Virus Glycoprotein Research Articles

Deep mutational scanning reveals functional constraints and antibody-escape potential of Lassa virus glycoprotein complex

Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of the Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we used pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affected cell entry and antibody neutralization. Our experiments defined functional constraints throughout GPC. We quantified how GPC mutations affected neutralization with a panel of monoclonal antibodies. All antibodies tested were escaped by mutations that existed among natural Lassa virus lineages. Overall, our work describes a biosafety-level-2 method to elucidate the mutational space accessible to GPC and shows how prospective characterization of antigenic variation could aid the design of therapeutics and vaccines.

Spray vaccination with a Newcastle disease virus (NDV)-vectored infectious laryngotracheitis (ILT) vaccine protects commercial chickens from ILT in the presence of maternally-derived antibodies

ABSTRACT Infectious laryngotracheitis (ILT) poses a significant threat to the poultry industry, and vaccines play an important role in protection. However, due to the increasing scale of poultry production, there is an urgent need to develop vaccines that are suitable for convenient immunization methods such as spraying. Previous studies have shown that Newcastle disease virus (NDV)-ILT vaccines administered via intranasal and intraocular routes to commercial chickens carrying maternally-derived antibodies (MDAs) are still protective against ILT. In this study, a recombinant NDV (rNDV) was generated to express infectious laryngotracheitis virus (ILTV) glycoprotein B (gB), named rLS-gB, based on a full-length cDNA clone of the LaSota strain. The protective effect of different doses of rLS-gB administered by spray vaccination to commercial chickens at 1 d of age (doa) was evaluated. The chickens were exposed to 160-μm aerosol particles for 10 min for spray vaccination, and no adverse reactions were observed after vaccination. Despite the presence of anti-NDV MDAs and anti-ILTV MDAs in chickens, the ILTV- and NDV-specific antibody titres were significantly greater in the vaccinated groups than in the unvaccinated group. After challenge with a virulent ILTV strain, no clinical signs were observed in the 107 EID50/ml group compared to the other groups. Furthermore, vaccination with 107 EID50/ml rLS-gB significantly reduced the ILTV viral load and ameliorated gross and microscopic lesions in the trachea of chickens. Overall, these results suggested that rLS-gB is a safe and efficient candidate spray vaccine for ILT and is especially suitable for scaled chicken farms.

Anti-hemagglutinin monomeric nanobody provides prophylactic immunity against H1 subtype influenza A viruses.

Influenza viruses constitute a major threat to human health globally. The viral surface glycoprotein hemagglutinin (HA) is the immunodominant antigen, contains the site for binding to the cellular receptor (RBS), and it is the major target of neutralizing antibody responses post-infection. We developed llama-derived single chain antibody fragments (VHHs) specific for type A influenza virus. Four VHHs were identified and further characterized. VHH D81 bound residues in the proximity of the C-terminal region of HA1 of H1 and H5 subtypes, and showed weak neutralizing activity, whereas VHH B33 bound residues in the proximity of the N-terminal region of the HA's stem domain (HA2) of H1, H5, and H9 subtypes, and showed no neutralizing activity. Of most relevance, VHHs E13 and G41 recognized highly conserved conformational epitopes on the H1 HA's globular domain (HA1) and showed high virus neutralizing activity (ranging between 0.94 to 0.01μM), when tested against several human H1N1 isolates. Additionally, E13 displayed abrogated virus replication of a panel of H1N1 strains spanning over 80 years of antigenic drift and isolated from human, avian, and swine origin. Interestingly, E13 conferred protection in vivo at a dose as low as 0.05 mg/kg. Mice treated with E13 intranasally resulted in undetectable virus challenge loads in the lungs at day 4 post-challenge. The transfer of sterilizing pan-H1 immunity, by a dose in the range of micrograms given intranasally, is of major significance for a monomeric VHH and supports the further development of E13 as an immunotherapeutic agent for the mitigation of influenza infections.

