Plant-derived single domain COVID-19 antibodies.

Data show a decrease in the risk of hospitalization and death from COVID-19. To date, global vaccinations for SARS-CoV-2 protections are underway, but additional treatments are urgently needed to prevent and cure infection among naïve and even vaccinated people. Neutralizing monoclonal antibodies are very promising for prophylaxis and therapy of SARS-CoV-2 infections. However, traditional large-scale methods of producing such antibodies are slow, extremely expensive and possess a high risk of contamination with viruses, prions, oncogenic DNA and other pollutants. The present study is aimed at developing an approach of producing monoclonal antibodies (mAbs) against SARS-CoV-2 spike (S) protein in plant systems which offers unique advantages, such as the lack of human and animal pathogens or bacterial toxins, relatively low-cost manufacturing, and ease of production scale-up. We selected a single N-terminal domain functional camelid-derived heavy (H)-chain antibody fragments (VHH, AKA nanobodies) targeted to receptor binding domain of SARS-CoV-2 spike protein and developed methods of their rapid production using transgenic plants and plant cell suspensions. Isolated and purified plant-derived VHH antibodies were compared with mAbs produced in traditional mammalian and bacterial expression systems. It was found that plant generated VHH using the proposed methods of transformation and purification possess the ability to bind to SARS-CoV-2 spike protein comparable to that of monoclonal antibodies derived from bacterial and mammalian cell cultures. The results of the present studies confirm the visibility of producing monoclonal single-chain antibodies with a high ability to bind the targeted COVID-19 spike protein in plant systems within a relatively shorter time span and at a lower cost when compared with traditional methods. Moreover, similar plant biotechnology approaches can be used for producing monoclonal neutralizing antibodies against other types of viruses.

Novel Therapeutic Target Critical for SARS-CoV-2 Infectivity and Induction of the Cytokine Release Syndrome.

We discovered a novel therapeutic target critical for SARS-CoV-2, cellular infectivity and the induction of the cytokine release syndrome. Here, we show that the mammalian enzyme neuraminidase-1 (Neu-1) is part of a highly conserved signaling platform that regulates the dimerization and activation of the ACE2 receptors and the Toll-like receptors (TLRs) implicated in the cytokine release syndrome (CRS). Activated Neu-1 cleaves glycosylated residues that provide a steric hindrance to both ACE2 and TLR dimerization, a process critical to both viral attachment to the receptor and entry into the cell and TLR activation. Blocking Neu-1 inhibited ACE2 receptor dimerization and internalization, TLR dimerization and activation, and the expression of several key inflammatory molecules implicated in the CRS and death from ARDS. Treatments that target Neu-1 are predicted to be highly effective against infection with SARS-CoV-2, given the central role played by this enzyme in viral cellular entry and the induction of the CRS.

Adaptive Evolution of the Spike Protein in Coronaviruses.

Coronaviruses are single-stranded, positive-sense RNA viruses that can infect many mammal and avian species. The Spike (S) protein of coronaviruses binds to a receptor on the host cell surface to promote viral entry. The interactions between the S proteins of coronaviruses and receptors of host cells are extraordinarily complex, with coronaviruses from different genera being able to recognize the same receptor and coronaviruses from the same genus able to bind distinct receptors. As the coronavirus disease 2019 pandemic has developed, many changes in the S protein have been under positive selection by altering the receptor-binding affinity, reducing antibody neutralization activities, or affecting T-cell responses. It is intriguing to determine whether the selection pressure on the S gene differs between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses due to the host shift from nonhuman animals to humans. Here, we show that the S gene, particularly the S1 region, has experienced positive selection in both SARS-CoV-2 and other coronaviruses. Although the S1 N-terminal domain exhibits signals of positive selection in the pairwise comparisons in all four coronavirus genera, positive selection is primarily detected in the S1 C-terminal domain (the receptor-binding domain) in the ongoing evolution of SARS-CoV-2, possibly owing to the change in host settings and the widespread natural infection and SARS-CoV-2 vaccination in humans.

High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike.

