SARS-CoV-2 envelope protein-derived extracellular vesicles act as potential media for viral spillover.

Extracellular vesicles (EVs) are shown to be a novel viral transmission model capable of increasing a virus's tropism. According to our earlier research, cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or transfected with envelope protein plasmids generate a novel type of EVs that are micrometer-sized and able to encase virus particles. Here, we showed the capacity of these EVs to invade various animals both in vitro and in vivo independent of the angiotensin-converting enzyme 2 receptor. First, via macropinocytosis, intact EVs produced from Vero E6 (monkey) cells were able to enter cells from a variety of animals, including cats, dogs, bats, hamsters, and minks, and vice versa. Second, when given to zebrafish with cutaneous wounds, the EVs showed favorable stability in aqueous environments and entered the fish. Moreover, infection of wild-type (WT) mice with heterogeneous EVs carrying SARS-CoV-2 particles led to a strong cytokine response and a notable amount of lung damage. Conversely, free viral particles did not infect WT mice. These results highlight the variety of processes behind viral transmission and cross-species evolution by indicating that EVs may be possible vehicles for SARS-CoV-2 spillover and raising risk concerns over EVs' potential for viral gene transfer.

Association between Ultradian Rhythms of Body Temperature in Small Mammals and Earth's Crust Stress.

Association was assessed between the data harvested by a long-baseline laser interference deformograph and the dynamics of body temperature (BT) in hamsters deprived of natural daily light-darkness changes. The power spectral data revealed the positive correlation between simultaneous time series of hamster BT and the Earth's crust deformation (ECD). The superposed epoch analysis established an association between abrupt upstrokes of hamster BT and ECD increments. Thus, the direct relationships between BT dynamics (reflecting predominance of sympathetic part of autonomic nervous system) and ECD (according to long-baseline laser interference deformography) were established. The study observed synchronization of the free-running circadian rhythm of hamster BT with the tidal stress in Earth's lithosphere. Further studies are needed to find the physical factor underlying the revealed relationships.

Three SARS-CoV-2 spike protein variants delivered intranasally by measles and mumps vaccines are broadly protective.

As the new SARS-CoV-2 Omicron variants and subvariants emerge, there is an urgency to develop intranasal, broadly protective vaccines. Here, we developed highly efficacious, intranasal trivalent SARS-CoV-2 vaccine candidates (TVC) based on three components of the MMR vaccine: measles virus (MeV), mumps virus (MuV) Jeryl Lynn (JL1) strain, and MuV JL2 strain. Specifically, MeV, MuV-JL1, and MuV-JL2 vaccine strains, each expressing prefusion spike (preS-6P) from a different variant of concern (VoC), were combined to generate TVCs. Intranasal immunization of IFNAR1-/- mice and female hamsters with TVCs generated high levels of S-specific serum IgG antibodies, broad neutralizing antibodies, and mucosal IgA antibodies as well as tissue-resident memory T cells in the lungs. The immunized female hamsters were protected from challenge with SARS-CoV-2 original WA1, B.1.617.2, and B.1.1.529 strains. The preexisting MeV and MuV immunity does not significantly interfere with the efficacy of TVC. Thus, the trivalent platform is a promising next-generation SARS-CoV-2 vaccine candidate.

In vitro experimental conditions and tools can influence the safety and biocompatibility results of antimicrobial electrospun biomaterials for wound healing.

Electrospun (ES) fibrous nanomaterials have been widely investigated as novel biomaterials. These biomaterials have to be safe and biocompatible; hence, they need to be tested for cytotoxicity before being administered to patients. The aim of this study was to develop a suitable and biorelevant in vitro cytotoxicity assay for ES biomaterials (e.g. wound dressings). We compared different in vitro cytotoxicity assays, and our model wound dressing was made from polycaprolactone and polyethylene oxide and contained chloramphenicol as the active pharmaceutical ingredient. Baby Hamster Kidney cells (BHK-21), human primary fibroblasts and MTS assays together with real-time cell analysis were selected. The extract exposure and direct contact safety evaluation setups were tested together with microscopic techniques. We found that while extract exposure assays are suitable for the initial testing, the biocompatibility of the biomaterial is revealed in in vitro direct contact assays where cell interactions with the ES wound dressing are evaluated. We observed significant differences in the experimental outcome, caused by the experimental set up modification such as cell line choice, cell medium and controls used, conducting the phosphate buffer washing step or not. A more detailed technical protocol for the in vitro cytotoxicity assessment of ES wound dressings was developed.

