Zika virus-like particles (VLPs): Stable cell lines and continuous perfusion processes as a new potential vaccine manufacturing platform

Alvim, Renata G. F.; Itabaiana, Ivaldo; Castilho, Leda R.

doi:10.1016/j.vaccine.2019.05.064

Cited by 30 publications

(43 citation statements)

References 32 publications

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“…1A, left panel). However, differently from previous studies that adopt transient protein expression techniques 3 , we focused on stable and constitutive gene expression because transgene integration in the cell genome enhances scalability and significantly decreases costs of recombinant protein production in mammalian cells 4 . Due to the challenges posed by the pandemic, such as the urgency and the disruption of international supply chains for reagents and synthetic gene constructs, we decreased time and costs by using an old-fashioned technique of co-transfecting the plasmid containing the S gene with an intellectual property-free plasmid containing an eukaryotic selection marker.…”

Section: Resultsmentioning

confidence: 99%

“…HEK293-3F6 cells (NRC Canada) growing in suspension in the chemically-defined, animal component-free HEK-TF (Xell AG) culture medium were stably transfected by lipofection using a broad-spectrum reagent (Lipofectamine 3000, Thermo Fisher) as described previously 4 . A total DNA concentration of 0.9 µg/mL was used, combining two vectors: the pαH vector (at 0.75 µg/mL) containing the sequence encoding the ectodomain (aminoacids 1-1208) of the spike protein in the prefusion conformation 1 (kindly provided by B. Graham, VRC/NIH, also available from BEI Resources under #NR-52563), and an empty vector containing the neomycin phosphotransferase gene (at 0.15 µg/mL).…”

Section: Methodsmentioning

confidence: 99%

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Development and large-scale validation of a highly accurate SARS-COV-2 serological test using regular test strips for autonomous and affordable finger-prick sample collection, transportation, and storage

Alvim

Lima

Rodrigues

et al. 2020

Preprint

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We describe a cost-effective, scalable technology to produce SARS-COV-2 spike (S) protein based on stable expression in HEK293 cells, and its use to develop a highly specific and sensitive ELISA test. The assay allows early detection of anti-S IgG seroconversion and endpoint titers correlate with virus neutralization. The low-cost S-antigen production, together with sample collection by finger prick and dried blood spots, allowed the development of a half-dollar test that fits the urgent need for large-scale serological surveillance in low-income countries.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Development and large-scale validation of a highly accurate SARS-COV-2 serological test using regular test strips for autonomous and affordable finger-prick sample collection, transportation, and storage

Alvim

Lima

Rodrigues

et al. 2020

Preprint

Self Cite

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“…BALB/c adult female mice, aged 6 to 8 weeks, were obtained from NAL-UFF, LAT-UFRJ produced and purified as previously described [37]. ZIKV and DENV NS1 proteins were produced and purified as reported previously.…”

Section: Mice and Treatmentsmentioning

confidence: 99%

“…1 E) followed by a peak of IgG at 14 d.p.i., (Fig. 1 F) as detected using Zika virus-like particles (VLPs) that display proteins E and M in their mature form [37]. Domain III of protein E (EDIII) is a target of neutralizing antibodies for different flaviviruses [35,[38][39][40] and we therefore also looked for serum IgG binding to this portion of protein E. We found that EDIII specific IgG peaked in serum at 14 d.p.i.…”

Section: Immunocompetent Balb/c Mice Develop a Specific Antibody Respmentioning

confidence: 99%

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Sustained antibody response to ZIKV infection induced by NS1 protein is accompanied by the progressive appearance of autoreactive antibodies and cross-reactive B cell clones

Cavazzoni

Bozza

Tostes

et al. 2020

Preprint

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Antibodies are key players in controlling viral infections. However, in addition to antigen-specific responses to viral antigens, humoral immune response can generate polyreactive and autoreactive antibodies of unknown function. Dengue and Zika virus infections have been linked to autoimmune disorders including Guillain-Barrè syndrome. A unique feature of flaviviruses is the secretion of non-structural protein 1 (NS1) by infected cells. NS1 is highly immunogenic and antibodies targeting NS1 can have both protective and pathogenic roles. In the present study, we investigated the humoral immune response to Zika virus NS1 and found NS1 to be an immunodominant viral antigen correlated to the presence of autoreactive antibodies. Through single B cell cultures, we coupled binding assays and BCR sequencing, confirming the immunodominance of NS1 and the presence of self-reactive clones in germinal centers both after infection and immunization, some of which were cross-reactivity with NS1. Anti-NS1 B cell clones showed features related to pathogenic autoreactive antibodies. Our findings demonstrate NS1 immunodominance at the cellular level as well as a potential role for NS1 in ZIKV associated autoimmune manifestations.

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Integrating high cell density cultures with adapted laboratory evolution for improved Gag‐HA virus‐like particles production in stable insect cell lines

Fernandes

Correia

Sousa

et al. 2021

Biotech & Bioengineering

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Stable insect cell lines are emerging as an alternative to the insect cell-baculovirus expression vector system (IC-BEVS) for protein expression, benefiting from being a virus-free, nonlytic system. Still, the titers achieved are considerably lower. In this study, stable insect (Sf-9 and High Five) cells producing Gag virus-like particles (VLPs) were first adapted to grow under hypothermic culture conditions (22°C instead of standard 27°C), and then pseudotyped with a model membrane protein (influenza hemagglutinin [HA]) for expression of Gag-HA VLPs. Adaptation to lower temperature led to an increase in protein titers of up to 12-fold for p24 (as proxy for Gag-VLP) and sixfold for HA, with adapted Sf-9 cells outperforming High Five cells.Resulting Gag-HA VLPs producer Sf-9 cells were cultured to high cell densities, that is, 100 × 10 6 cell/ml, using perfusion (ATF® 2) in 1 L stirred-tank bioreactors. Specific p24 and HA production rates were similar to those of batch culture, enabling to increase volumetric titers by 7-8-fold without compromising the assembly of Gag-HA VLPs. Importantly, the antigen (HA) quantity in VLPs generated using stable adapted cells in perfusion was ≈5-fold higher than that from IC-BEVS, with the added benefit of being a baculovirus-free system. This study demonstrates the potential of combining stable expression in insect cells adapted to hypothermic culture conditions with perfusion for improving Gag-HA VLPs production.

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Zika virus-like particles (VLPs): Stable cell lines and continuous perfusion processes as a new potential vaccine manufacturing platform

Cited by 30 publications

References 32 publications

Development and large-scale validation of a highly accurate SARS-COV-2 serological test using regular test strips for autonomous and affordable finger-prick sample collection, transportation, and storage

Development and large-scale validation of a highly accurate SARS-COV-2 serological test using regular test strips for autonomous and affordable finger-prick sample collection, transportation, and storage

Sustained antibody response to ZIKV infection induced by NS1 protein is accompanied by the progressive appearance of autoreactive antibodies and cross-reactive B cell clones

Integrating high cell density cultures with adapted laboratory evolution for improved Gag‐HA virus‐like particles production in stable insect cell lines

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