Designing prefusion-stabilized SARS-CoV-2 spike is critical for the effectiveness of COVID-19 vaccines. All COVID-19 vaccines in the US encode spike with K986P/V987P mutations to stabilize its prefusion conformation. However, contemporary methods on engineering prefusion-stabilized spike immunogens involve tedious experimental work and heavily rely on structural information. Here, we establish a systematic and unbiased method of identifying mutations that concomitantly improve expression and stabilize the prefusion conformation of the SARS-CoV-2 spike. Our method integrates a fluorescence-based fusion assay, mammalian cell display technology, and deep mutational scanning. As a proof-of-concept, we apply this method to a region in the S2 domain that includes the first heptad repeat and central helix. Our results reveal that besides K986P and V987P, several mutations simultaneously improve expression and significantly lower the fusogenicity of the spike. As prefusion stabilization is a common challenge for viral immunogen design, this work will help accelerate vaccine development against different viruses.

Identification of the regulatory mechanism of ACE2 in COVID-19-induced kidney damage with systems genetics approach.

Studies showed that SARS-CoV-2 can directly target the kidney and induce renal damage. As the cell surface receptor for SARS-CoV-2 infection, the angiotensin-converting enzyme 2 (ACE2) plays a pivotal role for renal physiology and function. Thus, it is important to understand ACE2 through which pathway influences the pathogenesis of renal damage induced by COVID-19. In this study, we first performed an eQTL mapping for Ace2 in kidney tissues in 53 BXD mice strains. Results demonstrated that Ace2 is highly expressed and strongly controlled by a genetic locus on chromosome 16 in the kidney, with six genes (Dnase1, Vasn, Usp7, Abat, Mgrn1, and Rbfox1) dominated as the upstream modulator, as they are highly correlated with Ace2 expression. Gene co-expression analysis showed that Ace2 co-variates are significantly involved in the renin-angiotensin system (RAS) pathway which acts as a reno-protector. Importantly, we also found that Ace2 is positively correlated with Pdgf family members, particularly Pdgfc, which showed the most association among the 76 investigated growth factors. Mammalian Phenotype Ontology enrichment indicated that the cognate transcripts for both Ace2 and Pdgfc were mainly involved in regulating renal physiology and morphology. Among which, Cd44, Egfr, Met, Smad3, and Stat3 were identified as hub genes through protein-protein interaction analysis. Finally, in aligning with our systems genetics findings, we found ACE2, pdgf family members, and RAS genes decreased significantly in the CAKI-1 kidney cancer cells treated with S protein and receptor binding domain structural protein. Collectively, our data suggested that ACE2 work with RAS, PDGFC, as well as their cognate hub genes to regulate renal function, which could guide for future clinical prevention and targeted treatment for COVID-19-induced renal damage outcomes. KEY MESSAGES: • Ace2 is highly expressed and strongly controlled by a genetic locus on chromosome 16 in the kidney. • Ace2 co-variates are enriched in the RAS pathway. • Ace2 is strongly correlated with the growth factor Pdgfc. • Ace2 and Pdgfc co-expressed genes involved in the regulation of renal physiology and morphology. • SARS-CoV-2 spike glycoprotein induces down-regulation of Ace2, RAS, and Pdgfc.

SARS-CoV-2 infection alkalinizes the ERGIC and lysosomes through the viroporin activity of the viral envelope protein.

The coronavirus SARS-CoV-2, the agent of the deadly COVID-19 pandemic, is an enveloped virus propagating within the endocytic and secretory organelles of host mammalian cells. Enveloped viruses modify the ionic homeostasis of organelles to render their intra-luminal milieu permissive for viral entry, replication and egress. Here, we show that infection of Vero E6 cells with the delta variant of the SARS-CoV-2 alkalinizes the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) as well as lysosomes, mimicking the effect of inhibitors of vacuolar proton ATPases. We further show the envelope protein of SARS-CoV-2 accumulates in the ERGIC when expressed in mammalian cells and selectively dissipates the ERGIC pH. This viroporin action is prevented by mutations of Val25 but not Asn15 within the channel pore of the envelope (E) protein. We conclude that the envelope protein acts as a proton channel in the ERGIC to mitigate the acidity of this intermediate compartment. The altered pH homeostasis of the ERGIC likely contributes to the virus fitness and pathogenicity, making the E channel an attractive drug target for the treatment of COVID-19.

Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2.

Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with kinact/KI = 9,600 M-1 s-1, achieves sub-µM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

Gain-of-glycosylation in breast multi-drug-resistant MCF-7 adenocarcinoma cells and cancer stem cells characterized by site- and structure-specific N-glycoproteomics.

N-linked glycosylation (N-glycosylation) is a common protein post-translational modification, occurring on more than half of mammalian proteins; in striking contract with small molecule modifications (such as methylation, phosphorylation) with only single structures, N-glycosylation has multiple dimensional structural features (monosaccharide composition, sequence, linkage, anomer), which generates enormous N-glycan structures; and these structures widely regulate protein structure and functions. For the modification site, N-glycosylation occurs on the Asn residue among the consensus N-X-S/T/C (X≠P) motif; mutation-originated amino acid change may lead to loss of such an original motif and thus loss-of-glycosylation (LoG) or gain of such a new motif and thus gain-of-glycosylation (GoG). Both LoG and GoG generates new structures and functions of glycoproteins, which has been observed in the S protein of SARS-Cov-2 as well as malignant diseases. Here we report our glycoproteome-wide qualitative N-glycoproteomics characterization of GoGs in breast cancer Adriamycin drug resistance (ADR) cells (MCF-7/ADR) and cancer stem cells (MCF-7/ADR CSCs); comprehensive N-glycosite and N-glycan structure information at the intact N-glycopeptide level were reported.

Potential Prion Involvement in Long COVID-19 Neuropathology, Including Behavior.

Prion' is a term used to describe a protein infectious particle responsible for several neurodegenerative diseases in mammals, e.g., Creutzfeldt-Jakob disease. The novelty is that it is protein based infectious agent not involving a nucleic acid genome as found in viruses and bacteria. Prion disorders exhibit, in part, incubation periods, neuronal loss, and induce abnormal folding of specific normal cellular proteins due to enhancing reactive oxygen species associated with mitochondria energy metabolism. These agents may also induce memory, personality and movement abnormalities as well as depression, confusion and disorientation. Interestingly, some of these behavioral changes also occur in COVID-19 and mechanistically include mitochondrial damage caused by SARS-CoV-2 and subsequenct production of reactive oxygen species. Taken together, we surmise, in part, long COVID may involve the induction of spontaneous prion emergence, especially in individuals susceptible to its origin may thus explain some of its manesfestions post-acute viral infection.

The Evolution and Biology of SARS-CoV-2 Variants.

Our understanding of the still unfolding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic would have been extremely limited without the study of the genetics and evolution of this new human coronavirus. Large-scale genome-sequencing efforts have provided close to real-time tracking of the global spread and diversification of SARS-CoV-2 since its entry into the human population in late 2019. These data have underpinned analysis of its origins, epidemiology, and adaptations to the human population: principally immune evasion and increasing transmissibility. SARS-CoV-2, despite being a new human pathogen, was highly capable of human-to-human transmission. During its rapid spread in humans, SARS-CoV-2 has evolved independent new forms, the so-called "variants of concern," that are better optimized for human-to-human transmission. The most important adaptation of the bat coronavirus progenitor of both SARS-CoV-1 and SARS-CoV-2 for human infection (and other mammals) is the use of the angiotensin-converting enzyme 2 (ACE2) receptor. Relaxed structural constraints provide plasticity to SARS-related coronavirus spike protein permitting it to accommodate significant amino acid replacements of antigenic consequence without compromising the ability to bind to ACE2. Although the bulk of research has justifiably concentrated on the viral spike protein as the main determinant of antigenic evolution and changes in transmissibility, there is accumulating evidence for the contribution of other regions of the viral proteome to virus-host interaction. Whereas levels of community transmission of recombinants compromising genetically distinct variants are at present low, when divergent variants cocirculate, recombination between SARS-CoV-2 clades is being detected, increasing the risk that viruses with new properties emerge. Applying computational and machine learning methods to genome sequence data sets to generate experimentally verifiable predictions will serve as an early warning system for novel variant surveillance and will be important in future vaccine planning. Omicron, the latest SARS-CoV-2 variant of concern, has focused attention on step change antigenic events, "shift," as opposed to incremental "drift" changes in antigenicity. Both an increase in transmissibility and antigenic shift in Omicron led to it readily causing infections in the fully vaccinated and/or previously infected. Omicron's virulence, while reduced relative to the variant of concern it replaced, Delta, is very much premised on the past immune exposure of individuals with a clear signal that boosted vaccination protects from severe disease. Currently, SARS-CoV-2 has proven itself to be a dangerous new human respiratory pathogen with an unpredictable evolutionary capacity, leading to a risk of future variants too great not to ensure all regions of the world are screened by viral genome sequencing, protected through available and affordable vaccines, and have non-punitive strategies in place for detecting and responding to novel variants of concern.