Hamster spermatozoa incorporate hypotaurine via TauT for self-protection.

In brief: Mammalian spermatozoa actively generate reactive oxygen species (ROS) during capacitation, a maturational process necessary for fertilization in vivo. This study shows that hypotaurine, a precursor of taurine present in the oviduct, is incorporated and concentrated in hamster sperm cells via the taurine transporter, TauT, for cytoprotection against self-produced ROS. Abstract: To achieve fertilization competence, mammalian spermatozoa undergo capacitation, during which they actively generate reactive oxygen species (ROS). Therefore, mammalian spermatozoa must protect themselves from these self-generated ROS. The mammalian oviductal fluid is rich in hypotaurine, a taurine precursor, which reportedly protects mammalian spermatozoa, including those of hamsters, from ROS; however, its precise mechanism remains unknown. This study aimed to elucidate the mechanism underlying hypotaurine-mediated protection of spermatozoa from ROS using hamsters, particularly focusing on the taurine/hypotaurine transporter TauT. The effect of hypotaurine on sperm motility and ROS levels was tested using sperm motility analysis and the CellROX dye and luminol assays. RNA sequencing analysis was performed to verify TauT expression. We found that hypotaurine was necessary for maintaining sperm motility and hyperactivated motility. Hypotaurine did not scavenge extracellular ROS but lowered intracellular ROS levels and was incorporated and concentrated in hamster spermatozoa. TauT was detected at both mRNA and protein levels. ß-Alanine blocked hypotaurine transport, increased intracellular ROS levels, and inhibited hyperactivation. Elimination of Na+ or Cl- ions inhibited hypotaurine transport and increased intracellular ROS levels. Thus, these results indicated that hamster spermatozoa incorporated and concentrated hypotaurine in sperm cells via TauT to protect themselves from self-generated ROS.

Protocol for microfluidic single-cell cultivation and live-cell imaging of Chinese hamster ovary suspension cell lines.

Microfluidic single-cell cultivation (MSCC) is a powerful tool for investigating the cellular behavior of various cell types at the single-cell level. Here, we present a protocol specifically developed for the reliable and reproducible MSCC of industrially relevant Chinese hamster ovary (CHO) suspension cell lines. We summarize critical experimental steps from the initial seed train up to the final MSCC experiment, with a special focus on pre-culture management and medium preparation, device inoculation, and the establishment of a constant medium perfusion.

Human parainfluenza virus 3 vaccine candidates attenuated by codon-pair deoptimization are immunogenic and protective in hamsters.

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.

Novel transcription and replication-competent virus-like particles system modelling the Nipah virus life cycle.

Nipah virus (NiV), a highly pathogenic Henipavirus in humans, has been responsible for annual outbreaks in recent years. Experiments involving live NiV are highly restricted to biosafety level 4 (BSL-4) laboratories, which impedes NiV research. In this study, we developed transcription and replication-competent NiV-like particles (trVLP-NiV) lacking N, P, and L genes. This trVLP-NiV exhibited the ability to infect and continuously passage in cells ectopically expressing N, P, and L proteins while maintaining stable genetic characteristics. Moreover, the trVLP-NiV displayed a favourable safety profile in hamsters. Using the system, we found the NiV nucleoprotein residues interacting with viral RNA backbone affected viral replication in opposite patterns. This engineered system was sensitive to well-established antiviral drugs, innate host antiviral factors, and neutralizing antibodies. We then established a high-throughput screening platform utilizing the trVLP-NiV, leading to the identification of tunicamycin as a potential anti-NiV compound. Evidence showed that tunicamycin inhibited NiV replication by decreasing the infectivity of progeny virions. In conclusion, this trVLP-NiV system provided a convenient and versatile molecular tool for investigating NiV molecular biology and conducting antiviral drug screening under BSL-2 conditions. Its application will contribute to the development of medical countermeasures against NiV infections.

