Cell-based passive immunization for protection against SARS-CoV-2 infection.

BACKGROUND: Immunologically impaired individuals respond poorly to vaccines, highlighting the need for additional strategies to protect these vulnerable populations from COVID-19. While monoclonal antibodies (mAbs) have emerged as promising tools to manage infectious diseases, the transient lifespan of neutralizing mAbs in patients limits their ability to confer lasting, passive prophylaxis from SARS-CoV-2. Here, we attempted to solve this problem by combining cell and mAb engineering in a way that provides durable immune protection against viral infection using safe and universal cell therapy. METHODS: Mouse embryonic stem cells equipped with our FailSafe™ and induced allogeneic cell tolerance technologies were engineered to express factors that potently neutralize SARS-CoV-2, which we call 'neutralizing biologics' (nBios). We subcutaneously transplanted the transgenic cells into mice and longitudinally assessed the ability of the cells to deliver nBios into circulation. To do so, we quantified plasma nBio concentrations and SARS-CoV-2 neutralizing activity over time in transplant recipients. Finally, using similar cell engineering strategies, we genetically modified FailSafe™ human-induced pluripotent stem cells to express SARS-CoV-2 nBios. RESULTS: Transgenic mouse embryonic stem cells engineered for safety and allogeneic-acceptance can secrete functional and potent SARS-CoV-2 nBios. As a dormant, subcutaneous tissue, the transgenic cells and their differentiated derivatives long-term deliver a supply of protective nBio titers in vivo. Moving toward clinical relevance, we also show that human-induced pluripotent stem cells, similarly engineered for safety, can secrete highly potent nBios. CONCLUSIONS: Together, these findings show the promise and potential of using 'off-the-shelf' cell products that secrete neutralizing antibodies for sustained protective immunity against current and future viral pathogens of public health significance.

Peptide-Antibody Fusions Engineered by Phage Display Exhibit an Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants.

The spread of COVID-19 has been exacerbated by the emergence of variants of concern (VoC). Many VoC contain mutations in the spike protein (S-protein) and are implicated in infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but they are limited by low potency and vulnerability to RBD mutations in VoC. To address these issues, we used naïve phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate-affinity nAb to generate tetravalent peptide-IgG fusions, and we showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD < 1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrated that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format, including the dominant B.1.1.529 (Omicron) variant that is resistant to most clinically approved therapeutic nAbs. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug and showed that it enabled the neutralization of a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.

Ultrapotent and broad neutralization of SARS-CoV-2 variants by modular, tetravalent, bi-paratopic antibodies.

Neutralizing antibodies (nAbs) that target the SARS-CoV-2 spike protein have received emergency use approval for treatment of COVID-19. However, with the emergence of variants of concern, there is a need for new treatment options. We report a format that enables modular assembly of bi-paratopic tetravalent nAbs with antigen-binding sites from two distinct nAbs. The tetravalent nAb purifies in high yield and exhibits biophysical characteristics that are comparable to those of clinically used therapeutic antibodies. The tetravalent nAb binds to the spike protein trimer at least 100-fold more tightly than bivalent IgGs (apparent KD < 1 pM) and neutralizes a broad array of SARS-CoV-2 pseudoviruses, chimeric viruses, and authentic viral variants with high potency. Together, these results establish the tetravalent diabody-Fc-Fab as a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that greatly exceed those of conventional bivalent IgGs.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations.

Neutralizing antibodies (nAbs) hold promise as therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and tetravalent versions block entry with a potency exceeding bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show increased tolerance to potential virus escape mutants and an emerging variant of concern. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for enhancing antiviral therapies against COVID-19 and related viral threats, and our strategy can be applied to virtually any antibody drug.

A Rationally Designed Bovine IgA Fc Scaffold Enhances in planta Accumulation of a VHH-Fc Fusion Without Compromising Binding to Enterohemorrhagic E. coli.

