Severe acute respiratory syndrome coronaviruses use the ACE2 receptor for viral entry while downregulating its activity, potentially leading to hypertension and major organ injuries. Dual-action technologies based on soluble ACE2 aimed to neutralize the virus while restoring ACE2's normal enzymatic activity. Here, we describe a novel general molecular design, VHHSpike-ACE2ECD-VHHAlbumin, in the toolbox of ACE2-centric therapeutic modalities. The optimized nanobody module VHHSpike afforded strong pan-specific binding against the entire sarbecoviral clade. This correlated with potent in vitro neutralization of pseudotyped virus variants of concern, with IC50 values in the picomolar range. Exogenous enzymatic activity was provided by the ACE2ECD module, which also contributed binding avidity via intrinsic homodimerization. Persistence of enzymatic activity in circulation was increased in vivo via the nanobody module VHHAlbumin optimized for serum albumin binding. Single-dose therapeutic administration of lead compound 72opt-ACE2-R28 demonstrated virus neutralization in lungs of hamsters at day 5 post-infection with SARS-CoV-2. In hypertensive mice maintained under continuous injection of angiotensin II, a single dose of 72opt-ACE2-R28 normalized systolic blood pressure, maintaining a 30 mmHg reduction after 24 h. Overall, encouraging coronavirus neutralization and hypertension reduction showed magnitudes and timeframes appropriate for treatment of typical acute infections. We discuss molecular bases of interactions with coronavirus spike protein molecules and future scale-up manufacturability toward clinical development of this modular design scaffold with high potential against emerging SARS-CoV-2 variants.

The development of robust Chinese Hamster Ovary (CHO) cell lines expressing high titers of monoclonal antibodies (MAbs) is central to bioprocess development. Following transfection and pool generation, clone selection is critical, as individual clones often behave differently in stirred-tank bioreactors. We propose a multivariate data analysis (MVDA) approach for clone selection that integrates productivity, growth, expression stability, and metabolism, with adaptable weighting based on process priorities. This method was applied to in-house data from CHO clones producing omalizumab. From 24 candidates, eight stable, high-performing clones were advanced for evaluation in 0.75-1 L bioreactors. MVDA revealed that including stability and metabolic parameters alters the ranking of lead clones compared with conventional screening. To assess scalability, cultures were run with or without air overlay to modulate dissolved CO2. Cultures without overlay reached up to 25% pCO2 (190 mmHg) and unexpectedly showed improved performance: 1.69-fold higher titer, 1.43-fold greater cell-specific productivity, 1.11-fold higher peak cell density, extended viability, and sustained product accumulation over 17-21 days. By integrating statistical tools and a historical dataset, our MVDA method identified a robust lead clone performing consistently across CO2 conditions, supporting its application in early upstream bioprocess development.

Concurrent circulation of SARS-CoV-2, influenza A (IAV), and respiratory syncytial virus (RSV) remains a major public health concern, driving the need for multivalent, combined vaccines. Here, we report the generation of trivalent enveloped virus-like particles (eVLPs) co-displaying SARS-CoV-2 spike (S), influenza H1, and RSV fusion (F) proteins produced in Chinese hamster ovary (CHO) cells. Using both transient and stable gene expression systems, we achieved intrinsic co-assembly of all three antigens within enveloped particles. Stable CHO eVLP expression yielded antigen volumetric titers of 50 mg/L. A prefusogenic F variant showed superior expression and incorporation efficiency compared to wild-type F and prefusion-stabilized forms. The adjuvanted trivalent eVLP vaccine elicited robust antigen-specific IgG and T-cell responses while inducing antibodies against a known protective RSV neutralizing site. Notably, the formulation provided complete protection against lethal influenza H1N1 challenge in mice. These results highlight the potential of CHO-derived eVLPs as a scalable multivalent vaccine platform capable of integrating multiple complex glycoproteins within a single product for broad protection against major respiratory viruses.

Surface plasmon resonance (SPR)-based biosensing enables the characterization of protein-protein interactions. Several SPR-based approaches have been designed to evaluate the binding mechanism between the angiotensin-converting enzyme 2 (ACE2) receptor and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein leading to a large range of kinetic and thermodynamic constants. This chapter describes a robust SPR assay based on the K5/E5 coiled-coil capture strategy that reduces artifacts. In this method, ACE2 receptors were produced with an E5-tag and immobilized as ligands in the SPR assay. This chapter details methods for high-yield production and purification of the studied proteins, functionalization of the sensor chip, conduction of the SPR assay, and data analysis.

