Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Submitted by Repositório Arca ([email protected]) on 2019-03-07T16:36:23Z No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) F115-10616_2014_Article_9794.pdf: 1313223 bytes, checksum: 9b92e60c8bfe1ff7c844719c932c4d62 (MD5)Approved for entry into archive by monique santos ([email protected]) on 2019-03-27T13:24:33Z (GMT) No. of bitstreams: 2 F115-10616_2014_Article_9794.pdf: 1313223 bytes, checksum: 9b92e60c8bfe1ff7c844719c932c4d62 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-03-27T13:24:33Z (GMT). No. of bitstreams: 2 F115-10616_2014_Article_9794.pdf: 1313223 bytes, checksum: 9b92e60c8bfe1ff7c844719c932c4d62 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016Universidade Federal Fluminense. Laboratório de Tecnologia de Produtos Naturai. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Monoclonal antibodies (mAb) are high added value glycoproteins recommended for immunotherapy, diagnosis, and also for the treatment of bacterial infections resistant to multiple drugs such as Methicillin Resistant Staphylococcus aureus (MRSA). In addition to environmental conditions related to cell cultures, the intrinsic characteristics of hybridoma cells, like the secretion stability of monoclonal antibodies by the cells through successive subcultures, are relevant for the characterization of cell lines related to the productivity of mAb. The rate of mAb production differs significantly between different cell lines and different passage numbers, and it is an important variable in characterization of cell lines. In order to find a more robust, faster-growing, and higher-productivity cell line of hybridoma, cultivations in 24-well plates were performed in different subculture periods, or cell passages (P), of hybridoma cells producing MRSA anti-PBP2a monoclonal antibodies [MRSA-antiPBP2a (mAb)]. The objective of this study was to study the effects of cell growth and production of MRSA-antiPBP2a mAb secreted by murine hybridoma cells grown in different passages as well as determine the which passages the hybridomas can be cultivated without harming their growth and productivity. So, cell growth profiles of hybridomas secreting MRSA-antiPBP2a (mAb) and the production of MRSA-antiPBP2a mAb in different subculture periods or cell passages (P) were studied. Cell growth tests, monoclonal antibody productivity, and metabolite characteristics revealed substantial differences in those cells kept between P10 and P50. Similarities in the secretion of monoclonal antibody, growth, and metabolic profiles, were noted in the MRSA-antiPBP2a mAb producing hybridoma cells kept between P10 and P20. Also, glucose consumption (g/L) and lactate production (g/L) in the latter cell cultures were monitored daily through biochemical analyzer. As of P30, it was observed a 4.4 times reduction in productivity, a 13 % reduction in metabolic yield, and a significant change in cell growth. Secretion of MRSA-antiPBP2a mAb should be obtained through the culture of hybridomas up to P20 in order to keep its stability

Monoclonal antibody anti-PBP2a protects mice against MRSA (methicillin-resistant Staphylococcus aureus) infections.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium responsible for serious nosocomial and community-acquired infections worldwide. Since few antibiotics are effective for treating MRSA infections, the development of new therapies is of great importance. Previous studies demonstrated that PBP2a is a target that generates protective antibodies against MRSA. A murine monoclonal antibody (MAb) that recognizes PBP2a from MRSA strains was previously isolated and characterized. In this report, we evaluated the biodistribution of this MAb in blood and tissues, as well as the extent of protection conferred using prophylactic and therapeutic assays compared to vancomycin treatment. Biodistribution was evaluated 12-96 h after MAb administration. It predominantly remained in the serum, but it was also detectable in the kidneys, lungs, and spleen at low concentrations (about 4.5% in the kidneys, 1.9% in the lungs, and 0.7% the spleen) at all observed timepoints. Prophylactic studies in a murine model demonstrated a significant bacterial load reduction in the kidneys of the groups treated with either with IgG (greater than 3 logs) or F(ab')2 (98%) when compared to that of the control groups (untreated). Mice were challenged with a lethal dose, and the survival rate was higher in the treated mice. Treatment with the MAb resulted in a bacterial load reduction in the kidneys similar to that of mice treated with vancomycin, and a MAb/vancomycin combination therapy was also effective. These results demonstrate that an anti-PBP2a MAb may be a promising therapeutic for treating MRSA infections

Identification of immunogenic proteins of the bacterium Acinetobacter baumannii using a proteomic approach

Made available in DSpace on 2015-06-08T14:01:53Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) anapaula_assefetal_IOC_2014.pdf: 1859957 bytes, checksum: 09c3b5c760f35acea9aafe7444daa579 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil.undação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.undação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa de Infecções Hospitalares. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.Purpose: Acinetobacter baumannii is an important opportunistic pathogen that causes pneumoniae, urinary tract infections, and/or septicemia in immunocompromised patients. This pathogen is frequently associated with nosocomial outbreaks worldwide and has become particularly problematic because of its prevalence and resistance patterns to several antibiotics. In the present study, we used an immunoproteome-based approach to identify immunogenic proteins located on the surface of A. baumannii for the development of a possible immunotherapy against this devastating bacterial infection. Experimental design: Sera from patients with A. baumannii infections (n = 50) and from a control group of healthy individuals (n = 3) were analyzed for reactivity against A. baumannii outer membrane proteins (OMPs) using Western blot analysis. To identify potential immunogenic proteins in A. baumannii,OMPs were separated by 2DE (2Delectrophoresis), and reactive sera from infected patients were randomly selected and divided into two different pools, each containing 15 sera. Finally, MALDI-TOF/TOF mass spectrometric analysis was employed to identify the corresponding proteins. Results: This analysis identified six immunoreactive proteins: OmpA, Omp34kDa, OprC, OprB-like, OXA-23, and ferric siderophore receptor protein. Notably, these proteins are highly abundant on the bacterial surface and involved in virulence, antibiotic resistance, and growth. Conclusions and clinical relevance: Our results support the notion that the proteins identified in the present immunoproteome study could serve as antigen candidates for the development of vaccines and passive immunotherapies against A. baumannii infections