NMR side-chain assignments of the Crimean–Congo hemorrhagic fever virus glycoprotein n cytosolic domain

Abstract. We assigned the side-chain resonances of the Crimean–Congo hemorrhagic fever virus (CCHFV) glycoprotein n (Gn) cytosolic domain that is 69 amino acids long to complete the backbone resonances previously published by Estrada et al. (2011). The process was facilitated by three factors. First, sample preparation using cell-free protein synthesis (CFPS) was completed in less than 2 d and allowed for correct zinc finger formation by adding zinc ions to the reaction. Second, access to NMR platforms with standardized pulse sequences allowed for data acquisition in 18 d. Third, data analysis using the online platform NMRtist allowed sequential resonance assignments to be made in a day, and assignments were verified and finalized in less than a week. Our work thus provides an example of how NMR assignments, including side chains, of small and well-behaved proteins can be approached in a rapid routine, at protein concentrations of 150 µM.

Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

Crimean-Congo hemorrhagic fever virus can cause lethal disease in humans yet there are no approved medical countermeasures. Viral glycoprotein GP38, exclusive to Nairoviridae, is a target of protective antibodies and is a key antigen in preclinical vaccine candidates. Here, we isolate 188 GP38-specific antibodies from human survivors of infection. Competition experiments show that these antibodies bind across 5 distinct antigenic sites, encompassing 11 overlapping regions. Additionally, we show structures of GP38 bound with 9 of these antibodies targeting different antigenic sites. Although these GP38-specific antibodies are non-neutralizing, several display protective efficacy equal to or better than murine antibody 13G8 in two highly stringent rodent models of infection. Together, these data expand our understanding regarding this important viral protein and may inform the development of broadly effective CCHFV antibody therapeutics.

Aluminum oxyhydroxide-Poly(I:C) combination adjuvant with balanced immunostimulatory potentials for prophylactic vaccines

The development of high-purity antigens promotes the urgent need of novel adjuvant with the capability to trigger high levels of immune response. Polyinosinic-polycytidylic (Poly(I:C)) is a synthetic double-stranded RNA (dsRNA) that can engage Toll-like receptor 3 (TLR3) to initiate immune responses. However, the Poly(I:C)-induced toxicity and inefficient delivery prevent its applications. In our study, combination adjuvants are formulated by aluminum oxyhydroxide nanorods (AlOOH NRs) and Poly(I:C), named Al-Poly(I:C), and the covalent interaction between the two components is further demonstrated. Al-Poly(I:C) mediates enhanced humoral and cellular immune responses in three antigen models, i.e., HBsAg virus-like particles (VLPs), human papilloma virus (HPV) VLPs and varicella-zoster virus (VZV) glycoprotein E (gE). Further mechanistic studies demonstrate that the dose and molecular weight (MW) of Poly(I:C) determine the physicochemical properties and adjuvanticity of the Al-Poly(I:C) combination adjuvants. Al-Poly(I:C) with higher Poly(I:C) dose promotes antigen-bearing dendritic cells (DCs) recruitment and B cells proliferation in lymph nodes. Al-Poly(I:C) formulated with higher MW Poly(I:C) induces higher activation of helper T cells, B cells, and CTLs. This study demonstrates that Al-Poly(I:C) potentiates the humoral and cellular responses in vaccine formulations. It offers insights for adjuvant design to meet the formulation requirements in both prophylactic and therapeutic vaccines.

