Design and characterization of chimeric Rabies-SARS-CoV-2 virus-like particles for vaccine purposes.

Due to the high number of doses required to achieve adequate coverage in the context of COVID-19 pandemics, there is a great need for novel vaccine developments. In this field, there have been research approaches that focused on the production of SARS-CoV-2 virus-like particles. These are promising vaccine candidates as their structure is similar to that of native virions but they lack the genome, constituting a biosafe alternative. In order to produce these structures using mammal cells, it has been established that all four structural proteins must be expressed. Here we report the generation and characterization of a novel chimeric virus-like particle (VLP) that can be produced by the expression of a single novel fusion protein that contains SARS-CoV-2 spike (S) ectodomain fused to rabies glycoprotein membrane anchoring region in HEK293 cells. This protein is structurally similar to native S and can autonomously bud forming enveloped VLPs that resemble native virions both in size and in morphology, displaying S ectodomain and receptor binding domain (RBD) on their surface. As a proof of concept, we analyzed the immunogenicity of this vaccine candidate in mice and confirmed the generation of anti-S, anti-RBD, and neutralizing antibodies. KEY POINTS: • A novel fusion rabies glycoprotein containing S ectodomain was designed. • Fusion protein formed cVLPs that were morphologically similar to SARS-CoV-2 virions. • cVLPs induced anti-S, anti-RBD, and neutralizing antibodies in mice.

A COVID-19 vaccine candidate based on SARS-CoV-2 spike protein and immune-stimulating complexes.

Spike protein from SARS-CoV-2, the etiologic agent of the COVID-19 pandemic disease, constitutes a structural protein that proved to be the main responsible for neutralizing antibody production. Thus, its sequence is highly considered for the design of candidate vaccines. Animal cell culture represents the best option for the production of subunit vaccines based on recombinant proteins since they introduce post-translational modifications that are important to mimic the natural antigenic epitopes. Particularly, the human cell line HEK293T has been explored and used for the production of biotherapeutics since the products derived from them present human-like post-translational modifications that are important for the protein's activity and immunogenicity. The aim of this study was to produce and characterize a potential vaccine for COVID-19 based on the spike ectodomain (S-ED) of SARS-CoV-2 and two different adjuvants: aluminum hydroxide (AH) and immune-stimulating complexes (ISCOMs). The S-ED was produced in sHEK293T cells using a 1-L stirred tank bioreactor operated in perfusion mode and purified. S-ED characterization revealed the expected size and morphology. High N-glycan content was confirmed. S-ED-specific binding with the hACE2 (human angiotensin-converting enzyme 2) receptor was verified. The immunogenicity of S-ED was evaluated using AH and ISCOMs. Both formulations demonstrated the presence of anti-RBD antibodies in the plasma of immunized mice, being significantly higher for the latter adjuvant. Also, higher levels of IFN-γ and IL-4 were detected after the ex vivo immune stimulation of spleen-derived MNCs from ISCOMs immunized mice. Further analysis confirmed that S-ED/ISCOMs elicit neutralizing antibodies against SARS-CoV-2. KEY POINTS: Trimeric SARS-CoV-2 S-ED was produced in stable recombinant sHEK cells in serum-free medium. A novel S-ED vaccine formulation induced potent humoral and cellular immunity. S-ED formulated with ISCOMs adjuvant elicited a highly neutralizing antibody titer.

Design and optimization of an IgG human ELISA assay reactive to recombinant RBD SARS-CoV-2 protein.