We previously isolated a single domain antibody (VHH) that binds Enterohemorrhagic Escherichia coli (EHEC) with the end-goal being the enteromucosal passive immunization of cattle herds. To improve the yield of a chimeric fusion of the VHH with an IgA Fc, we employed two rational design strategies, supercharging and introducing de novo disulfide bonds, on the bovine IgA Fc component of the chimera. After mutagenizing the Fc, we screened for accumulation levels after transient transformation in Nicotiana benthamiana leaves. We identified and characterized five supercharging and one disulfide mutant, termed '(5 + 1)Fc', that improve accumulation in comparison to the native Fc. Combining all these mutations is associated with a 32-fold increase of accumulation for the Fc alone, from 23.9 mg/kg fresh weight (FW) to 599.5 mg/kg FW, as well as a twenty-fold increase when fused to a VHH that binds EHEC, from 12.5 mg/kg FW tissue to 236.2 mg/kg FW. Co-expression of native or mutated VHH-Fc with bovine joining chain (JC) and bovine secretory component (SC) followed by co-immunoprecipitation suggests that the stabilizing mutations do not interfere with the capacity of VHH-Fc to assemble with JC and FC into a secretory IgA. Both the native and the mutated VHH-Fc similarly neutralized the ability of four of the seven most prevalent EHEC strains (O157:H7, O26:H11, O111:Hnm, O145:Hnm, O45:H2, O121:H19 and O103:H2), to adhere to HEp-2 cells as visualized by immunofluorescence microscopy and quantified by fluorometry. These results collectively suggest that supercharging and disulfide bond tethering on a Fc chain can effectively improve accumulation of a VHH-Fc fusion without impacting VHH functionality.

Human ACE2 receptor polymorphisms and altered susceptibility to SARS-CoV-2.

COVID-19 is a respiratory illness caused by a novel coronavirus called SARS-CoV-2. The viral spike (S) protein engages the human angiotensin-converting enzyme 2 (ACE2) receptor to invade host cells with ~10-15-fold higher affinity compared to SARS-CoV S-protein, making it highly infectious. Here, we assessed if ACE2 polymorphisms can alter host susceptibility to SARS-CoV-2 by affecting this interaction. We analyzed over 290,000 samples representing >400 population groups from public genomic datasets and identified multiple ACE2 protein-altering variants. Using reported structural data, we identified natural ACE2 variants that could potentially affect virus-host interaction and thereby alter host susceptibility. These include variants S19P, I21V, E23K, K26R, T27A, N64K, T92I, Q102P and H378R that were predicted to increase susceptibility, while variants K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L and D509Y were predicted to be protective variants that show decreased binding to S-protein. Using biochemical assays, we confirmed that K31R and E37K had decreased affinity, and K26R and T92I variants showed increased affinity for S-protein when compared to wildtype ACE2. Consistent with this, soluble ACE2 K26R and T92I were more effective in blocking entry of S-protein pseudotyped virus suggesting that ACE2 variants can modulate susceptibility to SARS-CoV-2.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations.

Neutralizing antibodies (nAbs) hold promise as effective therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and its tetravalent versions can block entry with a potency that exceeds the bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, observations consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show much increased tolerance to potential virus escape mutants. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for developing potent antiviral therapies against COVID-19 and related viral threats, and our strategy can be readily applied to any antibody drug currently in development.

Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells.

Enterohemorrhagic Escherichia coli (EHEC) has consistently been one of the foremost foodborne pathogen threats worldwide based on the past 30 years of surveillance. EHEC primarily colonizes the bovine gastrointestinal (GI) tract from which it can be transmitted to nearby farm environments and remain viable for months. There is an urgent need for effective and easily implemented pre-harvest interventions to curtail EHEC contamination of the food and water supply. In an effort to address this problem, we isolated single-domain antibodies (VHHs) specific for intimin, an EHEC adhesin required for colonization, and designed chimeric VHH fusions with secretory IgA functionality intended for passive immunotherapy at the mucosal GI surface. The antibodies were produced in leaves of Nicotiana benthamiana with production levels ranging between 1 and 3% of total soluble protein. in vivo assembly of all subunits into a hetero-multimeric complex was verified by co-immunoprecipitation. Analysis of multivalent protection across the most prevalent EHEC strains identified one candidate antibody, VHH10-IgA, that binds O145:Hnm, O111:Hnm, O26:H11, and O157:H7. Fluorometric and microscopic analysis also indicated that VHH10-IgA completely neutralizes the capacity of the latter three strains to adhere to epithelial cells in vitro. This study provides proof of concept that a plant-produced chimeric secretory IgA can confer cross-serotype inhibition of bacterial adhesion to epithelial cells.

