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.

Production and Characterization of F(Ab')2 Fragments Obtained by Enzymatic Digestion from Murine Anti-MRSA PBP2a Monoclonal Antibodies.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a worldwide health problem. In a previous study, a murine monoclonal antibody (mMAB), capable of binding to PBP2a within MRSA strains, was generated. F(ab')2 antibody fragments are widely described in the literature as immunochemical tools and reagents for diagnostics and therapeutics, particularly because of their low immunogenicity and rapid pharmacokinetics. In this study, F(ab')2 fragments from mMAB were generated by enzymatic digestion, using pepsin. They were purified by affinity chromatography using protein A and concentrated by a MWCO 50 kDa filtration unit. The results indicate that it is possible to obtain F(ab')2 fragments by pepsin digestion. ELISA, western blotting, and fluorescence microscopy data demonstrated that F(ab')2 affinity for PBP2a is not lost even after the enzymatic digestion process. As expected, in the pharmacokinetics tests, F(ab')2 presented a faster elimination (between 12 and 18 h) compared to IgG. These F(ab')2 fragments could be used in future immunodiagnostic applications, including in vitro or in situ radiolabeling and in the treatment of infections caused by this important pathogen.

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

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.

Identificação de Anticorpos Anti-PBP2a em Pacientes Colonizados e Infectados por Staphylococcus aureus Resistente a Meticilina / Identification of Anti-PBP2a Antibodies in Patients Colonized and Infected by Methicillin Resistant Staphylococcus aureus (MRSA)

Infecções causadas por Staphylococcus aureus resistente a Meticilina (MRSA) são especialmente problemáticas devido às dificuldades de tratamento e alta taxa de mortalidade associada. O principal determinante do amplo espectro de resistência aos beta-lactâmicos em cepas de MRSA é a proteína de ligação à penicilina 2a (PBP2a). A PBP2a está localizada na superfície exterior, que seriam acessíveis ao sistema imune. Não está totalmente caracterizado se o hospedeiro pode produzir anticorpos anti- PBP2a durante infecções por MRSA ou se estes anticorpos seriam protetores. O objetivo deste estudo foi investigar a presença de anticorpos anti- PBP2a em um grupo de 60 pacientes colonizados ou infectados por MRSA. Também foi investigado se estes anticorpos poderiam reduzir o crescimento bacteriano em um ensaio in vitro. Através da técnica de ELISA, os resultados mostraram que cerca de 70% das amostras apresentaram anticorpos anti- PBP2a (colonizados: 68,6% versus doentes infectados: 72 %), confirmado pelo teste Western Blot. Também foi avaliado o efeito bactericida do soro contendo anticorpos anti- PBP2a e soros controle. Uma redução do crescimento bacteriano foi observada em soros de anticorpos anti - PBP2a, em comparação com os controles. Os resultados indicam que pacientes infectados ou colonizados por MRSA produzem anticorpos contra PBP2a e que estes anticorpos pode conferir proteção contra a MRSA.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are especially troublesome because of treatment difficulties and high mortality rate. The major determinant of the broad-spectrum beta-lactam resistance in MRSA strains is the penicillin-binding protein 2a (PBP2a). Since PBP2a is located on the outer surface, it would be accessible to the immune system. It is not fully characterized whether the host can produce anti-PBP2a antibodies during MRSA infections or whether these antibodies would be protective. The aim of this study was to investigate the presence of anti-PBP2a antibodies in a group of 60 patients colonized or infected by MRSA. It was also investigated whether these antibodies can reduce bacterial growth in an in vitro assay. Via the ELISA technique, the results showed that approximately 70% of the samples had anti-PBP2a antibodies (colonized: 68.6% versus infected patients: 72%), confirmed by the Western blotting assay. The bactericidal effect of serum containing anti-PBP2a antibodies and control sera were also evaluated. A reduction in bacterial growth was observed in the anti-PBP2a antibody sera as compared to the controls. The results indicated that MRSA-colonized or-infected patients produce antibodies against PBP2a, which can confer protection against MRSA.