Serology assays are essential tools to mitigate the effect of COVID-19, help to identify previous SARS-CoV-2 infections or vaccination, and provide data for surveillance and epidemiologic studies. In this study, we report the production and purification process of the receptor-binding domain (RBD) of SARS-CoV-2 in HEK293 cells, which allowed the design, optimization, and validation of an indirect ELISA (iELISA) for the detection of human anti-RBD antibodies. To find the optimal conditions of this iELISA, a multivariate strategy was performed throughout design of experiments (DoE) and response surface methodology (RSM), one of the main tools of quality by design (QbD) approach. The adoption of this strategy helped to reduce the time and cost during the method development stage and to define an optimum condition within the analyzed design region. The assay was then validated, exhibiting a sensitivity of 94.24 (86.01-98.42%; 95% CI) and a specificity of 95.96% (89.98-98.89%; 95% CI). Besides, the degree of agreement between quality results assessed using kappa's value was 0.92. Hence, this iELISA represents a high-throughput technique, simple to perform, reliable, and feasible to be scaled up to satisfy the current demands. Since RBD is proposed as the coating antigen, the intended use of this iELISA is not only the detection of previous exposure to the virus, but also the possibility of detecting protective immunity. KEY POINTS: • RBD was produced in 1-L bioreactor and highly purified. • An iELISA assay was optimized applying QbD concepts. • The validation procedure demonstrated that this iELISA is accurate and precise.

Gelatin and Tannic Acid Based Iongels for Muscle Activity Recording and Stimulation Electrodes.

Iongels are soft ionic conducting materials, usually composed of polymer networks swollen with ionic liquids (ILs), which are being investigated for applications ranging from energy to bioelectronics. The employment of iongels in bioelectronic devices such as bioelectrodes or body sensors has been limited by the lack of biocompatibility of the ILs and/or polymer matrices. In this work, we present iongels prepared from solely biocompatible materials: (i) a biobased polymer network containing tannic acid as a cross-linker in a gelatin matrix and (ii) three different biocompatible cholinium carboxylate ionic liquids. The resulting iongels are flexible and elastic with Young's modulus between 11.3 and 28.9 kPa. The morphology of the iongels is based on a dual polymer network system formed by both chemical bonding due to the reaction of the gelatin's amines with the polyphenol units and physical interactions between the tannic acid and the gelatin. These biocompatible iongels presented high ionic conductivity values, from 0.003 and up to 0.015 S·cm-1 at room temperature. Furthermore, they showed excellent performance as a conducting gel in electrodes for electromyography and electrocardiogram recording as well as muscle stimulation.

Screening of CHO-K1 endogenous promoters for expressing recombinant proteins in mammalian cell cultures.

For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, new endogenous promoters were discovered based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico-derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.

Pharmacokinetics Versus In Vitro Antiproliferative Potency to Design a Novel Hyperglycosylated hIFN-α2 Biobetter.

PURPOSE: IFN4N is a glycoengineered version of recombinant human interferon alpha 2 (rhIFN-α2) that was modified to exhibit four N-glycosylation sites. It shows reduced in vitro specific biological activity (SBA) mainly due to R23 mutation by N23. However, it has improved pharmacokinetics and led to a high in vivo antitumor activity in mice. In order to prepare a new IFN-based biobetter, this work compares the influence of glycosylation (affecting pharmacokinetics) with the in vitro antiproliferative SBA on the in vivo efficacy. METHODS: Based on IFN4N, three groups of muteins were designed, produced, and characterized. Group A: variants with the same glycosylation degree (4N) but higher in vitro antiproliferative SBA (R23 restored); group B: muteins with higher glycosylation degree (5N) but similar in vitro antiproliferative activity; and group C: variants with improved glycosylation (5N and 6N) and in vitro antiproliferative bioactivity. RESULTS: Glycoengineering was successful for improving pharmacokinetics, and R23 restoration considerably increased in vitro antiproliferative activity of new muteins compared to IFN4N. Hyperglycosylation was able to improve the in vivo efficacy similarly to or even better than R23 restoration. Additionally, the highest glycosylated mutein exhibited the lowest immunogenicity. CONCLUSIONS: Hyperglycosylation constitutes a successful strategy to prepare a novel IFN biobetter.

Effect of ANITVNITV peptide fusion on the bioactivity and pharmacokinetics of human IFN-α2b and a hyper-N-glycosylated variant.

