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

Improved production of viru like particles of Cowpea Chlorotic Mottle virus using the baculovirus expression system

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Transcriptional and metabolic response of CHO cells to different carbon dioxide concentrations

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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

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

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Molecular and process design for rotavirus-like particle production in Saccharomyces cerevisiae

Background: Virus-like particles (VLP) have an increasing range of applications including vaccination, drug delivery, diagnostics, gene therapy and nanotechnology. These developments require large quantities of particles that need to be obtained in efficient and economic processes. Production of VLP in yeast is attractive, as it is a low-cost protein producer able to assemble viral structural proteins into VLP. However, to date only single-layered VLP with simple architecture have been produced in this system. In this work, the first steps required for the production of rotavirus-like particles (RLP) in S. cerevisiae were implemented and improved, in order to obtain the recombinant protein concentrations required for VLP assembly. Results: The genes of the rotavirus structural proteins VP2, VP6 and VP7 were cloned in four Saccharomyces cerevisiae strains using different plasmid and promoter combinations to express one or three proteins in the same cell. Performance of the best constructs was evaluated in batch and fed-batch cultures using a complete synthetic media supplemented with leucine, glutamate and succinate. The strain used had an important effect on recombinant protein concentration, while the type of plasmid, centromeric (YCp) or episomal (YEp), did not affect protein yields. Fed-batch culture of the PD.U-267 strain resulted in the highest concentration of rotavirus proteins. Volumetric and specific productivities increased 28.5- and 11-fold, respectively, in comparison with batch cultures. Expression of the three rotavirus proteins was confirmed by immunoblotting and RLP were detected using transmission electron microscopy. Conclusions: We present for the first time the use of yeast as a platform to express multilayered rotavirus-like particles. The present study shows that the combined use of molecular and bioprocess tools allowed the production of triple-layered rotavirus RLP. Production of VLP with complex architecture in yeasts could lead to the development of new vaccine candidates with reduced restrictions by regulatory agencies, using the successful experience with other yeast-based VLP vaccines commercialized worldwide

Design of a vaccine against dengue and Zika viruses based on a mimotope of the envelope dimer epitope

Zika 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

Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters

The temperature inducible expression system, based on the pL and/or pR phage lambda promoters regulated by the thermolabile cI857 repressor has been widely use to produce recombinant proteins in prokariotic cells. In this expression system, induction of heterologous protein is achieved by increasing the culture temperature, generally above 37°C. Concomitant to the overexpression of heterologous protein, the increase in temperature also causes a variety of complex stress responses. Many studies have reported the use of such temperature inducible expression system, however only few discuss the simultaneous stress effects caused by recombinant protein production and the up-shift in temperature. Understanding the integral effect of such responses should be useful to develop improved strategies for high yield protein production and recovery. Here, we describe the current status of the heat inducible expression system based on the pL and/or pR λ phage promoters, focusing on recent developments on expression vehicles, the stress responses at the molecular and physiological level that occur after heat induction, and bioprocessing factors that affect protein overexpression, including culture operation variables and induction strategies

Virus like particles expressed in insect cells and mammalian cells as a plataform for the development of a Zika vaccine

The Zika virus (ZIKV) is an emergent mosquito-borne virus of the flaviviridae family that has caused severe challenges to global health since 2015. It causes Guillain-Barré syndrome and congenital malformations. Although case numbers have decreased, it is important to develop a vaccine for outbreaks. One alternative is the use of virus like particles (VLP) as vaccines. ZIKV is enveloped and is composed of three main structural proteins: enveloped (E), pre-membrane (M), and capsid. The main target of neutralizing antibodies is the E glycoprotein, which is glycosylated in some strains. The N-glycosylation profile is determined by the producer host cell. ZIKV has both insect and human hosts, and the N-glycosylation profile of the E protein produced by each host is expected to be different. It can be expected that glycosylation pattern has an impact on immune response against the E protein, but its effect on the immunogenicity against VLP of ZIKV has not been determined. For this reason, the present work seeks in the first instance, to design and produce VLP of ZIKV (ZIK VLP) in insect and human cells. To produce ZIK VLP, a chimeric gene was designed containing the M and E ZIKV genes fused to the transmembrane (TM) domain of Japanese encephalitis virus (JEV), ss- M-E (minus) ZIKV, TM JEV. After that, a recombinant baculovirus that contains the chimeric sequence was generated for VLP expression in insect cells. Production kinetics were followed, and the best conditions for VLP production were determined. For expression in human cells, the chimera was introduced into lentiviral vectors and was produced in HEK-293T/17 cells and used for the stable transfection of HEK-293 cells producing ZIKV VLP. High producing clones were selected by flow citometry. ZIK VLP were purified and characterized. In this work, strategies were developed for the efficient production of PPV in both systems, which can be used for further research. Ongoing studies are focused on determining the glycosylation profile of VLP expressed in both systems and on investigating the impact of glycosylation pattern of ZIK VLP immunogenicity in an animal model

