Development of recombinant vaccine candidates composed of ltka from mannheimia haemolytica A1 and PlpEC AND OmpH from Pasteurella Multocida A:3 against bovine respiratory disease

Çırçır, Ayça
Mannheimia haemolytica A1 and Pasteurella multocida serotype A:3 are gramnegative bacterial pathogens which are considered the causative agents of bovine respiratory disease (BRD) or shipping fever in cattle. The foremost of several virulence factors of M. haemolytica A1 which allows lung colonization and establishment of infection is leukotoxin (Lkt). The others include adhesin, capsule, outer membrane proteins, and various proteases used for attachment, spreading and evading innate and adaptive host immune responses. Similarly, the major virulence factors of P. multocida are capsule, lipopolysaccharide (LPS), toxin (PMT), outer membrane proteins (OMPs), especially PlpE and OmpH, adhesins and type IV fimbriae (pili). BRD has considerable economic importance to the global cattle and feedlot industry. Effective control of BRD is challenging due to inconsistencies in diagnosis and treatment regimes. Extensive antibiotic usage in cattle industry, both prophylactically and therapeutically, leads to development of antibiotic resistance. Therefore, development of vaccine strategies against BRD to demonstrate protection becomes an important issue for cattle industry. P. multocida A:3 lipoprotein E (PlpE) and outer membrane protein H (OmpH) and M. haemolytica A1 leukotoxin (LtkA) have been selected as the antigens for vaccine development studies in our laboratory. PlpEC-OmpH and OmpH-LktA recombinant fusion proteins were previously constructed, expressed and purified. When PlpEC-OmpH was used as an universal vaccine candidate in combination with a mineral oil-based CpG adjuvant, it conferred 100% protection against P. multocida A:3 in BALB/c mice. In the present study, OmpH-Lkt recombinant protein was formulated as an oil-based vaccine candidate and BALB/c mice were immunized with this vaccine formulation. In two independent sets of mice challenge-protective potency experiments for each pathogen, %100 and %50 protection was demonstrated against M. haemolytica A1 and P. multocida A:3, respectively. Additionally, lktA fragment was cloned from the genomic DNA of M. haemolytica A1 at the upstream of plpEC-ompH fusion and the resulting lktA-plpEC-ompH triple fusion was constructed. In vitro expression of this fusion was shown in E.coli BL21 (D3) cells and the purified protein was used for vaccine preparation. The vaccine candidate comprising LktA-PlpECOmpH was formulated with a mineral oil-based adjuvant and the sera collected from the immunized mice were used for the determination of specific antibody and serum IFN-γ titers. Although the antigen-specific IgG1 and IgG2a levels were higher in immunized group, there was no significant difference between control and immunized mice groups in terms of IFN-γ levels. Protective capacity (potency) of this vaccine candidate was also evaluated via mice challenge experiments with lethal doses of P. multocida A:3 and M. haemolytica A1 separately, which did not give consistent results in two independent sets of experiments for each pathogen.


Immunogenicity and protective efficacy of the recombinant Pasteurella lipoprotein E and outer membrane protein H from Pasteurella multocida A:3 in mice
Okay, Sezer; Ozcengiz, Erkan; GÜRSEL, İHSAN; Özcengiz, Gülay (2012-12-01)
Pasteurella multocida serotype A:3 is a Gram-negative bacterial pathogen, one of the causative agents of shipping fever of cattle. In this study, outer membrane protein H (ompH) and Pasteurella lipoprotein E (plpE) genes were cloned and plpEC-ompH fusion was constructed and expressed in Escherichia coli. Recombinant PlpE, OmpH and PlpEC-OmpH fusion proteins were purified and formulated with oil-based and oil-based CpG ODN adjuvants. Antibody responses in mice vaccinated with recombinant PlpE and PlpEC-OmpH ...
Development of an experimental recombinant vaccine formulation composed of LKTA from Mannheimia Haemolytica A1 and P31 and LPPB from Histophilus Somni 8025 against Hovine Respiratory Disease
Türkmen, Nazlı Hilal; Özcengiz, Gülay; Department of Molecular Biology and Genetics (2020-9)
Two major bacterial pathogens of Bovine Respiratory Disease (BRD), Mannheimia haemolytica and Histophilus somni are Gram-negative, opportunistic bacteria that live in upper respiratory tracts of ruminants commensally. The one of the most important virulence factor of M. haemolytica, leukotoxin, is responsible for lung colonization and establishment of infection. On the other hand, H. somni OMPs have a significant contribution to the pathogenicity of the organism. As the disease results in a destroyed animal...
Development of recombinant vaccines composed of PlpE and OmpH from pasteurella multocida A:3
Okay, Sezer; Özcengiz, Gülay; Department of Biology (2011)
Pasteurella multocida serotype A:3 is a gram-negative bacterial pathogen which is one of the causative agents of shipping fever in cattle. In this study, ompH and two fragments of plpE gene (plpEN and plpEC) were cloned from the genomic DNA of P. multocida P-1062 (ATCC 15743, serotype A:3) and plpEN-ompH and plpEC-ompH fusions were constructed. In vitro expression of the genes was shown in HEK-293 cells. Later, full-length plpE gene was cloned and the recombinant proteins were expressed in E. coli and purif...
Investigation of the Ligand Binding Characteristics of Staphylococcus aureus NorA Efflux Pump
Işık , Esra Büşra; Serçinoğlu, Onur (Orta Doğu Teknik Üniversitesi Enformatik Enstitüsü; 2022-10)
Staphylococcus aureus is a gram-positive bacterial pathogen which is highly adaptive to environmental conditions and causes various disorders. Excessive usage of antibiotics may result in development of antibiotic resistance in S. aureus. One of the resistance mechanisms is increase in the activity of transmembrane multi-drug efflux pumps. NorA is the most studied efflux pump in S. aureus, which belongs to Major Facilitator Superfamily (MFS). NorA has been shown to contribute to resistance against a variety...
Multiple drug resistance patterns and plasmid profiles of non-typhi salmonellae in Turkey
Yildirmak, T; Yazgan, A; Özcengiz, Gülay (1998-10-01)
A total of 259 clinical isolates of nonrepetitive non-typhi salmonellae (NTS) were examined for antibiotic resistance patterns and plasmid content. The antibiotics used were amoxicillin-clavulanic acid (AMC), ampicillin (AM), aztreonam (ATM), carbenicillin (CB), cefixime (CFM), cefotaxime (CTX), cefoxitin (FOX), ceftazidime (CAZ), ceftriaxone (CRO), chloramphenicol (C), ciprofloxacin (CIP), gentamicin (GM), imipenem (IPM), ofloxacin (OFX), tetracycline (TE), trimethoprim-sulfomethoxazole (SXT). Multi-drug r...
Citation Formats
A. Çırçır, “Development of recombinant vaccine candidates composed of ltka from mannheimia haemolytica A1 and PlpEC AND OmpH from Pasteurella Multocida A:3 against bovine respiratory disease,” M.S. - Master of Science, Middle East Technical University, 2014.