_____________________________________________________________________________________________________ *Corresponding author: E-mail: ibrahimshnawa3@gmail.com; Cite as: IMS, Shnawa, Algerbori HSH, and Thewaini QNO. 2024. “Immunogenicity of Bacterial Protoplasmic Sonicate Proteins”. Asian Journal of Immunology 7 (1):175-80. https://journalaji.com/index.php/AJI/article/view/141. Asian Journal of Immunology Volume 7, Issue 1, Page 175-180, 2024; Article no.AJI.123078 Immunogenicity of Bacterial Protoplasmic Sonicate Proteins Shnawa IMS a,b*, Algerbori HSH c and Thewaini QNO a a Department of Medical Biotechnology, College of Biotechnology, AL-Qasim Green University, Qasim, Babylon Province, Iraq. b College of Nursing Technology, University of Hilla, Babylon Province, Iraq. c Department of Basic Sciences, College of Dentistry, University of Babylon, Babylon Provine, Iraq. Authors’ contributions This work was carried out in collaboration among all authors. All authors read and approved the final manuscript. Article Information DOI: https://doi.org/10.9734/aji/2024/v7i1141 Open Peer Review History: This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/123078 Received: 04/07/2024 Accepted: 06/09/2024 Published: 20/09/2024 ABSTRACT Bacterial Protoplasmic Sonicate Proteins BPSP were recovered from skin burn Pseudomonas aeruginosa and Klebsiella oxytoca infections. These proteins were considerd as immunogens in rabbits. Mucosal and systemic, humoral and cellular immune immune responses were matched. The P. aeruginosa protoplasmic sonicate proteins induces an increase in; NBT % neutorphil phagocytosis, significant leukocyte migration inhibition cytokines, IL6 cytokine elevation and rise up of humoral agglutinins responses. While, K. oxytoca protoplasmic sonicate proteins induces increase of; humoral agglutinins and IL6 cytokine as well as non-signifcant increase in leukocyte inhibitory cytokines. Both at mucosal and systemic compartments. The study highlights the potentials of these proteins as candidates for developing vaccines against multidrug resistance infections, particularly in burn patients. These findings underscore the importance of immunogenicity in advancing bacterin development in clinical use. Original Research Article https://doi.org/10.9734/aji/2024/v7i1141 https://www.sdiarticle5.com/review-history/123078 Shnawa et al.; Asian J. Immunol., vol. 7, no. 1, pp. 175-180, 2024; Article no.AJI.123078 176 Keywords: Antigens; bacterin; cellular; humoral; immunogen; protein; vaccine. 1. INTRODUCTION Bacterial antigens [BAGs] were being investigated to be of use in, bacterial diagnosis, bacterial infection diagnosis, vaccine development and vaccine production. Immunogenicity of BAGs holds a backbone position in vaccine development strategies [1–7]. The objective of the present work was the investigation of immunogenicity of protoplasmic sonicate protein antigens of skin burn infections with P. aeruginosa and K. oxytoca. 2. MATERIALS AND METHODS 2.1 Protoplasmic Sonicate Protein Antigens P SPA The PSPA separation was in direct way for P. aeruginosa [8], while, by in direct way as it needs removal of the capsule [9,10] before processing for obtaining PSPs. The method for separation, identification, purification and quantitation of these PSPs were as in the method described by Bjorn et al. [8]. The details of the processing method was as in the followings; Six mls of tris buffer 0.01 N and PH 8 was added to the surface of 24 hrs growth of P. aeruginosa and K. oxytoca (after suspending bacteria then removal of capsule [9,10]) on the nutrient agar plates. Growth were vortoxed in vortox test tubes for thee minutes. Suspensions were centrifuged at 5000 rpm for ten minutes. Supernatant were discarded and pellets Ps were kept. Ps suspensions were tubbed and washed three times with tris buffer 0.01N.Ps were reconstituted with 6 mls, and were tubbed in the cell disintegrator tubes. Then jacketed with cooled ice. The best sonication conditions