Coating Nanoparticles with Plant-Produced Transferrin-Hydrophobin Fusion Protein Enhances Their Uptake in Cancer Cells.

The encapsulation of drugs to nanoparticles may offer a solution for targeted delivery. Here, we set out to engineer a self-assembling targeting ligand by combining the functional properties of human transferrin and fungal hydrophobins in a single fusion protein. We showed that human transferrin can be expressed in Nicotiana benthamiana plants as a fusion with Trichoderma reesei hydrophobins HFBI, HFBII, or HFBIV. Transferrin-HFBIV was further expressed in tobacco BY-2 suspension cells. Both partners of the fusion protein retained their functionality; the hydrophobin moiety enabled migration to a surfactant phase in an aqueous two-phase system, and the transferrin moiety was able to reversibly bind iron. Coating porous silicon nanoparticles with the fusion protein resulted in uptake of the nanoparticles in human cancer cells. This study provides a proof-of-concept for the functionalization of hydrophobin coatings with transferrin as a targeting ligand.

Protein Bodies in Leaves Exchange Contents through the Endoplasmic Reticulum.

Protein bodies (PBs) are organelles found in seeds whose main function is the storage of proteins that are used during germination for sustaining growth. PBs can also be induced to form in leaves when foreign proteins are produced at high levels in the endoplasmic reticulum (ER) and when fused to one of three tags: Zera®, elastin-like polypeptides (ELP), or hydrophobin-I (HFBI). In this study, we investigate the differences between ELP, HFBI and Zera PB formation, packing, and communication. Our results confirm the ER origin of all three fusion-tag-induced PBs. We show that secretory pathway proteins can be sequestered into all types of PBs but with different patterns, and that different fusion tags can target a specific protein to different PBs. Zera PBs are mobile and dependent on actomyosin motility similar to ELP and HFBI PBs. We show in vivo trafficking of proteins between PBs using GFP photoconversion. We also show that protein trafficking between ELP or HFBI PBs is faster and proteins travel further when compared to Zera PBs. Our results indicate that fusion-tag-induced PBs do not represent terminally stored cytosolic organelles, but that they form in, and remain part of the ER, and dynamically communicate with each other via the ER. We hypothesize that the previously documented PB mobility along the actin cytoskeleton is associated with ER movement rather than independent streaming of detached organelles.

Green to red photoconversion of GFP for protein tracking in vivo.

A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.

Protein body formation in leaves of Nicotiana benthamiana: a concentration-dependent mechanism influenced by the presence of fusion tags.

Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles originally found in seeds whose function is to accumulate seed storage proteins. It has been shown that PB formation is not limited to seeds and green fluorescent protein (GFP) fused to either elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags induce the formation of PBs in leaves of N. benthamiana. In this study, we compared the ELP- and HFBI-induced PBs and showed that ELP-induced PBs are larger than HFBI-induced PBs. The size of ELP- and HFBI-induced PBs increased over time along with the accumulation levels of their fused protein. Our results show that PB formation is a concentration-dependent mechanism in which proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. Our results show that the presence of fusion tags is not necessary for the formation of PBs, but affects the distribution pattern and size of PBs. This was confirmed by PBs induced by fluorescent proteins as well as fungal xylanases. We noticed that in the process of PB formation, secretory and ER-resident molecules are passively sequestered into the lumen of PBs. We propose to use this property of PBs as a tool to increase the accumulation levels of erythropoietin and human interleukin-10 by co-expression with PB-inducing proteins.

Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins.

BACKGROUND: Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. RESULTS: The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. CONCLUSION: The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags increase the accumulation levels of the recombinant protein and induce the formation of PBs regardless of the cultivar used. However, a specific level of recombinant protein accumulation needs to be reached for PBs to form.

The SET domain protein, Set3p, promotes the reliable execution of cytokinesis in Schizosaccharomyces pombe.