Ferritin - a multifaceted protein scaffold for biotherapeutics.

The ferritin nanocage is an endogenous protein that exists in almost all mammals. Its hollow spherical structure that naturally stores iron ions has been diversely exploited by researchers in biotherapeutics. Ferritin has excellent biosafety profiles, and the nanosized particles exhibit rapid dispersion and controlled/sustained release pharmacokinetics. Moreover, the large surface-to-volume ratio and the disassembly/reassembly behavior of the 24 monomer subunits into a sphere allow diverse modifications by chemical and genetic methods on the surface and inner cage of ferritin. Here, we critically review ferritin and its applications. We (i) introduce the application of ferritin in drug delivery; (ii) present an overview of the use of ferritin in imaging and diagnosis for biomedical purposes; (iii) discuss ferritin-based vaccines; and (iv) review ferritin-based agents currently in clinical trials. Although there are no currently approved drugs based on ferritin, this multifunctional protein scaffold shows immense potential in drug development in diverse categories, and ferritin-based drugs have recently entered phase I clinical trials. This golden shortlist of recent developments will be of immediate benefit and interest to researchers studying ferritin and other protein-based biotherapeutics.

Leukotrienes in Innate Immunity: Still Underappreciated after All These Years?

Leukotrienes (LTs) are lipid mediators derived from the 5-lipoxygenase pathway of arachidonate metabolism. Though best known for their role in asthma, they have broad actions that touch on virtually every aspect of mammalian biology. In a Brief Review published in the journal in 2005, we presented the existing evidence supporting a role for LTs in host defense. In this updated Brief Review, we focus on selected advances since then. We detail new insights into mechanisms and regulation of LT biosynthesis; the protective roles of LTs in the host response to diverse classes of pathogens, with an emphasis on viruses, including SARS-CoV-2; the phagocyte signal transduction mechanisms by which LTs exert their antimicrobial actions; the capacity for overexuberant LT production to promote tissue damage; and roles of LTs in the noninfectious immune-relevant conditions neuroinflammation and cancer.

Scalable agroinfiltration-based production of SARS-CoV-2 antigens for use in diagnostic assays and subunit vaccines.

Agroinfiltration is a method used in biopharming to support plant-based biosynthesis of therapeutic proteins such as antibodies and viral antigens involved in vaccines. Major advantages of generating proteins in plants is the low cost, massive scalability and the rapid yield of the technology. Herein, we report the agroinfiltration-based production of glycosylated SARS-CoV-2 Spike receptor-binding domain (RBD) protein. We show that it exhibits high-affinity binding to the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) and displays folding similar to antigen produced in mammalian expression systems. Moreover, our plant-expressed RBD was readily detected by IgM, IgA, and IgG antibodies from the serum of SARS-CoV-2 infected and vaccinated individuals. We further demonstrate that binding of plant-expressed RBD to ACE2 is efficiently neutralized by these antibodies. Collectively, these findings demonstrate that recombinant RBD produced via agroinfiltration exhibits suitable biochemical and antigenic features for use in serological and neutralization assays, and in subunit vaccine platforms.

The mechanism and significance of the conversion of xanthine dehydrogenase to xanthine oxidase in mammalian secretory gland cells.