were five times for five minutes at 20 oscillation ampiltude. The sonicated cell suspensions were centrifuged at 5000 rpm for ten minutes. Supernats were collected and ultrafiltred with 0.22 um millipore filter. Fiterates were collected in sterile plastic tubes, then proteins were separated with PEG 6000 ,6% as inShnawa and AlSadi [11]. The protein concentration determination was measured by Biurt test [12]. To this end the preparations can be designated as PAPSP and KOPSP both P. aeruginosa and K.oxytoca respectively. These PSPs were distributed into alliqoutes of 0.5 mls. In an appendroff plastic tubes and kept at - 20C,till use. 2.2 Immune Reagents Specific immune priming of rabbits were done with the PSP concentrations of 2.71 mg/l for PAPSP and 1.8 md/l for KOPSP [12]. The somatic antigens for both bacteria were prepared as heat killed as in [13].Complete Freund Ajuvant that from Difco, Co.Ltd. The test proposed immunogens were made as; one volume of CFA mixed with one volume the test proteins Immunization Protocols 2.3 Immunization Protocols A mounthwise twice dosage of PSP-CFA in 2 mls amounts were dosaged in the first and second months to the rabbits. The specific immune priming was that of multisite injection protocol [14]. 2.4 Rabbits A local breed rabbits brought from the local market were checked for ecto and endoparasites. As well as for pyrogens and found to be free off. They were acclimatized for housing conditions two weeks befor experimentation at and libitum conditions. Among which nine were elected and subdivided into two test and one control group each of three rabbits. 2.5 Samplings and Immune Function Tests At the temination of the specific immune priming protocols, blood with and with out heparin were collected from the test and control rabbits by cardiac puncture for humoral and cellular immune tests. Sera were saved for serology and cytokine studies. Heparinized blood were used for leukocyte inhibitory factor [15] and for NBT phagocytosis [16]. Appendix for test and control rabbits were collected and open up, washed from digesta and processed for separation of mucosal globulins [17]. Mucosal leukocytes were separated by dextran 2% as in [18]. IL 6 determinations were made as in the recommendation of the instruction of the manufacturer. Standared tube agglutination test were made as in [19]. 3. RESULTS The NBT neutrophil phagocytosis percentages in PAPSPA (56.75 % for mucosal and36.5 for Shnawa et al.; Asian J. Immunol., vol. 7, no. 1, pp. 175-180, 2024; Article no.AJI.123078 177 systemic) and KOPSPA (48.5% for mucosal, 41% for systemic) primed rabbits were higher than that of control rabbits (20% for mucosal,18% for the systemic). Leukocyte inhibitory cytoknine LIF % in PAPSP primed rabbits were 56.6% for mucosal and 58.45 for systemic as compared to control ,the mucosal was 90% and systemic was 86%.While for KOPSPA primed rabbits were; 88% for mucosal and 87.5% for systemic LIF as compared to normal control were 93% for mucosal and 89% for systemic responses. The IL6 concentration determinations were shownig that PAPSPA and KOPSPA primed rabbits have got higher IL6 concentration means than normal control rabbits. PAPSPA primed rabbits IL6 concentration means were 92.8 for mucosal and 72.7 for systemic responses. In KOPSPA primed rabbits the mucosal concentration means were 83.26 and for systemic were 76.79 as compared to normal control were 8.83 for mucosal and 9.25 for systemic responses. The humoral specific agglutinin titre levels for PAPSPA were 128 for mucosal and 320 for the systemic responses and KOPSP primed rabbits were 64 for mucosal and 640 for systemic responses as compared to control rabbits were 4 for mucosal and 20 for systemic responses. Mucosal agglutinins were resistant to treatment with 2ME, Tables 1 and 2. 