Defining the multiplicity and type of infection for the production of Zaire Ebola virus-like particles in the insect cell baculovirus expression system

Ebola virus hemorrhagic fever affects thousands of people worldwide with high mortality rates. The Ebola virus has a short incubation time between 2-21 days and death usually occurs within 4-10 days1. Ebola virus disease is characterized by a sudden onset of fever, weakness, headache, diarrhea and vomiting, internal and external bleeding2. In the Filovirus family, Zaire Ebola virus (ZEBOV) is the most aggressive and virulent species, its fatality rates have been reported to be up to 90%3. Even when important advances in vaccine development have occurred, the need of safe and effective vaccines persists4. An alternative is the production of virus-like particles, which are formed by the recombinant virus structural proteins that self-assemble into highly immunogenic structures5. The ZEBOV contains three main structural proteins: the glycoprotein (GP), the viral matrix protein 40 (VP40) and the nucleoprotein (NP). GP induces humoral and cellular responses by itself but when VP40 is co-expressed, the immune response increases in a mouse model6. NP determines the structure of the resulting VLP. To our knowledge, there is no information about the production conditions that result in coexpression and assembly of ZEBOV recombinant proteins. In this work, a multifactorial experimental design was used to evaluate 32 different conditions for the production of the ZEBOV structural proteins utilizing the insect cell-baculovirus expression system technology (BEST). Multiplicity (MOI = 0.1 or 5 ufp/cell) and consecutive times of infection (0 or 6 hours after the first infection) were the principal factors, and the production of each recombinant protein and assembly of VLP were the evaluated responses. We observed that multiplicity of infection had an impact over expression of the recombinant proteins, higher multiplicities increased yield and VLP assembly. In contrast, later times of infection reduced the production of each protein. The initial presence of VP40 resulted in a higher concentration of NP. The conditions where the simultaneous expression of the three structural proteins and where VLP were detected were identified. The highest MOIs for bacVP40 and bacGP were needed. bacNP should be added during the initial infection with an MOI of 0.1, or at 6 hpi at MOI of 5. The obtained ZEBOV-VLPs were similar to native virus. The obtained VLP are a candidate vaccine under evaluation. Research performed thanks to the financial support of PAPIIT-UNAM IT200418 and CONACyT 247101. References: 1. Shuaib F, Gunala R, Musa EO, Mahoney FJ, et al., 2014. Ebola virus disease outbreak-Nigeria, July–September 2014. Morb. Mortal. Wkly. Rep. 63 (39),867–872. 2. Qiu X, Audet J, Wong G. Fernando L, et al., 2013. Sustained protection virus infection following treatment of infected nonhuman primates with ZMAb. Sci. Rep. 3, 3. Richardson JS, Wong G, Pillet S, Schindle S, et al., 2011. Evaluation of different strategies for post-exposure treatment of Ebola virus infection in rodents. J.Bioterror. Biodef. S1, 007 4. Ige, Ohimain E, 2016. Recent advances in the development of vaccines for Ebola virus disease. Virus Research 211: 174-185. 5. Palomares LA, Ramírez OT, 2009. Challenges for the production of virus-like particles in insect cells: The case of rotavirus-like particles. Biochem. Eng. J. 45: 158-167. 6. Wahl-Jensen, V. et al (2005). Role of Ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages. Journal of Virology, 79(4), 2413-241