In response to perturbation of the cell division machinery fission yeast cells activate regulatory networks that ensure the faithful completion of cytokinesis. For instance, when cells are treated with drugs that impede constriction of the actomyosin ring (low doses of Latrunculin A, for example) these networks ensure that cytokinesis is complete before progression into the subsequent mitosis. Here, we identify three previously uncharacterized genes, hif2, set3, and snt1, whose deletion results in hyper-sensitivity to LatA treatment and in increased rates of cytokinesis failure. Interestingly, these genes are orthologous to TBL1X, MLL5, and NCOR2, human genes that encode components of a histone deacetylase complex with a known role in cytokinesis. Through co-immunoprecipitation experiments, localization studies, and phenotypic analysis of gene deletion mutants, we provide evidence for an orthologous complex in fission yeast. Furthermore, in light of the putative role of the complex in chromatin modification, together with our results demonstrating an increase in Set3p levels upon Latrunculin A treatment, global gene expression profiles were generated. While this analysis demonstrated that the expression of cytokinesis genes was not significantly affected in set3Δ backgrounds, it did reveal defects in the ability of the mutant to regulate genes with roles in the cellular response to stress. Taken together, these findings support the existence of a conserved, multi-protein complex with a role in promoting the successful completion of cytokinesis.

Global gene expression analysis of fission yeast mutants impaired in Ser-2 phosphorylation of the RNA pol II carboxy terminal domain.

In Schizosaccharomyces pombe the nuclear-localized Lsk1p-Lsc1p cyclin dependent kinase complex promotes Ser-2 phosphorylation of the heptad repeats found within the RNA pol II carboxy terminal domain (CTD). Here, we first provide evidence supporting the existence of a third previously uncharacterized Ser-2 CTD kinase subunit, Lsg1p. As expected for a component of the complex, Lsg1p localizes to the nucleus, promotes Ser-2 phosphorylation of the CTD, and physically interacts with both Lsk1p and Lsc1p in vivo. Interestingly, we also demonstrate that lsg1Δ mutants--just like lsk1Δ and lsc1Δ strains--are compromised in their ability to faithfully and reliably complete cytokinesis. Next, to address whether kinase mediated alterations in CTD phosphorylation might selectively alter the expression of genes with roles in cytokinesis and/or the cytoskeleton, global gene expression profiles were analyzed. Mutants impaired in Ser-2 phosphorylation display little change with respect to the level of transcription of most genes. However, genes affecting cytokinesis--including the actin interacting protein gene, aip1--as well as genes with roles in meiosis, are included in a small subset that are differentially regulated. Significantly, genetic analysis of lsk1Δ aip1Δ double mutants is consistent with Lsk1p and Aip1p acting in a linear pathway with respect to the regulation of cytokinesis.

The Arabidopsis tt19-4 mutant differentially accumulates proanthocyanidin and anthocyanin through a 3' amino acid substitution in glutathione S-transferase.

The Arabidopsis transparent testa (tt) mutant tt19-4 shows reduced seed coat colour, but stains darkly with DMACA and accumulates anthocyanins in aerial tissues. Positional cloning showed that tt19-4 was allelic to tt19-1 and has a G-to-T mutation in a conserved 3'-domain in the TT19-4 gene. Soluble and unextractable seed proanthocyanidins and hydrolysis of unextractable proanthocyanidin differ between wild-type Col-4 and both mutants. However, seed quercetins, unextractable proanthocyanidin hydrolysis, and seedling anthocyanin content, and flavonoid gene expression differ between tt19-1 and tt19-4. Transformation of tt19-1 with a TT19-4 cDNA results in vegetative anthocyanins, whereas TT19-4 cDNA cannot complement the proanthocyanidin and pale seed coat phenotype of tt19-1. Both recombinant TT19 and TT19-4 enzymes are functional GSTs and are localized in the cytosol, but TT19 did not function with wide range of flavonoids and natural products to produce conjugation products. We suggest that the dark seed coat of Arabidopsis is related to soluble proanthocyanidin content and that quercetin holds the key to the function of TT19. In addition, TT19 appears to have a 5' GSH-binding domain influencing both anthocyanin and proanthocyanidin accumulation and a 3' domain affecting proanthocyanidin accumulation by a single amino acid substitution.