An efficient plasmid-based system for the recovery of recombinant vesicular stomatitis virus encoding foreign glycoproteins

Viral glycoproteins mediate entry into host cells, thereby dictating host range and pathogenesis. In addition, they constitute the principal target of neutralizing antibody responses, making them important antigens in vaccine development. Recombinant vesicular stomatitis virus (VSV) encoding foreign glycoproteins can provide a convenient and safe surrogate system to interrogate the function, evolution, and antigenicity of viral glycoproteins from viruses that are difficult to manipulate or those requiring high biosafety level containment. However, the production of recombinant VSV can be technically challenging. In this work, we present an efficient and robust plasmid-based system for the production of recombinant VSV encoding foreign glycoproteins. We validate the system using glycoproteins from different viral families, including arenaviruses, coronaviruses, and hantaviruses, as well as highlight their utility for studying the effects of mutations on viral fitness. Overall, the methods described herein can facilitate the study of both native and recombinant VSV encoding foreign glycoproteins and can serve as the basis for the production of VSV-based vaccines.

A broadly-neutralizing antibody against Ebolavirus glycoprotein that potentiates the breadth and neutralization potency of other antibodies.

Ebolavirus disease (EVD) is caused by multiple species of Ebolavirus. Monoclonal antibodies (mAbs) against the virus glycoprotein (GP) are the only class of therapeutic approved for treatment of EVD caused by Zaire ebolavirus (EBOV). Therefore, mAbs targeting multiple Ebolavirus species may represent the next generation of EVD therapeutics. Broadly reactive anti-GP mAbs were produced; among these, mAbs 11886 and 11883 were broadly neutralizing in vitro. A 3.0 Å cryo-electron microscopy structure of EBOV GP bound to both mAbs shows that 11886 binds a novel epitope bridging the glycan cap (GC), 310 pocket and GP2 N-terminus, whereas 11883 binds the receptor binding region (RBR) and GC. In vitro, 11886 synergized with a range of mAbs with epitope specificities spanning the RBR/GC, including 11883. Notably, 11886 increased the breadth of neutralization by partner mAbs against different Ebolavirus species. These data provide a strategic route to design improved mAb-based next-generation EVD therapeutics.

Absolute quantification of viral proteins from pseudotyped VSV-GP using UPLC-MRM.

The development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (VSV-GP) is a new biotherapeutic emerging in the oncolytic viral therapy platform. Novel analytical assays that can accurately and precisely quantify the viral proteins are a necessity for the successful development of viral vector as a biotherapeutic. We developed an ultra-high performance liquid chromatography multiple reaction monitoring-based assay to quantify the absolute concentrations of the different structural proteins of VSV-GP. The complete processing of GP complex (GPC) is a prerequisite for the infectivity of the virus. The assay extends the potential for quantifying full-length GPC, which provides an understanding of the processing of GPC (along with the quantification of GP1 and GP2 separately). We used this assay in tracking GPC processing in HEK-293-F production cell lines infected with VSV-GP.

Anticoronavirus activity of rhizome of Dryopteris crassirhizoma via multistage targeting of virus entry and viral proteases, Mpro and PLpro

Ethnopharmacological relevanceThe rhizome of Dryopteris crassirhizoma Nakai (Dryopteridaceae, RDC), a traditional East Asian herbal medicine, possesses a broad spectrum of medicinal properties, including anti-inflammatory, anticancer, antibacterial, and antiviral activities. Aim of the studyThis study investigates the 30% ethanolic extract of RDC's antiviral potential against human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and its variants infections. Materials and methodsA 30% ethanolic extract of RDC or its components, filixic acid ABA (PubChem CID: 15081408) and dryocrassin ABBA (PubChem CID: 3082025) were treated with Human Coronavirus infection (HCoV-OC43, SARS-CoV-2 and its variants). The base peak chromatogram of RDC was evaluated using UPLC-Q/TOF Mass to identify the RDC, and the quantitative analysis of RDC compounds was performed using LC-MS/MS. A cytopathic effect (CPE) reduction assay, Western blot, immunofluorescence staining of viral protein expression, and qRT-PCR were performed to quantify the viral RNA copy numbers and determine the antiviral activity. The time-of-addition assay, the virus attachment, penetration, and virucidal assays, and SARS-CoV-2 Mpro and PLpro activity assay were used to elucidate the mode of action. ResultsRDC exhibited dose-dependent inhibition of HCoV-OC43–induced cytopathic effects, reducing viral RNA copy numbers and viral protein levels. Time-of-addition assays indicated that RDC targets the early stages of the HCoV-OC43 life cycle, inhibiting virion attachment and penetration with virucidal activity. Notably, filixic acid ABA and dryocrassin ABBA, constituents of RDC, reduced HCoV-OC43 viral RNA loads. Furthermore, RDC effectively blocked viral entry in pseudotyped lentivirus assays, involving spike proteins of SARS-CoV-2 Delta plus and South Africa variants, as well as control lentiviral particles expressing vesicular stomatitis virus glycoprotein G. Additionally, RDC demonstrated inhibition of SARS-CoV-2 infection and its variants by targeting viral proteases, namely main protease (Mpro) and papain-like protease (PLpro). ConclusionsThese findings underscore RDC's multistage approach to targeting viral infections by impeding virus entry and inhibiting viral protease activity. Therefore, RDC holds promise as a potent, broad-spectrum anticoronaviral therapeutic agent.