The recent COVID-19 pandemic revealed an urgent need to develop robust cell culture platforms which can react rapidly to respond to this kind of global health issue. Chinese hamster ovary (CHO) stable pools can be a vital alternative to quickly provide gram amounts of recombinant proteins required for early-phase clinical assays. In this study, we analyze early process development data of recombinant trimeric spike protein Cumate-inducible manufacturing platform utilizing CHO stable pool as a preferred production host across three different stirred-tank bioreactor scales (0.75, 1, and 10 L). The impact of cell passage number as an indicator of cell age, methionine sulfoximine (MSX) concentration as a selection pressure, and cell seeding density was investigated using stable pools expressing three variants of concern. Multivariate data analysis with principal component analysis and batch-wise unfolding technique was applied to evaluate the effect of critical process parameters on production variability and a random forest (RF) model was developed to forecast protein production. In order to further improve process understanding, the RF model was analyzed with Shapley value dependency plots so as to determine what ranges of variables were most associated with increased protein production. Increasing longevity, controlling lactate build-up, and altering pH deadband are considered promising approaches to improve overall culture outcomes. The results also demonstrated that these pools are in general stable expressing similar level of spike proteins up to cell passage 11 (~31 cell generations). This enables to expand enough cells required to seed large volume of 200-2000 L bioreactor.

The COVID-19 pandemic has emphasised the importance of vaccines and preparedness against viral threats crossing species barriers. In response, a worldwide vaccination campaign targeting SARS-CoV-2 was implemented, which provides some cross-protective immunological memory to other coronavirus species with zoonotic potential. Following a vaccination regimen against SARS-CoV-2 spike in a preclinical mouse model, we were able to demonstrate the induction of neutralizing antibodies towards multiple human ACE2 (hACE2)-binding sarbecovirus spikes. Importantly, compared to vaccines based on the SARS-CoV-2 Reference strain, vaccines based on Omicron spike sequences induced drastically less broadly cross-protective neutralizing antibodies against other hACE2-binding sarbecoviruses. This observation remained true whether the vaccination regimens were based on protein subunit or mRNA / LNP vaccines. Overall, while it may be necessary to update vaccine antigens to combat the evolving SARS-CoV-2 virus for enhanced protection from COVID-19, Reference-based vaccines may be a more valuable tool to protect against novel coronavirus zoonoses.

The continuous improvement of expression platforms is necessary to respond to the increasing demand for recombinant proteins that are required to carry out structural or functional studies as well as for their characterization as biotherapeutics. While transient gene expression (TGE) in mammalian cells constitutes a rapid and well-established approach, non-clonal stably transfected cells, or "pools," represent another option, which is especially attractive when recurring productions of the same protein are required. From a culture volume of just a few liters, stable pools can provide hundreds of milligrams to gram quantities of high-quality secreted recombinant proteins.In this chapter, we describe a highly efficient and cost-effective procedure for the generation of Chinese Hamster Ovary cell stable pools expressing secreted recombinant proteins using commercially available serum-free media and polyethylenimine (PEI) as the transfection reagent. As a specific example of how this protocol can be applied, the production and downstream purification of recombinant His-tagged trimeric SARS-CoV-2 spike protein ectodomain (SmT1) are described.

PDF file, 297KB, TGF-B1 neutralization using a (TBRII)2 trap having a natural or artificial linker sequence.

PDF file, 971KB, Assessment of the effects of TGF-B and (TBRII)2 trap on 4T1 cells in vitro.

BACKGROUND:As the COVID-19 pandemic continues to evolve, novel vaccines need to be developed that are readily manufacturable and provide clinical efficacy against emerging SARS-CoV-2 variants. Virus-like particles (VLPs) presenting the spike antigen at their surface offer remarkable benefits over other vaccine antigen formats; however, current SARS-CoV-2 VLP vaccines candidates in clinical development suffer from challenges including low volumetric productivity, poor spike antigen density, expression platform-driven divergent protein glycosylation and complex upstream/downstream processing requirements. Despite their extensive use for therapeutic protein manufacturing and proven ability to produce enveloped VLPs, Chinese Hamster Ovary (CHO) cells are rarely used for the commercial production of VLP-based vaccines.METHODS:Using CHO cells, we aimed to produce VLPs displaying the full-length SARS-CoV-2 spike. Affinity chromatography was used to capture VLPs released in the culture medium from engineered CHO cells expressing spike. The structure, protein content, and glycosylation of spikes in VLPs were characterized by several biochemical and biophysical methods. In vivo, the generation of neutralizing antibodies and protection against SARS-CoV-2 infection was tested in mouse and hamster models.RESULTS:We demonstrate that spike overexpression in CHO cells is sufficient by itself to generate high VLP titers. These VLPs are evocative of the native virus but with at least three-fold higher spike density. In vivo, purified VLPs elicit strong humoral and cellular immunity at nanogram dose levels which grant protection against SARS-CoV-2 infection.CONCLUSIONS:Our results show that CHO cells are amenable to efficient manufacturing of high titers of a potently immunogenic spike protein-based VLP vaccine antigen.

PDF file, 832KB, Schematics and molecular models of single-chain activin and BMP traps.

PDF file, 243KB, Improved neutralization of BMP-2 using single-chain bivalent trap (BMPR1a)2 compared to monovalent receptor BMPR1a-ED.

