transferred to a membrane. The membrane is probed with an anti-His antibody, allowing for the identification of the His-tagged recombinant proteins. The presence of distinct bands on the membrane confirmed the successful expression of the desired proteins.

[0166] • Protein Quantification: To ensure consistent protein expression levels across different batches, the quantification of the expressed recombinant proteins is performed using established protein quantification assays. This step aimed to verify the reproducibility and reliability of the expression system.

[0167] • Confirmation through Replication: To further establish the reliability of the expression system, the entire confirmation process, including plasmid extraction, digestion, and Western blotting, is replicated across multiple independent experiments. Consistent and reproducible results are obtained, substantiating the reliability of the developed recombinant vaccine construct.

[0168] The confirmation of recombinant protein expression involved a multi-step process employing molecular biology techniques, sequencing, and protein analysis, ensuring the robustness and reliability of the developed recombinant vaccine. These detailed confirmatory steps enhance the credibility of the disclosed technology and its potential for successful commercialization.

[0169] Vaccine Dosage Optimization

[0170] To ascertain the optimal vaccine dosage, a meticulous dosage optimization study is conducted on five groups of day-old broiler chicks. Each group, comprising 5 chicks, received varying doses of the recombinant vaccine construct (105, 106, 107, 108, 109CFU) on days 1, 7, and 14 post-hatch. This multi-dose regimen is designed to evaluate the impact of different concentrations on the immune response and survival rates.

[0171] The chicks are closely monitored for any adverse reactions or abnormalities throughout the vaccination period. After the third dose, the chicks are challenged with a lethal dose of Salmonella gallinarum (108CFU) to assess the vaccine's protective efficacy under realistic conditions.

[0172] Results indicated that the 107CFU dosage emerged as the most effective in conferring robust protection, as all the immunized birds survived the lethal Salmonella challenge. This dosage is selected based on a balance between achieving a sufficiently strong immune response and minimizing any potential side effects.

[0173] Further analysis included the assessment of antibody titers, cytokine profiles, and histopathological examinations to gain insights into the immune response elicited by different dosages. The optimal dosage not only demonstrated the highest survival rates but also exhibited a robust and balanced immune response, suggesting an ideal balance between immunogenicity and safety.

[0174] The dosage optimization study not only determined the most effective concentration but also provided valuable insights into the dynamics of the immune response triggered by the recombinant vaccine. This comprehensive approach ensures that the selected dosage is not only protective but also safe for the targeted poultry population. The 107CFU dosages are subsequently utilized in subsequent large-scale trials, demonstrating consistent efficacy and laying the foundation for broader applications in the poultry industry.

[0175] Large-scale Trial

[0176] Building on the promising results from initial trials, a comprehensive large-scale trial is conducted to further assess the vaccine's efficacy in a more representative setting. A cohort of around 100-day-old broiler chicks is divided into experimental and control groups, each consisting of 50 birds. The experimental group received oral administration of the recombinant Lactobacillus plantarum vaccine at the optimized dosage of 107CFU on days 1, 7, and 14, while the control group received a placebo of natural Lactobacillus at the same dosage and schedule.

[0177] The trial encompassed a meticulous monitoring and data collection protocol:

[0178] Antibody Titration:

[0179] • Blood samples are collected from both groups on days 1, 6, 13, and 20 post-initial vaccinations.

[0180] • Antibody titers against Salmonella are determined using established serological assays to validate the humoral immune response in the experimental group.

[0181] Challenge and Survival Assessment:

[0182] • Following the vaccination regimen, all birds are challenged with a lethal dose of Salmonella gallinarum (108CFU).

[0183] • Survival rates are monitored daily, with particular emphasis on the critical postchallenge period.

[0184] Post-mortem Examinations and Histopathology:

[0185] • Birds that succumbed to the challenge are subjected to post-mortem examinations to isolate and analyze Salmonella presence in liver, spleen, cecum, and intestines.

[0186] • Histopathological examinations are conducted to assess tissue damage, hemorrhage, and immune response in both the experimental and control groups.

