Implementation of a strategy to produce a broadly neutralizing monoclonal antibody against Zika and dengue viruses

Dengue 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, expression, and characterization of an asymmetric bispecific antibody fused to a scFv shuttle for brain uptake

Please click Additional Files below to see the full abstract

Population kinetics during simultaneous infection of insect cells with two different recombinant baculoviruses for the production of rotavirus-like particles

<p>Abstract</p> <p>Background</p> <p>The simultaneous production of various recombinant proteins in every cell of a culture is often needed for the production of virus-like particles (VLP) or vectors for gene therapy. A common approach for such a purpose is the coinfection of insect cell cultures with different recombinant baculoviruses, each containing one or more recombinant genes. However, scarce information exists regarding kinetics during multiple infections, and to our knowledge, no studies are available on the behavior of the different populations that arise during coinfections. Such information is useful for designing infection strategies that maximize VLP or vector yield. In this work, kinetics of cell populations expressing rotavirus GFPVP2 (infected with bacGFPVP2), VP6 (infected with bacVP6), or both proteins simultaneously (coinfected with both baculoviruses) were followed by flow cytometry.</p> <p>Results</p> <p>In single infections, the population infected with any of the recombinant baculoviruses followed the Poisson distribution, as the population expressing a recombinant protein exhibited a hyperbolic-type function with respect to the multiplicity of infection (MOI) up to 5 pfu/cell. In coinfections, the population fraction expressing each recombinant protein could not be anticipated from results of single infections, as in some cases interference and synergistic effects were found. Only cultures with a total MOI below 5 pfu/cell followed the Poisson distribution. For cultures with a MOI of bacGFPVP2 above that of bacVP6 (overall MOI above 5 pfu/cell), the total population expressing one or both recombinant proteins was as low as 50% below that predicted by Poisson. In contrast, the population fraction expressing VP6 increased in coinfections, compared to that in single infections. The largest population fraction simultaneously expressing both recombinant proteins was 58%, and corresponded to cultures infected at a MOI of 5 and 1 pfu/cell of bacGFPVP2 and bacVP6, respectively.</p> <p>Conclusion</p> <p>The infection conditions that maximize the cell population simultaneously expressing two recombinant proteins were determined. Such conditions could not have been anticipated from population kinetics in individual infections. This information should be taken into account for improved simultaneous production of various recombinant proteins in any work dealing with coinfections.</p

Overexpression of the mitochondrial pyruvate carrier increases CHO cell and recombinant protein productivity and reduces lactate production

The metabolism of CHO cells is characterized by a low efficiency of glucose metabolism, resulting in lactate production. We hypothesized that the cause of such low efficiency is a slow transportation of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (mpc), responsible of introducing pyruvate into the mitochondria, is formed of two subunits, mpc1 and mpc2. We constructed stable CHO cell lines overexpressing both genes in order to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Overexpression of both genes was verified by qPCR and Western Blot, showing a significant increase compared to the basal level on the control cells. Kinetic evaluation of the CHO-mpc cells showed a 50% reduction of the lactate concentration respect to the control. Cell growth rate and maximum concentration were also increased, and an increase of 40% on the production of recombinant secreted placental alkaline phosphatase was observed. We show that transport of pyruvate into the mitochondria limits the efficiency of oxidation of glucose, which can be overcome by a cell engineering approach. Financial support by UNAM-DGAPA-PAPIIT IT-200315 and SEP-CONACyT Ciencia Básica 255445

Design of mimotopes of a conserved epitope in dengue and Zika viruses for the obtention of broadly neutralizing antibodies

Zika 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 mimotopes of a conserved epitope in dengue and Zika viruses for the obtention of broadly neutralizing antibodies

Zika 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

Methylation inhibition for coping with epigenetic-based heterogeneity of CHO cell subclones and improving recombinant MAB production

Please click Additional Files below to see the full abstract

Measuring Golgi\u27s redox potential in CHO cells and its response to hypoxic conditions

Please click Additional Files below to see the full abstract