A hierarchical structure in the N-glycosylation process governs the N-glycosylation output: prolonged cultivation induces glycoenzymes expression variations that are reflected in the cellular N-glycome but not in the protein and site-specific glycoprofile of CHO cells.

N-glycosylation is a central component in the modification of secretory proteins. One characteristic of this process is a heterogeneous output. The heterogeneity is the result of both structural constraints of the glycoprotein as well as the composition of the cellular glycosylation machinery. Empirical data addressing correlations between glycosylation output and glycosylation machinery composition are seldom due to the low abundance of glycoenzymes. We assessed how differences in the glycoenzyme expression affected the N-glycosylation output at a cellular as well as at a protein-specific level. Our results showed that cellular N-glycome changes could be correlated with the variation of glycoenzyme expression, whereas at the protein level differential responses to glycoenzymes alterations were observed. We therefore identified a hierarchical structure in the N-glycosylation process: the enzyme levels in this complex pathway determine its capacity (reflected in the N-glycome), while protein-specific parameters determine the glycosite-specificity. What emerges is a highly variable and adaptable protein modification system that represents a hallmark of eukaryotic cells.

Increased stable integration efficiency in CHO cells through enhanced nuclear localization of Bxb1 serine integrase.

BACKGROUND: Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase. METHODS: In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration. RESULTS: The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion. CONCLUSIONS: This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.

AZD3152 neutralizes SARS-CoV-2 historical and contemporary variants and is protective in hamsters and well tolerated in adults.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in variants that can escape neutralization by therapeutic antibodies. Here, we describe AZD3152, a SARS-CoV-2-neutralizing monoclonal antibody designed to provide improved potency and coverage against emerging variants. AZD3152 binds to the back left shoulder of the SARS-CoV-2 spike protein receptor binding domain and prevents interaction with the human angiotensin-converting enzyme 2 receptor. AZD3152 potently neutralized a broad panel of pseudovirus variants, including the currently dominant Omicron variant JN.1 but has reduced potency against XBB subvariants containing F456L. In vitro studies confirmed F456L resistance and additionally identified T415I and K458E as escape mutations. In a Syrian hamster challenge model, prophylactic administration of AZD3152 protected hamsters from weight loss and inflammation-related lung pathologies and reduced lung viral load. In the phase 1 sentinel safety cohort of the ongoing SUPERNOVA study (ClinicalTrials.gov: NCT05648110), a single 600-mg intramuscular injection of AZD5156 (containing 300 mg each of AZD3152 and cilgavimab) was well tolerated in adults through day 91. Observed serum concentrations of AZD3152 through day 91 were similar to those observed with cilgavimab and consistent with predictions for AZD7442, a SARS-CoV-2-neutralizing antibody combination of cilgavimab and tixagevimab, in a population pharmacokinetic model. On the basis of its pharmacokinetic characteristics, AZD3152 is predicted to provide durable protection against symptomatic coronavirus disease 2019 caused by susceptible SARS-CoV-2 variants, such as JN.1, in humans.

Dynamics of Trypanosoma cruzi infection in hamsters and novel association with progressive motor dysfunction.