[0187] The large-scale trial results are consistent with earlier findings, affirming the efficacy of the recombinant vaccine. The experimental group demonstrated a 95% survival rate, while the control group exhibited less than 40% survival. Post-mortem examinations confirmed the absence of Salmonella in the tissues of the immunized birds, further supported by histopathological assessments indicating healthy organs.

[0188] These robust results from the large-scale trial provide strong evidence for the reproducibility and scalability of the developed vaccine, reinforcing its potential for practical implementation in commercial poultry farming. The comprehensive data collected during this trial enhances the depth of evidence supporting the novel vaccine's effectiveness and its capacity to address Salmonella infections in diverse poultry populations.

[0189] Immunization of Layers

[0190] Layer hens, crucial contributors to egg production, represent a vital sector of the poultry industry. In this phase of the study, the recombinant vaccine's efficacy is assessed in layer hens, focusing on immunization protocol, Salmonella challenge, and subsequent analysis of egg safety.

[0191] Immunization Protocol:

[0192] • Layers are primed with a dosage of 107CFU of the recombinant vaccine construct on day 1, followed by booster doses on days 5 and 10.

[0193] • Each dose is administered orally, ensuring efficient delivery and absorption within the gastrointestinal tract. Egg Safety Assessment:

[0194] • Concurrent with the immunization schedule, eggs laid by immunized layers are systematically examined for the presence of Salmonella contamination.

[0195] • Rigorous testing procedures, including bacterial culture and PCR assays, are employed to ensure the eggs remained free of Salmonella.

[0196] Salmonella gallinarum Challenge

[0197] • Following the completion of the immunization schedule, layers are challenged with a lethal dose of Salmonella gallinarum (108CFU) via intraperitoneal injection.

[0198] • This mode of challenge aimed to simulate real-world scenarios and assess the resilience of the immunized layers against systemic Salmonella infection.

[0199] Survival and Comparative Analysis:

[0200] • Immunized layers demonstrated exceptional resilience, with 100% survival postSalmonella challenge. This starkly contrasted with the control group, where only 65% of layers survived the infection.

[0201] • Statistical analyses are performed to validate the significance of the observed differences in survival rates between the immunized and control groups.

[0202] Egg Quality and Salmonella Presence:

[0203] • Eggs laid by immunized layers are subjected to extensive quality assessments. Parameters such as shell integrity, yolk color, and albumen consistency are examined.

[0204] • Notably, eggs from the immunized group are consistently Salmonella-free, highlighting the efficacy of the recombinant vaccine in preventing vertical transmission.

[0205] Post-Challenge Analysis:

[0206] • Post-Salmonella challenge, all layers are subjected to post-mortem examinations. Immunized layers exhibited clear organs — liver, spleen, cecum, and intestines — indicating the successful prevention of Salmonella dissemination.

[0207] • In contrast, survivors from the control group showed Salmonella isolation primarily from the liver and cecum, along with histopathological signs of hemorrhage, congestion, and neutrophil infiltration.

[0208] Challenge Studies with S. Typhimurium and S. Enteritidis

[0209] To comprehensively assess the efficacy of the recombinant vaccine, challenge studies are conducted using two additional Salmonella serovars — S. Typhimurium and S. Enteritidis. The goal is to evaluate the vaccine's protective capacity against diverse Salmonella strains commonly associated with poultry infections.

[0210] Study Design:

[0211] • Broiler chicks are divided into immunized and control groups.

[0212] • Immunized groups received the recombinant vaccine (107CFU) on days 1, 7, and 14.

[0213] • Control groups are administered natural Lactobacillus (107CFU) following the same schedule.

[0214] Results:

[0215] • Post-Salmonella challenge, the immunized groups exhibited notable resistance to both S. Typhimurium and S. Enteritidis.

[0216] • Immunized chickens remained salmonella-free and displayed normal growth rates, indicative of a robust immune response.

[0217] • Control chickens, conversely, exhibited diminished growth and became carriers of Salmonella, highlighting the efficacy of the recombinant vaccine in preventing both infection and colonization.

[0218] Health Assessments: • Post-mortem examinations confirmed the absence of Salmonella in vital organs of immunized chickens.

[0219] • Control chickens displayed signs of infection, including organ pathology and the isolation of Salmonella from the liver, spleen, cecum, and intestines.