Different strategies have been developed and successfully applied to biotherapeutics in order to improve their in vivo efficacy. The genetic fusion to natural or synthetic glycosylated peptides constitutes a promising strategy since it conserves the protein sequence and results in the improvement of the pharmacokinetic properties. The ANITVNITV peptide described by Perlmann and coworkers presents 9 amino acids and 2 potential N-glycosylation sites. Its fusion to FSH resulted in the increase of the molecular mass and negative charge of the protein. Consequently, the pharmacokinetics was considerably improved. The aim of the present study was to compare the influence of ANITVNITV peptide fusion on the physicochemical, biological and pharmacokinetic properties of native hIFN-α2b (IFNwt), which contains a single O-glycosylation site, and a hyperglycosylated variant (IFN4N), that bears, in addition, 4 N-linked glycans. The resulting molecules, IFNwtNter and IFN4NNter, evidenced a higher molecular mass and negative charge compared to IFNwt and IFN4N, respectively. Therefore, the pharmacokinetic properties of the new molecules were significantly improved. The molecules obtained by the synthetic peptide fusion strategy evidenced a decrease in their in vitro antiviral specific biological activities (SBA). However, in vitro antiproliferative SBA was differentially modified for IFNwtNter and IFN4NNter in comparison with the parental molecules. For IFNwtNter, a reduction in the antiproliferative SBA was also observed. Remarkably, the addition of the ANITVNITV peptide to the N-terminus of IFN4N had a positive impact on its growth-inhibitory activity. This feature together with its improved pharmacokinetics encourages the development of IFN4NNter as an IFN-α based biobetter.

Strategies to Develop Therapeutic N- and O-Hyperglycosylated Proteins.

Glycoengineering by N- and/or O-hyperglycosylation represents a procedure to introduce potential sites for adding N- and/or O-glycosyl structures to proteins with the aim of producing biotherapeutics with improved pharmacodynamic and pharmacokinetic properties. In this chapter, a detailed description of the steps routinely performed to generate new proteins having high content of N- and/or O-glycosyl moieties is carried out. The rational strategy involves the initial stage of designing N- and/or O-hyperglycosylated muteins to be expressed by mammalian cells and includes the upstream and downstream processing stages necessary to develop hyperglycosylated versions of the proteins of interest with the purpose of beginning the long road toward producing biobetters.

Glycosylation and antiproliferative activity of hyperglycosylated IFN-α2 potentiate HEK293 cells as biofactories.

Both CHO and HEK cells are interesting hosts for the production of biotherapeutics due to their ability to introduce post-translational modifications such as glycosylation. Even though oligosaccharide structures attached to proteins are conserved among eukaryotes, many differences have been found between therapeutic glycoproteins expressed in hamster and human derived cells. In this work, a hyperglycosylated IFN-α2b mutein (IFN4N) was produced in CHO and HEK cell lines and an extensive characterization of their properties was performed. IFN4NCHO exhibited a higher average molecular mass and more acidic isoforms compared to IFN4NHEK. In agreement with these results, a 2-times higher sialic acid content was found for IFN4NCHO in comparison with the HEK-derived protein. This result was in agreement with monosaccharide quantification and glycan's analysis using WAX chromatography and HILIC coupled to mass spectrometry; all methods supported the existence of highly sialylated and also branched structures for IFN4NCHO glycans, in contrast with smaller and truncated structures among IFN4NHEK glycans. Unexpectedly, those remarkable differences in the glycosylation pattern had not a considerable impact on the clearance rate of both molecules in rats. In fact, although IFN4NHEK reached maximum plasma concentration 3-times faster than IFN4NCHO, their elimination profile did not differ significantly. Also, despite the in vitro antiviral specific biological activity of both proteins was the same, IFN4NHEK was more efficient as an antiproliferative agent in different tumor-derived cell lines. Accordingly, IFN4NHEK showed a higher in vivo antitumor activity in animal models. Our results show the importance of an appropriate host selection to set up a bioprocess and potentiate the use of HEK293 cells for the production of a new hyperglycosylated protein-based pharmaceutical.

Epigenetic modulations rendering cell-to-cell variability and phenotypic metastability.