Chagas disease is a zoonosis caused by the protozoan parasite Trypanosoma cruzi. Clinical outcomes range from long-term asymptomatic carriage to cardiac, digestive, neurological and composite presentations that can be fatal in both acute and chronic stages of the disease. Studies of T. cruzi in animal models, principally mice, have informed our understanding of the biological basis of this variability and its relationship to infection and host response dynamics. Hamsters have higher translational value for many human infectious diseases, but they have not been well developed as models of Chagas disease. We transposed a real-time bioluminescence imaging system for T. cruzi infection from mice into female Syrian hamsters (Mesocricetus auratus). This enabled us to study chronic tissue pathology in the context of spatiotemporal infection dynamics. Acute infections were widely disseminated, whereas chronic infections were almost entirely restricted to the skin and subcutaneous adipose tissue. Neither cardiac nor digestive tract disease were reproducible features of the model. Skeletal muscle had only sporadic parasitism in the chronic phase, but nevertheless displayed significant inflammation and fibrosis, features also seen in mouse models. Whereas mice had normal locomotion, all chronically infected hamsters developed hindlimb muscle hypertonia and a gait dysfunction resembling spastic diplegia. With further development, this model may therefore prove valuable in studies of peripheral nervous system involvement in Chagas disease.

Characterization of a natural 'dead-end' variant of Schmallenberg virus.

Schmallenberg virus (SBV) belongs to the Simbu serogroup within the family Peribunyaviridae, genus Orthobunyavirus and is transmitted by Culicoides biting midges. Infection of naïve ruminants in a critical phase of gestation may lead to severe congenital malformations. Sequence analysis from viremic animals revealed a very high genome stability. In contrast, sequence variations are frequently described for SBV from malformed fetuses. In addition to S segment mutations, especially within the M segment encoding the major immunogen Gc, point mutations or genomic deletions are also observed. Analysis of the SBV_D281/12 isolate from a malformed fetus revealed multiple point mutations in all three genome segments. It also has a large genomic deletion in the antigenic domain encoded by the M segment compared to the original SBV reference strain 'BH80/11' isolated from viremic blood in 2011. Interestingly, SBV_D281/12 showed a marked replication deficiency in vitro in Culicoides sonorensis cells (KC cells), but not in standard baby hamster kidney cells (BHK-21). We therefore generated a set of chimeric viruses of rSBV_D281/12 and wild-type rSBV_BH80/11 by reverse genetics, which were characterized in both KC and BHK-21 cells. It could be shown that the S segment of SBV_D281/12 is responsible for the replication deficit and that it acts independently from the large deletion within Gc. In addition, a single point mutation at position 111 (S to N) of the nucleoprotein was identified as the critical mutation. Our results suggest that virus variants found in malformed fetuses and carrying characteristic genomic mutations may have a clear 'loss of fitness' for their insect hosts in vitro. It can also be concluded that such mutations lead to virus variants that are no longer part of the natural transmission cycle between mammalian and insect hosts. Interestingly, analysis of a series of SBV sequences confirmed the S111N mutation exclusively in samples of malformed fetuses and not in blood from viremic animals. The characterization of these changes will allow the definition of protein functions that are critical for only one group of hosts.

A highly effective ferritin-based divalent nanoparticle vaccine shields Syrian hamsters against lethal Nipah virus.

The Nipah virus (NiV), a highly deadly bat-borne paramyxovirus, poses a substantial threat due to recurrent outbreaks in specific regions, causing severe respiratory and neurological diseases with high morbidity. Two distinct strains, NiV-Malaysia (NiV-M) and NiV-Bangladesh (NiV-B), contribute to outbreaks in different geographical areas. Currently, there are no commercially licensed vaccines or drugs available for prevention or treatment. In response to this urgent need for protection against NiV and related henipaviruses infections, we developed a novel homotypic virus-like nanoparticle (VLP) vaccine co-displaying NiV attachment glycoproteins (G) from both strains, utilizing the self-assembling properties of ferritin protein. In comparison to the NiV G subunit vaccine, our nanoparticle vaccine elicited significantly higher levels of neutralizing antibodies and provided complete protection against a lethal challenge with NiV infection in Syrian hamsters. Remarkably, the nanoparticle vaccine stimulated the production of antibodies that exhibited superior cross-reactivity to homologous or heterologous henipavirus. These findings underscore the potential utility of ferritin-based nanoparticle vaccines in providing both broad-spectrum and long-term protection against NiV and emerging zoonotic henipaviruses challenges.