[0220] The challenge studies with S. Typhimurium and S. Enteritidis further underscore the broadspectrum protection conferred by the recombinant vaccine. Immunized chickens not only resisted infection but also remained healthy and Salmonella-free. The contrasting outcomes in the control groups emphasize the vaccine's role in preventing Salmonella-induced health complications and transmission.

[0221] This comprehensive report solidifies the development and efficacy of the recombinant vaccine against Salmonella in poultry. The successful trials conducted on broiler chicks and layer hens underscore the potential for widespread application and commercial viability of the disclosed technology. The robust protection observed, particularly with the 107CFU dosage, not only validates the effectiveness of the vaccine but also positions it as a promising solution for mitigating Salmonella infections in poultry. The extensive trials, spanning both small-scale and large-scale studies, consistently demonstrate the vaccine's ability to confer high levels of protection, with a 95% survival rate in immunized groups compared to control groups. The post-mortem examinations, histopathological analyses, and Salmonella isolation results provide a thorough understanding of the vaccine's impact on poultry health.

[0222] The successful immunization of layer hens, resulting in Salmonella-free eggs, adds an additional layer of significance to the vaccine's potential impact on food safety. The robustness and versatility of the vaccine, showcased through varying dosages and challenges, further affirm its potential as a transformative solution for the poultry industry.

[0223] The drawings and the forgoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of embodiments is at least as broad as given by the following claims.

[0224] Benefits, other advantages, and solutions to problems have been described above about specific embodiments. However, the benefits, advantages, solutions to problems, and any component(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all the claims.

Claims

CLAIMS1. A process for preparing Lactobacillus-based recombinant vaccine candidate against multiple Salmonella Serovar in poultry, the process comprises: providing a recombinant vaccine construct, wherein said construct comprises genetically modified Lactobacillus plantarum NC8 as a live vector; modifying the genetic structure of said Lactobacillus plantarum NC8 to express conserved Salmonella antigens, including PagN, SopE2, and FliC, anchored by a ptrk 892 backbone with a constitutive promoter, phosphoglycerate mutase (PGM); incorporating Signal Lp_2145 and cAM12 Anchor sequences into said genetic construct to enhance surface expression of recombinant proteins on Lactobacillus plantarum NC8; administering said recombinant vaccine orally to poultry, leveraging the probiotic properties of Lactobacillus plantarum NC8 for effective colonization of the poultry gastrointestinal tract; inducing a prolonged and intensified immune response by ensuring sustained high-level expression of target antigens through the utilization of the robust constitutive promoter, phosphoglycerate mutase (PGM); and optimizing immunogenicity through the surface expression of recombinant proteins on Lactobacillus plantarum NC8, fostering a robust and precisely targeted immune response.

2. The process as claimed in claim 1, further comprises preventing vertical transmission of Salmonella in poultry using a recombinant vaccine, comprising: administering the vaccine to poultry to induce immunity against Salmonella-, ensuring vaccinated flocks produce Salmonella-btee eggs, thereby preventing vertical transmission of the pathogen; conducting trials to validate the efficacy of the vaccine in preventing vertical transmission; and monitoring vaccinated laying hens for the absence of Salmonella contamination in their eggs, compared to unvaccinated groups.

3. The process as claimed in claim 1, further comprises synthesizing a plasmid containing Salmonella antigen sequences, comprising: designing and codon optimizing nucleotide sequences encoding PagN, SopE2, and FliC antigens for expression in Lactobacillus plantarum, with strategic addition of Furin cleavage sites between each antigen; synthesizing the codon-optimized expression cassette containing PagN, SopE2, and FliC antigens with Furin cleavage sites; selecting the plasmid vector Ptrk892 as the backbone for the expression construct; digesting Ptrk892 with BstXI and Notl enzymes to create compatible ends for ligation; ligating the synthesized expression cassette into the digested Ptrk892 plasmid vector to integrate the cassette; adding a His tag during ligation to facilitate validation of recombinant protein expression; incorporating Signal Lp_2145 and cAM12 Anchor sequences into the genetic construct to enable surface expression of recombinant proteins on Lactobacillus plantarum NC8, enhancing antigen presentation and immune response induction; verifying the constructed plasmid through sequencing to confirm correct insertion of the expression cassette and presence of the His tag; and validating expression of the recombinant proteins through assays such as Western blotting and immunofluorescence, utilizing the His tag for detection.