Phenylboronic Acid-Modified Membrane-Like Magnetic Quantum Dots Enable the Ultrasensitive and Broad-Spectrum Detection of Viruses by Lateral Flow Immunoassay.

Although lateral flow immunochromatographic assay (LFIA) is an effective point-of-care testing technology, it still cannot achieve broad-spectrum and ultrasensitive detection of viruses. Herein, we propose a multiplex LFIA platform using a two-dimensional graphene oxide (GO)-based magnetic fluorescent nanofilm (GF@DQD) as a multifunctional probe and 4-aminophenylboronic acid (APBA) as a broad-spectrum recognition molecule for viral glycoprotein detection. GF@DQD-APBA with enhanced magnetic/fluorescence properties and universal capture ability for multiple viruses was easily prepared through the electrostatic adsorption of one layer of density-controlled Fe3O4 nanoparticles (NPs) and thousands of small CdSe/ZnS-MPA quantum dots (QDs) on a monolayer GO sheet followed by chemical coupling with APBA on the QD surface. The GF@DQD-APBA probe enabled the universal capture and specific determination of different target viruses on the test strip through an arbitrary combination with the antibody-modified LFIA strip, thus greatly improving detection efficiency and reducing the cost and difficulty of multiplex LFIA for viruses. The proposed technique can simultaneously and sensitively diagnose three newly emerged viruses within 20 min with detection limits down to the pg/mL level. The excellent practicability of GF@DQD-APBA-LFIA was also demonstrated in the detection of 34 clinical specimens positive for SARS-CoV-2, revealing its potential for epidemic control and on-site viral detection.

Silicon or Calcium Doping Coordinates the Immunostimulatory Effects of Aluminum Oxyhydroxide Nanoadjuvants in Prophylactic Vaccines.

Aluminum salts still remain as the most popular adjuvants in marketed human prophylactic vaccines due to their capability to trigger humoral immune responses with a good safety record. However, insufficient induction of cellular immune responses limits their further applications. In this study, we prepare a library of silicon (Si)- or calcium (Ca)-doped aluminum oxyhydroxide (AlOOH) nanoadjuvants. They exhibit well-controlled physicochemical properties, and the dopants are homogeneously distributed in nanoadjuvants. By using Hepatitis B surface antigen (HBsAg) as the model antigen, doped AlOOH nanoadjuvants mediate higher antigen uptake and promote lysosome escape of HBsAg through lysosomal rupture induced by the dissolution of the dopant in the lysosomes in bone marrow-derived dendritic cells (BMDCs). Additionally, doped nanoadjuvants trigger higher antigen accumulation and immune cell activation in draining lymph nodes. In HBsAg and varicella-zoster virus glycoprotein E (gE) vaccination models, doped nanoadjuvants induce high IgG titer, activations of CD4+ and CD8+ T cells, cytotoxic T lymphocytes, and generations of effector memory T cells. Doping of aluminum salt-based adjuvants with biological safety profiles and immunostimulating capability is a potential strategy to mediate robust humoral and cellular immunity. It potentiates the applications of engineered adjuvants in the development of vaccines with coordinated immune responses.