PEI-Mediated Transient Gene Expression in CHO Cells.

We describe a method for polyethyleneimine (PEI)-mediated transient transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells for protein expression applicable at scales from 2 mL to 2 L. The method involves transfection at a high cell density (5 × 106 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31 °C with agitation by orbital shaking. This method requires 0.3 mg/L of coding pDNA, 2.7 mg/L of nonspecific (filler) DNA, and 15 mg/L of PEI. The production phase is performed at 31 °C in the presence of 0.25% N,N-dimethylacetamide (DMA). If desired, the method can be modified to avoid use of DMA by increasing the amount of coding DNA. We also provide information on culture vessel options, recommended working volumes, and recommended shaking speeds for transfections at scales from 2 mL to 2 L.

Recombinant Protein Production from Stable CHO Cell Pools.

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.

Generation of Monoclonal Chinese Hamster Ovary Cell Lines Using DISPENCELL-S3.

Single-cell isolation is a key step in the manufacturing of therapeutic proteins, which relies on the development of monoclonal cell lines. It increases production safety and consistency. It also ensures higher manufacturing performances thanks to the selection of the rare clonally derived cell lines with optimal growth and production capacities. DISPENCELL-S3 is a small format single-cell dispenser whose technology is based on impedance spectroscopy. Here, we provide a detailed protocol for generating Chinese hamster ovary (CHO) monoclonal cell lines using DISPENCELL-S3. Production and characterization of an adequate cell sample for single-cell isolation, as well as the optimization of the DISPENCELL-S3 dispensing parameters are described. Monoclonal outgrowth assessment and the use of the recorded impedance signal as evidence of clonality are also outlined.

Bench-Scale Stirred-Tank Bioreactor for Recombinant Protein Production in Chinese Hamster Ovary (CHO) Cells in Suspension.

Most pharmaceutical biotechnology companies use stirred-tank bioreactors (STR) for recombinant protein manufacturing. These bioreactors are used at a variety of different scales ranging from bench to production scales, with working volumes from 10 mL to 25,000 L. Bench-scale STRs are commonly used to culture mammalian cells for process development, to troubleshoot production scale bioreactors using scale-down models (SDM), or to conduct fundamental research. In this chapter, we describe the operations of a bench-scale STR for the production of recombinant proteins with suspension-adapted Chinese hamster ovary (CHO) cells. These operations include bioreactor setup and configuration, batching media, inoculation of the seed cell culture, production phase, and harvest of cell-free fluids.

Development of Responsive Promoters and their Utilization for Stable CHO Sensor Cell Lines.

Chinese hamster ovary (CHO) cells are the most important mammalian expression systems to produce recombinant proteins. To ensure a proper expression of the desired molecule, it is important to monitor and adjust bioprocess parameters like oxygen concentration as well as osmolality. However, the observation of crucial cultivation parameters can be an elaborate procedure requiring lots of hands-on work. In addition, for emerging modeling approaches for bioprocesses, a model cell line responding with a measurable signal to an external influence would be highly valuable. This protocol describes in detail the procedure to generate responsive promoters reacting to limiting conditions as well as the generation of stable sensor cell lines communicating with the operator. Thereby, hypoxia and osmolality sensing response elements established in CHO cells will be utilized to trigger the expression of a minimal CMV promoter. To assess the activity of the responsive promoter in close to real time, unstable variants of GFP and BFP will be expressed, which can be analyzed via flow cytometry. Finally, an automated sampling system coupled to a fluorescence microscope enables a continuous observation of CHO cells and reports emerging limiting conditions by detecting increasing amounts of a specific fluorescent protein.

Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells.

Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of α1,6-fucose significantly enhances antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) approach with different formats to disrupt the α-1,6-fucosyltransferase (FUT8) gene and subsequently inhibit α-1,6 fucosylation on antibodies expressed in CHO cells.