4. The process as claimed in claim 3, wherein the design and codon optimization of nucleotide sequences are performed using bioinformatics tools to maximize expression efficiency in Lactobacillus plantarum, wherein the synthesized expression cassette is obtained through gene synthesis techniques utilizing commercially available services, wherein Ptrk892 plasmid vector ischosen based on its compatibility with Lactobacillus plantarum and suitability for gene expression applications, wherein the addition of a His tag during ligation enables purification and detection of the expressed recombinant proteins for downstream applications, wherein Signal Lp_2145 and cAM12 Anchor sequences are strategically incorporated into the genetic construct to promote efficient surface expression of recombinant proteins on Lactobacillus plantarum NC8.

5. The process as claimed in claim 1, further comprises transforming and expressing a construct in Lactobacillus plantarum, comprising: preparing Lactobacillus plantarum cells and rendering them competent for transformation; mixing purified plasmid DNA containing the construct with competent Lactobacillus plantarum cells and subjecting them to transformation techniques such as electroporation or chemical transformation; allowing the transformed cells to recover in a suitable growth medium; plating the transformed cells on selective agar plates containing appropriate antibiotics to select cells harboring the expression construct; and allowing the transformed Lactobacillus plantarum cells to express the antigen sequences under suitable growth conditions.

6. The process as claimed in claim 5, wherein transformation is achieved by subjecting the mixture of purified plasmid DNA and competent Lactobacillus plantarum cells to an electric field in the case of electroporation or utilizing chemical agents to facilitate DNA uptake in the case of chemical transformation, wherein recovery of transformed cells involves incubating them in a growth medium that supports cell growth and allows for the expression of resistance genes carried by the expression construct, wherein selection of transformed cells is performed by plating them on agar plates supplemented with antibiotics that selectively kill cells lacking the expression construct while allowing transformed cells to survive and grow, wherein expression of the antigen sequences by transformed Lactobacillus plantarum cells is achieved by providing suitable growth conditions such as temperature, pH, and nutrient availability conducive to protein synthesis.

7. The process as claimed in claim 3, further comprises detecting expressed antigens by Western blotting, comprising: extracting proteins from transformed Lactobacillus plantarum cells expressing the Salmonella antigens; separating the extracted proteins using SDS-PAGE gel electrophoresis; transferring the separated proteins from the gel to a nitrocellulose or PVDF membrane; blocking non-specific binding sites on the membrane using a blocking agent such as BSA or milk; incubating the membrane with primary antibodies specific to the Salmonella antigens; washing the membrane to remove unbound primary antibodies; incubating the membrane with secondary antibodies conjugated with enzymes such as HRP; and detecting the presence of antigen-antibody complexes using suitable detection methods like chemiluminescence.

8. The process as claimed in claim 7, wherein protein extraction is performed using methods such as sonication, mechanical disruption, or enzymatic digestion to release proteins from the transformed Lactobacillus plantarum cells, wherein SDS-PAGE gel electrophoresis is conducted to separate the extracted proteins based on their molecular weights.

9. The process as claimed in claim 7, wherein transfer of separated proteins to a nitrocellulose or PVDF membrane is achieved through techniques such as electroblotting or capillary transfer, wherein blocking of non-specific binding sites on the membrane is carried out by incubating the membrane with blocking agents such as BSA or milk to prevent false positive signals, wherein primary antibody incubation involves incubating the membrane with specific primary antibodies raised against the Salmonella antigens of interest.

10. The process as claimed in claim 7, wherein washing of the membrane is performed to remove unbound primary antibodies and reduce background noise, wherein secondary antibody incubation entails incubating the membrane with secondary antibodies conjugated with enzymes such as HRP to amplify the signal, wherein detection of antigen-antibody complexes is achieved using suitable detection methods such as chemiluminescence, fluorescence, or colorimetric assays.

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