(+)-fenchol and (−)-isopinocampheol derivatives targeting the entry process of filoviruses

The increasing frequency of filovirus outbreaks in African countries has led to a pressing need for the development of effective antifilovirus agents. In continuation of our previous research on the antifilovirus activity of monoterpenoid derivatives, we synthesized a series of (+)-fenchol and (−)-isopinocampheol derivatives by varying the type of heterocycle and linker length. Derivatives with an N-alkylpiperazine cycle proved to be the most potent antiviral compounds, with half-maximal inhibitory concentration (IC50) 1.4–20 μМ against Lenti-EboV-GP infection and 11.3–47 μМ against Lenti-MarV-GP infection. Mechanism-of-action experiments revealed that the compounds may exert their action by binding to surface glycoproteins (GPs). It was demonstrated that the binding of the synthesized compounds to the Marburg virus GP is less efficient as compared to the Ebola virus GP. Furthermore, it was shown that the compounds possess lysosomotropic properties. Thus, the antiviral activity may be due to dual effects. This study offers new antiviral agents that are worthy of further exploration.

Targeting host O-linked glycan biosynthesis affects Ebola virus replication efficiency and reveals differential GalNAc-T acceptor site preferences on the Ebola virus glycoprotein.

Ebola virus glycoprotein (EBOV GP) is one of the most heavily O-glycosylated viral glycoproteins, yet we still lack a fundamental understanding of the structure of its large O-glycosylated mucin-like domain and to what degree the host O-glycosylation capacity influences EBOV replication. Using tandem mass spectrometry, we identified 47 O-glycosites on EBOV GP and found similar glycosylation signatures on virus-like particle- and cell lysate-derived GP. Furthermore, we performed quantitative differential O-glycoproteomics on proteins produced in wild-type HEK293 cells and cell lines ablated for the three key initiators of O-linked glycosylation, GalNAc-T1, -T2, and -T3. The data show that 12 out of the 47 O-glycosylated sites were regulated, predominantly by GalNAc-T1. Using the glycoengineered cell lines for authentic EBOV propagation, we demonstrate the importance of O-linked glycan initiation and elongation for the production of viral particles and the titers of progeny virus. The mapped O-glycan positions and structures allowed to generate molecular dynamics simulations probing the largely unknown spatial arrangements of the mucin-like domain. The data highlight targeting GALNT1 or C1GALT1C1 as a possible way to modulate O-glycan density on EBOV GP for novel vaccine designs and tailored intervention approaches.IMPORTANCEEbola virus glycoprotein acquires its extensive glycan shield in the host cell, where it is decorated with N-linked glycans and mucin-type O-linked glycans. The latter is initiated by a family of polypeptide GalNAc-transferases that have different preferences for optimal peptide substrates resulting in a spectrum of both very selective and redundant substrates for each isoform. In this work, we map the exact locations of O-glycans on Ebola virus glycoprotein and identify subsets of sites preferentially initiated by one of the three key isoforms of GalNAc-Ts, demonstrating that each enzyme contributes to the glycan shield integrity. We further show that altering host O-glycosylation capacity has detrimental effects on Ebola virus replication, with both isoform-specific initiation and elongation playing a role. The combined structural and functional data highlight glycoengineered cell lines as useful tools for investigating molecular mechanisms imposed by specific glycans and for steering the immune responses in future vaccine designs.

Novel insights into the host cell glycan binding profile of human metapneumovirus.

Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galβ1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates.IMPORTANCEAll cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease.