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) occurs only in mammalian species. In fresh bovine milk, the enzyme exists predominantly as the oxidase form, in contrast to various normal organs where it is found primarily as the dehydrogenase: the mechanism of conversion to the oxidase in milk remains obscure. A systematic search for the essential factors for conversion from XDH to XO has been performed within fresh bovine milk using the highly purified dehydrogenase form after removal endogenous oxidase form by fractionation analysis. We find that conversion to the oxidase form requires four components under air: lactoperoxidase (LPO), XDH, SCN-, and substrate hypoxanthine or xanthine; the contribution of sulfhydryl oxidase appears to be minor. Disulfide bond formation between Cys-535 and Cys-995 is principally involved in the conversion, consistent with the result obtained from previous work with transgenic mice. In vitro reconstitution of LPO and SCN- results in synergistic conversion of the dehydrogenase to the oxidase the presence of xanthine, indicating the conversion is autocatalytic. Milk from an LPO knockout mouse contains a significantly greater proportion of the dehydrogenase form of the enzyme, although some oxidase form is also present, indicating that LPO contributes principally to the conversion, but that sulfhydryl oxidase (SO) may also be involved to a minor extent. All the components XDH/LPO/SCN- are necessary to inhibit bacterial growth in the presence of xanthine through disulfide bond formation in bacterial protein(s) required for replication, as part of an innate immunity system in mammals. Human GTEx Data suggest that mRNA of XDH and LPO are highly co-expressed in the salivary gland, mammary gland, mucosa of the airway and lung alveoli, and we have confirmed these human GTEx Data experimentally in mice. We discuss the possible roles of these components in the propagation of SARS-CoV-2 in these human cell types.

Regulation of ribonucleoprotein condensates by RNase L during viral infection.

In response to viral infection, mammalian cells activate several innate immune pathways to antagonize viral gene expression. Upon recognition of viral double-stranded RNA, protein kinase R (PKR) phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α) on serine 51. This inhibits canonical translation initiation, which broadly antagonizes viral protein synthesis. It also promotes the assembly of cytoplasmic ribonucleoprotein complexes termed stress granules (SGs). SGs are widely thought to promote cell survival and antiviral signaling. However, co-activation of the OAS/RNase L antiviral pathway inhibits the assembly of SGs and promotes the assembly of an alternative ribonucleoprotein complex termed an RNase L-dependent body (RLB). The formation of RLBs has been observed in response to double-stranded RNA, dengue virus infection, or SARS-CoV-2 infection. Herein, we review the distinct biogenesis pathways and properties of SGs and RLBs, and we provide perspective on their potential functions during the antiviral response. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Turnover and Surveillance > Regulation of RNA Stability RNA Export and Localization > RNA Localization.

Host protein kinases required for SARS-CoV-2 nucleocapsid phosphorylation and viral replication.

Multiple coronaviruses have emerged independently in the past 20 years that cause lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated or who experience breakthrough infections. Understanding the common host factors necessary for the life cycles of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that coordinately phosphorylate a cluster of serine and threonine residues in the viral N protein, which is required for viral replication. We also showed that loss or inhibition of SRPK1/2, which we propose initiates the N protein phosphorylation cascade, compromised the viral replication cycle. Because these phosphorylation sites are highly conserved across coronaviruses, inhibitors of these protein kinases not only may have therapeutic potential against COVID-19 but also may be broadly useful against coronavirus-mediated diseases.

Microalgae as an Efficient Vehicle for the Production and Targeted Delivery of Therapeutic Glycoproteins against SARS-CoV-2 Variants.