PDF file, 2243KB, Molecular dynamics analysis of the (TBRII)2 trap in complex with TGF-B3.

PDF file, 293KB, Quantification of proliferating cells in 4T1 tumors.

SARS-CoV-2 subunit vaccines continue to be the focus of intense clinical development worldwide. Protein antigens in these vaccines most commonly consist of the spike ectodomain fused to a heterologous trimerization sequence, designed to mimic the compact, prefusion conformation of the spike on the virus surface. Since 2020, we have produced dozens of such constructs in CHO cells, consisting of spike variants with different mutations fused to different trimerization sequences. This set of constructs displayed notable conformational heterogeneity, with two distinct trimer species consistently detected by analytical size exclusion chromatography. A recent report showed that spike ectodomain fusion constructs can adopt an alternative trimer conformation consisting of loosely associated ectodomain protomers. Here, we applied multiple biophysical and immunological techniques to demonstrate that this alternative conformation is formed to a significant extent by several SARS-CoV-2 variant spike proteins. We have also examined the influence of temperature and pH, which can induce inter-conversion of the two forms. The substantial structural differences between these trimer types may impact their performance as vaccine antigens.

Monitoring antibody response to SARS-CoV-2 is critical for assessing the humoral response, especially important considering the emergence of multiple SARS-CoV-2 variants of concern (VOCs).

Several key mutations in the Spike protein receptor binding domain (RBD) have been identified to influence its affinity for the human Angiotensin-Converting Enzyme 2 (ACE2). Here, we perform a comparative study of the ACE2 binding to the wild type (Wuhan) RBD and some of its variants: Alpha B.1.1.7, Beta B.1.351, Delta B.1.617.2, Kappa B.1.617.1, B.1.1.7 + L452R and Omicron B.1.1.529. Using a coiled-coil mediated tethering approach of ACE2 in a novel surface plasmon resonance (SPR)-based assay, we measured interactions at different temperatures. Binding experiments at 10 °C enhanced the kinetic dissimilarities between the RBD variants and allowed a proper fit to a Langmuir 1:1 model with high accuracy and reproducibility, thus unraveling subtle differences within RBD mutants and ACE2 glycovariants. Our study emphasizes the importance of SPR-based assay parameters in the acquisition of biologically relevant data and offers a powerful tool to deepen our understanding of the role of the various RBD mutations in ACE2 interaction binding parameters.

Inactivated Foot-and-Mouth Disease (FMD) vaccine has proven to be effective in the control of the disease. However, its production has some disadvantages, including the costly biosafety facilities required for the production of huge amounts of growing live virus, the need of an exhaustive purification process to eliminate non-structural proteins of the virus in the final formulations in order to differentiate infected from vaccinated animals and variable local regulatory restrictions to produce and commercialize the vaccine. Thus, a novel vaccine against FMD that overcome these restrictions is desirable. Although many developments have been made in this regard, most of them failed in terms of efficacy or when considering their transferability to the industry. We have previously reported the use of transient gene expression in mammalian cells to produce FMD virus-like particles (VLPs) as a novel vaccine for FMD and demonstrated the immunogenicity of the recombinant structures in animal models. Here, we report the optimization of the production system by assaying different DNA:polyethylenimine concentrations, cell densities, and direct and indirect protocols of transfection. Also, we evaluated the reproducibility and scalability of the technology to produce high yields of recombinant VLPs in a cost-effective and scalable system compatible with industrial tech-transfer of an effective and safe vaccine.

Abstract Deregulation of TGF-β superfamily signaling is a causative factor in many diseases. Here we describe a protein engineering strategy for the generation of single-chain bivalent receptor traps for TGF-β superfamily ligands. Traps were assembled using the intrinsically disordered regions flanking the structured binding domain of each receptor as "native linkers" between two binding domains. This yields traps that are approximately threefold smaller than antibodies and consists entirely of native receptor sequences. Two TGF-β type II receptor-based, single-chain traps were designed, termed (TβRII)2 and (TβRIIb)2, that have native linker lengths of 35 and 60 amino acids, respectively. Both single-chain traps exhibit a 100 to 1,000 fold higher in vitro ligand binding and neutralization activity compared with the monovalent ectodomain (TβRII-ED), and a similar or slightly better potency than pan–TGF-β–neutralizing antibody 1D11 or an Fc-fused receptor trap (TβRII-Fc). Despite its short in vivo half-life (<1 hour), which is primarily due to kidney clearance, daily injections of the (TβRII)2 trap reduced the growth of 4T1 tumors in BALB/c mice by 50%, an efficacy that is comparable with 1D11 (dosed thrice weekly). In addition, (TβRII)2 treatment of mice with established 4T1 tumors (100 mm3) significantly inhibited further tumor growth, whereas the 1D11 antibody did not. Overall, our results indicate that our rationally designed bivalent, single-chain traps have promising therapeutic potential. Mol Cancer Ther; 11(7); 1477–87. ©2012 AACR.