Enhanced brain-targeting and efficacy of cannabidiol via RVG-Exo/CBD nanodelivery system

This study introduces an innovative brain-targeted drug delivery system, RVG-Exo/CBD, utilizing rabies virus glycoprotein (RVG)-engineered exosomes for encapsulating cannabidiol (CBD). The novel delivery system was meticulously characterized, confirming the maintenance of exosomal integrity, size, and successful drug encapsulation with a high drug loading rate of 83.0 %. Evaluation of the RVG-Exo/CBD's brain-targeting capability demonstrated superior distribution and retention in brain tissue compared to unmodified exosomes, primarily validated through in vivo fluorescence imaging. The efficacy of this delivery system was assessed using a behavioral sensitization model in mice, where RVG-Exo/CBD notably suppressed methamphetamine-induced hyperactivity more effectively than CBD alone, indicating a reduction in effective dose and enhanced bioavailability. Overall, the RVG-Exo/CBD system emerges as a promising strategy for enhancing the therapeutic efficacy and safety of CBD, particularly for neurological applications, highlighting its potential for addressing the limitations associated with traditional CBD administration in clinical settings.

Drug design of new anti-EBOV inhibitors: QSAR, homology modeling, molecular docking and molecular dynamics studies

Ebola virus disease is a deadly pathogenic disease with a fatality rate of 25–90 % as recorded in previous outbreaks. The Ebola Virus glycoprotein (EBOV-GP) plays a crucial role in the entry of viruses into human cells, making it an interesting target for therapeutic discovery. Therefore, inhibiting this protein can directly limit the virus replication and disease progression at an early stage of infection. The present study focuses on the design of novel potent EBOV-GP inhibitors using multiple computational techniques. In this context, two QSAR models were built from a set of 86 amodiaquine derivatives as anti-EBOV-GP using Monte Carlo and genetic algorithm multiple linear regression methods. Both models confirmed their predictive performance with satisfactory statistical parameters of the validation (R2 = 0.9129; Q2 = 0.8848 for the CORAL model and R2 = 0.8848; Q2 = 0.8148 for the GA-MLR model). From the outputs of the CORAL model, the structural fragments responsible for increasing and decreasing the inhibition activity were extracted and interpreted. This molecular information was used to design 26 new potentially safe and active candidate drugs. Molecular docking and dynamics simulations have affirmed the efficacy of the designed compounds. Specifically, compounds D2 (pIC50_coral = 7.12; pIC50_GA-MLR = 7.07), D3 (pIC50_coral = 7.83; pIC50_GA-MLR = 7.10), and D5 (pIC50_coral = 7.26; pIC50_GA-MLR = 7.55) displayed notable predicted inhibitory activity, according to both models. These compounds also exhibited conformational and structural stability, as well as a favorable binding profile. Furthermore, these potential drug candidates were found to be non-toxicity and have acceptable pharmacological properties.

Blood brain barrier-targeted lipid nanoparticles improved the neuroprotection of Ferrostatin-1 against cerebral ischemic damage in an experimental stroke model