Severe acute respiratory syndrome-Coronavirus 2 (SARS-CoV-2) can infect various human organs, including the respiratory, circulatory, nervous, and gastrointestinal ones. The virus is internalized into human cells by binding to the human angiotensin-converting enzyme 2 (ACE2) receptor through its spike protein (S-glycoprotein). As S-glycoprotein is required for the attachment and entry into the human target cells, it is the primary mediator of SARS-CoV-2 infectivity. Currently, this glycoprotein has received considerable attention as a key component for the development of antiviral vaccines or biologics against SARS-CoV-2. Moreover, since the ACE2 receptor constitutes the main entry route for the SARS-CoV-2 virus, its soluble form could be considered as a promising approach for the treatment of coronavirus disease 2019 infection (COVID-19). Both S-glycoprotein and ACE2 are highly glycosylated molecules containing 22 and 7 consensus N-glycosylation sites, respectively. The N-glycan structures attached to these specific sites are required for the folding, conformation, recycling, and biological activity of both glycoproteins. Thus far, recombinant S-glycoprotein and ACE2 have been produced primarily in mammalian cells, which is an expensive process. Therefore, benefiting from a cheaper cell-based biofactory would be a good value added to the development of cost-effective recombinant vaccines and biopharmaceuticals directed against COVID-19. To this end, efficient protein synthesis machinery and the ability to properly impose post-translational modifications make microalgae an eco-friendly platform for the production of pharmaceutical glycoproteins. Notably, several microalgae (e.g., Chlamydomonas reinhardtii, Dunaliella bardawil, and Chlorella species) are already approved by the U.S. Food and Drug Administration (FDA) as safe human food. Because microalgal cells contain a rigid cell wall that could act as a natural encapsulation to protect the recombinant proteins from the aggressive environment of the stomach, this feature could be used for the rapid production and edible targeted delivery of S-glycoprotein and soluble ACE2 for the treatment/inhibition of SARS-CoV-2. Herein, we have reviewed the pathogenesis mechanism of SARS-CoV-2 and then highlighted the potential of microalgae for the treatment/inhibition of COVID-19 infection.

The JAK-STAT pathway at 30: Much learned, much more to do.

The discovery of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway arose from investigations of how cells respond to interferons (IFNs), revealing a paradigm in cell signaling conserved from slime molds to mammals. These discoveries revealed mechanisms underlying rapid gene expression mediated by a wide variety of extracellular polypeptides including cytokines, interleukins, and related factors. This knowledge has provided numerous insights into human disease, from immune deficiencies to cancer, and was rapidly translated to new drugs for autoimmune, allergic, and infectious diseases, including COVID-19. Despite these advances, major challenges and opportunities remain.

Stress granules and mTOR are regulated by membrane atg8ylation during lysosomal damage.

We report that lysosomal damage is a hitherto unknown inducer of stress granule (SG) formation and that the process termed membrane atg8ylation coordinates SG formation with mTOR inactivation during lysosomal stress. SGs were induced by lysosome-damaging agents including SARS-CoV-2ORF3a, Mycobacterium tuberculosis, and proteopathic tau. During damage, mammalian ATG8s directly interacted with the core SG proteins NUFIP2 and G3BP1. Atg8ylation was needed for their recruitment to damaged lysosomes independently of SG condensates whereupon NUFIP2 contributed to mTOR inactivation via the Ragulator-RagA/B complex. Thus, cells employ membrane atg8ylation to control and coordinate SG and mTOR responses to lysosomal damage.

Characterization and immunogenicity of SARS-CoV-2 spike proteins with varied glycosylation.

The ongoing coronavirus disease-19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drastically changed our way of life and continues to have an unmitigated socioeconomic impact across the globe. Research into potential vaccine design and production is focused on the spike (S) protein of the virus, which is critical for virus entry into host cells. Yet, whether the degree of glycosylation in the S protein is associated with vaccine efficacy remains unclear. Here, we first optimized the expression of the S protein in mammalian cells. While we found no significant discrepancy in purity, homogeneity, or receptor binding ability among S proteins derived from 293F cells (referred to as 293F S-2P), 293S GnTI- cells (defective in N-acetylglucosaminyl transferase I enzyme; 293S S-2P), or TN-5B1-4 insect cells (Bac S-2P), there was significant variation in the glycosylation patterns and thermal stability of the proteins. Compared with the partially glycosylated 293S S-2P or Bac S-2P, the fully glycosylated 293F S-2P exhibited higher binding reactivity to convalescent sera. In addition, 293F S-2P induced higher IgG and neutralizing antibody titres than 293S or Bac S-2P in mice. Furthermore, a prime-boost-boost regimen, using a combined immunization of S-2P proteins with various degrees of glycosylation, elicited a more robust neutralizing antibody response than a single S-2P alone. Collectively, this study provides insight into ways to design a more effective SARS-CoV-2 immunogen.