4. DISSCUSSION The concept, application and continuity of the immunogenicity theme for bacterial protein antigens are still in the current mode of researchers allover the world [1-7]. Immunogenicity appeared to have two main facets.First that of theoritical immunologisets which advocate that immunogenicity is denoted to self-nonself recognition theme [6]. While the second facets was that for most of the proper immunologists which can be summerized as the ability of an antigen to initiate humoral and/or cellular conversion from the normal baseline immune functions to an optimized cellular immune reactions outcomes that are finalized by the optimest synthesis and production of cellular secretory proteins (antibodies, cytokines) concentrations and /or optimised depression of such secretory proteins [1-5,7]. Immunogenicity appeared to be essential for diagnosis and prophylaction of human infections as well as in cancer personalized prophylactic and therpeutic medicine. Hence the presnt study for Pseudomonas and Klesiella immunogenicity may participate in developing of a porototype bacterial protein based vaccine and/ or in development for an autovaccine for multidrug resistant skin burn infection [20]. The antigenic make up of bacteria [1-5,7] like that of P. aeruginosa are formed from; flagella, pili, exottoxin A, exoppolysaachride, LPS, OMP, hemolysin, elastases, proteasis, heat stable phospholipid, heat stable glycolipid, exoenzyme S and ribosome [21-23]. While that of Klebsiella oxytoca are; capsule, somatic antigens and LPS [24]. Hence, PAPSP and KOPSP protein Table 1. The immunogenicity of PAPSP in primed rabbits and controls Rabbits groups NBT% LIF% IL6 pg/ml. Agglutinin titres PAPSPA M S 46.75 36.6 56.6 58.45 2.8 72.7 128 320 Control M 20 93 92.8 4 S 18 89 72.7 20 Table 2. The immunogenicity of KOPSP in primed and control rabbits Rabbit groups NBT% LIF % IL6 pg/ml Agglutinin titres KOPSPA M S 48.5 41 88 87 48 72 64 640 Controls M S 20 18 93 89 8.83 9.25 4 20 Shnawa et al.; Asian J. Immunol., vol. 7, no. 1, pp. 175-180, 2024; Article no.AJI.123078 178 Table 3. The immune features of the study bacterial proteins Features PAPSP [25] KOPSP [25] 1-Chemical nature 2-Origin 3-Location 4-Processing method 5-Non-specific immune function 6- Specific cellular immune function 7-Induction of cytokine network 8-Humoral Immune responses 9-Functional Epitope Mapping [26,27] 10-Immuen system compartments 11-Expected Immune potentials [20] Protein Bacterial Intracellular Ultrasonication Rise up of phagocytosis By NBT% Significant inhibition of LIF cytokine Rise up of IL6 cytokine Rise up of specific agglutinins T cell dependent and T cell in- ndepedent epitopes Mucosal and Systemic Prototype protein based vaccine and an autovaccine Protein Bacterial Intracellular Ultrasonication Rise up of phagocytosis by NBT % Nonsignificant inhibition of LIF cytokines Rise up of IL6 cytokines Rise up of specific agglutinins T cell independent and /or, TH2 dependent B cell epitope Mucosal and systemic Prototype protein based vaccine and an autovaccine. antigens are novel antigens prepared in this experimental settings. PAPS was proved to be immunogenic in rabbits model, Table 1, promoting humoral and cellular immune responses both at mucosal and systemic copartments. These findings were in contradiction with that of [24]. While, KOPSP immunogenicity, Table 2, were by promoting humoral immune responses both at mucosal and systemic level [21]. Different burn infecting bacterial protein preparations have shown different immune potential features, Table 3,[25]. The functional epitope mapping for PAPSP may be T cell depndent and T cell independent, while that of KOPSP may be of T cell independent or Th2 cell depent B cell epitopes [26,27]. Since immunogenicity is an integral part for developing bacterins. Such findings holds the position of novelity, based upon the need for an autogenous bacterins for multidrug resistant skin burn bacterial infections [28,29]. 5. CONCLUSIONS P. aeruginosa PAPSP and K. oxytoca KOPSP protoplasmic sonicate proteins were found as lapin immunogens. 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