Cerebral ischemic stroke is a serious disease with high mortality and disability rates. However, few neuroprotective drugs have been used for ischemic stroke in the clinic. Two main reasons may be responsible for this failure: difficulty in penetrating the blood-brain barrier (BBB) and easily inactivated in the blood circulation. Ferroptosis, a lipid oxidation-related cell death, plays significant roles in cerebral ischemia-reperfusion injury. We utilized RVG29, a peptide derived from Rabies virus glycoprotein, to obtain BBB-targeted lipid nanoparticles (T-LNPs) in order to investigate whether T-LNPs improved the neuroprotective effects of Ferrostatin-1 (Fer1, an inhibitor of ferroptosis) against cerebral ischemic damage. T-LNPs significantly increased BBB penetration following oxygen/glucose deprivation exposure in an in vitro BBB model and enhanced the fluorescence distribution in brain tissues at 6 h post-administration in a cerebral ischemic murine model. Moreover, T-LNPs encapsulated Fer1 (T-LNPs-Fer1) significantly enhanced the inhibitory effects of Fer1 on ferroptosis by maintaining the homeostasis of NADPH oxidase 4 (NOX4) and glutathione peroxidase 4 (GPX4) signals in neuronal cells after cerebral ischemia. T-LNPs-Fer1 significantly suppressed oxidative stress [heme oxygenase-1 expression and malondialdehyde (the product of lipid ROS reaction)] in neurons and alleviated ischemia-induced neuronal cell death, compared to Fer1 alone without encapsulation. Furthermore, T-LNPs-Fer1 significantly reduced cerebral infarction and improved behavior functions compared to Fer1-treated cerebral ischemic mice after 45-min ischemia/24-h reperfusion. These findings showed that the T-LNPs helped Fer1 penetrate the BBB and improved the neuroprotection of Fer1 against cerebral ischemic damage in experimental stroke, providing a feasible translational strategy for the development of clinical drugs for the treatment of ischemic stroke.

Revisiting the interaction between complement lectin pathway protease MASP-2 and SARS-CoV-2 nucleoprotein.

Complement activation is considered to contribute to the pathogenesis of severe SARS-CoV-2 infection, mainly by generating potent immune effector mechanisms including a strong inflammatory response. Involvement of the lectin complement pathway, a major actor of the innate immune anti-viral defense, has been reported previously. It is initiated by recognition of the viral surface Spike glycoprotein by mannose-binding lectin (MBL), which induces activation of the MBL-associated protease MASP-2 and triggers the proteolytic complement cascade. A role for the viral nucleoprotein (N) has also been reported, through binding to MASP-2, leading to protease overactivation and potentiation of the lectin pathway. In the present study, we reinvestigated the interactions of the SARS-CoV-2 N protein, produced either in bacteria or secreted by mammalian cells, with full-length MASP-2 or its catalytic domain, in either active or proenzyme form. We could not confirm the interaction of the N protein with the catalytic domain of MASP-2 but observed N protein binding to proenzyme MASP-2. We did not find a role of the N protein in MBL-mediated activation of the lectin pathway. Finally, we showed that incubation of the N protein with MASP-2 results in proteolysis of the viral protein, an observation that requires further investigation to understand a potential functional significance in infected patients.

Identification of residues in Lassa virus glycoprotein 1 involved in receptor switch

Lassa virus (LASV) is an enveloped, negative-sense RNA virus that causes Lassa hemorrhagic fever. Successful entry of LASV requires the viral glycoprotein 1 (GP1) to undergo a receptor switch from its primary receptor alpha-dystroglycan (α-DG) to its endosomal receptor lysosome-associated membrane protein 1 (LAMP1). A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch. To test the hypothesis that other non-conserved residues also contribute to receptor switch, we constructed a series of mutant LASV GP1 proteins and tested them for binding to LAMP1. Four residues, L84, K88, L107, and H170, were identified as critical for receptor switch. Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus (LCMV) residue (L84 ​N, K88E, L10F, and H170S) reduced the binding affinity of LASV GP1 for LAMP1. Moreover, all mutations caused decreases in glycoprotein precursor (GPC)-mediated membrane fusion at both pH 4.5 and 5.2. The infectivity of pseudotyped viruses bearing either GPCL84N or GPCK88E decreased sharply in multiple cell types, while L107F and H170S had only mild effects on infectivity. Using biolayer light interferometry assay, we found that all four mutants had decreased binding affinity to LAMP1, in the order of binding affinity being L84 ​N ​> ​L107F ​> ​K88E ​> ​H170S. The four amino acid loci identified for the first time in this study have important reference significance for the in-depth investigation of the mechanism of receptor switching and immune escape of LASV occurrence and the development of reserve anti-LASV infection drugs.