Tumor cells display phenotypic plasticity and heterogeneity due to genetic and epigenetic variations which limit the predictability of therapeutic interventions. Chromatin modifications can arise stochastically but can also be a consequence of environmental influences such as the microenvironment of cancer cells. A better understanding of the impact and dynamics of epigenetic modulation at defined chromosomal sites is required to get access to the underlying mechanisms. We investigated the epigenetic modulations leading to cell-to-cell heterogeneity in a tumor cell line model. To this end, we analyzed expression variance in 80 genetically uniform cell populations having a single-copy reporter randomly integrated in the genome. Single-cell analysis showed high intraclonal heterogeneity. Epigenetic characterization revealed that expression heterogeneity was accompanied by differential histone marks whereas contribution of DNA methylation could be excluded. Strikingly, some clones revealed a highly dynamic, stochastically altered chromatin state of the transgene cassette which was accompanied with a metastable expression pattern. In contrast, other clones represented a robust chromatin state of the transgene cassette with a stable expression pattern. Together, these results elucidate locus-specific epigenetic modulation in gene expression that contributes to phenotypic heterogeneity of cells and might account for cellular plasticity.

Improvement of in vitro stability and pharmacokinetics of hIFN-α by fusing the carboxyl-terminal peptide of hCG ß-subunit.

Improving in vivo half-life and in vitro stability of protein-based therapeutics is a current challenge for the biopharmaceutical industry. In particular, recombinant human interferon alpha-2b (rhIFN-α2b), which belongs to a group of cytokines extensively used for the treatment of viral diseases and cancers, shows a poor stability in solution and an extremely short plasma half-life which determines a strict therapeutic regimen comprising high and repeated doses. In this work, we have used a strategy based on the fusion of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG) ß-subunit, bearing four O-linked oligosaccharide recognition sites, to each or both N- and C-terminal ends of rhIFN-α2b. Molecules containing from 5 (CTP-IFN and IFN-CTP) to 9 (CTP-IFN-CTP) O-glycosylation sites were efficiently expressed and secreted to CHO cells supernatants, and exhibited antiviral and antiproliferative bioactivities in vitro. Significant improvements in pharmacokinetics in rats were achieved through this approach, since the doubly CTP-modified IFN variant showed a 10-fold longer elimination half-life and a 19-fold decreased plasma apparent clearance compared to the wild-type cytokine. Moreover, CTP-IFN-CTP demonstrated a significant increase in in vitro thermal resistance and a higher stability against plasma protease inactivation, both features attributed to the stabilizing effects of the O-glycans provided by the CTP moiety. These results constitute the first report that postulates CTP as a tag for improving both the in vitro and in vivo stability of rhIFN-α2b which, in turn, would positively influence its in vivo bioactivity.

Stability of single copy transgene expression in CHOK1 cells is affected by histone modifications but not by DNA methylation.

Intraclonal heterogeneity of genetically modified mammalian cells has been observed as a phenomenon that has a strong impact on overall transgene expression levels and that limits the predictability of transgene expression in genetically modified cells, thereby hampering single cell based screening approaches. The underlying mechanism(s) leading to this variance are poorly understood. To study the dynamics and mechanisms of heterogeneity of early stage silencing we analyzed the expression in more than 100 independent clones of CHOK1 cells that harbour genetically stable integrates of single copy reporter cassettes driven by EF1α and CMV promoters. Single cell analysis showed intraclonal variability with heterogeneity in expression in genetically uniform populations. DNA methylation is a well known mechanism responsible for silencing of gene expression. Interestingly, loss of expression was not associated with DNA methylation of the CMV promoter. However, in most of the clonal populations expression could be increased by inhibitors of the histone deacetylases (HDACi) suggesting that heterogeneity of transgene expression is crucially governed by histone modifications. Further, to determine if the epigenetic status of transgene expression is governed by the chromosomal integration locus we targeted heterologous expression cassettes into two chromosomal sites using recombinase mediated cassette exchange (RMCE). The expression status of a particular clone was faithfully re-established when the same promoter used. In this way the problem of early stage cell clone instability can be bypassed. However, upon introduction of an unrelated promoter methylation-independent silencing was observed. Together, these results suggest that histone modifications are the relevant mechanisms by which epigenetic modulation of transgene expression cassettes is governed in the early phase of clone generation.