5 research outputs found
Implementation of a strategy to produce a broadly neutralizing monoclonal antibody against Zika and dengue viruses
Get PDFDengue and Zika viruses are Flavivirus transmitted by a mosquito bite that cause dengue and Zika fever, respectively, with symptoms including fever, rash and headache. The association of Zika virus infection of pregnant women with the development of microcefalia in the fetus lead to the declaration of Zika as a Public Health Emergency of International Concern by the WHO in 2016. The rapid development of methods of detection, quantification and purification of the virus, among others, is necessary for studying the virus at the laboratory and eventually develop a vaccine. Using molecular biology and cell culture techniques, we implemented a strategy to produce a broadly neutralizing monoclonal antibody against dengue and Zika viruses at the lab.
Signal peptides were selected for high level expression of antibodies in CHO cells, according to previous reports (Haryadi et al. 2015). The sequence reported for the human anti-dengue/Zika IgG1 EDE1 C8, including the sequences of the optimized signal peptides for each chain, were cloned into the Freedom™ pCHO 1.0 vector and CHO-S cells were transfected with the resulting construction. Transfection efficiency was low (13%), however, transfected cells were submitted to selection using methotrexate (MTX) and puromycin (PUR) as selection reagents. Two pools of transfected cells were selected using two concentrations of MTX and PUR, and after selection, 100% cells showed expression of the protein of interest, as determined by a parallel control EGFP transfection. Concentration of selection reagents had no effect in productivity in 6-days batch cultures; therefore, the pool of transfected cells growing in the lowest concentration of MTX and PUR was assessed for stability and productivity in presence and absence of selection reagents. Cells growing in medium with and without MTX and PUR showed stable production of the antibody in 10-days cultures, however, differences were found in µmax, Xmax, and productivity, with the highest values of µmax and Xmax for the cultures without selection reagents (µmax = 0.04 h-1, Xmax = 2.68x107 cells/mL), in comparison with cultures with selection reagents (µmax = 0.03 h-1, Xmax = 2.19x107 cells/mL). Productivity was higher for cells growing in medium with MTX and PUR (0.159x10-6 ug/cell h) than for cells without selection pressure (0.104 pg/cell h), corroborating the importance of maintaining the selection pressure for optimal expression of the protein of interest in this system. The purified antibody recognized the Zika virus and three serotypes of dengue virus, as observed by dot blot, and according to previous reports that demonstrate that the EDE1 C8 antibody recognizes a quaternary epitope conserved in both viruses (Barba-Spaeth et al. 2016). A more exhaustive evaluation of cell pools is necessary to determine the stability of the expression of the antibody for longer periods of time and to optimize its productivity. This antibody will be used for future research and methods development in our lab. Also, the methodology described here could be used as a start point in the production of other therapeutic antibodies and vaccines.
References:
Barba-Spaeth G, Dejnirattisai W, Rouvinski A, Vaney MC, Medits I, Sharma A, Simon-Loriere E, Sakuntabhai A, Cao-Lormeau VM, Haouz A, England P, Stiasny K, Mongkolsapaya, J, Heinz FX, Screaton, GR, Rey FA (2016) Structural basis of potent Zika-dengue virus antibody cross-neutralization. Nature 536: 48-53.
Haryadi R, Ho S, Kok YJ, Pu HX, Zheng L, Pereira NA, Li B, Bi X, Goh LT, Yang Y, Song Z (2015) Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells. PLoS ONE 10: e0116878.
Financial support by DGAPA PAPIIT UNAM IT-200418
Design of mimotopes of a conserved epitope in dengue and Zika viruses for the obtention of broadly neutralizing antibodies
Get PDFZika and dengue viruses are members of the Flavivirus genus that share many structural and pathological characteristics. They cause mild fever, rash and general body pain but can cause severe reactions, as hemorrhages (dengue virus), congenital syndrome (Zika virus), or even death. After an infection, virus-specific antibodies are generated by the immune system; however, because of the structural similarity between these viruses, some antibodies can cross-react with different members of the flavivirus family. After a secondary infection, the cross-reactive antibodies can lead to the more severe forms of the disease, through a mechanism named antibody-dependent enhancement of infection (ADE). Recently, some broadly neutralizing antibodies (antibodies that neutralize both, dengue and Zika, viruses), have been isolated and it has been demonstrated that they do not induce ADE. These antibodies are directed to a discontinuous quaternary epitope named the Envelope Dimer Epitope (EDE)1, located in the envelope (E) protein of both viruses. To obtain EDE, it is necessary to express the complete E protein, which contains other epitopes that induce ADE. This study aims to generate peptides that emulate the EDE epitope structure (mimotopes) without inducing ADE, and study its capacity to elicit broadly neutralizing antibodies against dengue and Zika viruses, to obtain a vaccine candidate for both viruses.
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Design of mimotopes of a conserved epitope in dengue and Zika viruses for the obtention of broadly neutralizing antibodies
Get PDFZika and dengue viruses are members of the Flavivirus genus that share many structural and pathological characteristics. They cause mild fever, rash and general body pain but can cause severe reactions, as hemorrhages (dengue virus), congenital syndrome (Zika virus), or even death. After an infection, virus-specific antibodies are generated by the immune system; however, because of the structural similarity between these viruses, some antibodies can cross-react with different members of the flavivirus family. After a secondary infection, the cross-reactive antibodies can lead to the more severe forms of the disease, through a mechanism named antibody-dependent enhancement of infection (ADE). Recently, some broadly neutralizing antibodies (antibodies that neutralize both, dengue and Zika, viruses), have been isolated and it has been demonstrated that they do not induce ADE. These antibodies are directed to a discontinuous quaternary epitope named the Envelope Dimer Epitope (EDE)1, located in the envelope (E) protein of both viruses. To obtain EDE, it is necessary to express the complete E protein, which contains other epitopes that induce ADE. This study aims to generate peptides that emulate the EDE epitope structure (mimotopes) without inducing ADE, and study its capacity to elicit broadly neutralizing antibodies against dengue and Zika viruses, to obtain a vaccine candidate for both viruses.
Please click Download on the upper right corner to see the full abstract
Design of a vaccine against dengue and Zika viruses based on a mimotope of the envelope dimer epitope
Get PDFZika and dengue viruses are members of the Flavivirus genus that cause mild fever, rash and general body pain; but can cause severe reactions, as hemorrhages (dengue virus), congenital syndrome (Zika virus), or even death. Because of the structural similarity between these viruses, some antibodies generated after an infection can cross-react with different members of the flavivirus family. After a secondary infection, the cross-reactive antibodies can lead to more severe forms of the disease, through a mechanism named antibody-dependent enhancement of infection (ADE). Broadly neutralizing antibodies are antibodies that neutralize both, dengue and Zika viruses; and it has been demonstrated that they do not induce ADE. These antibodies are directed to a discontinuous quaternary epitope named the Envelope Dimer Epitope (EDE)1, located in the envelope (E) protein. To obtain the EDE, it is necessary to express the complete E protein, which contains other epitopes that induce ADE. This study aims to generate a peptide that emulates the EDE epitope structure (mimotope) in order to be used as a dual vaccine against dengue and Zika viruses; without causing ADE.
Please click Download on the upper right corner to see the full abstract
