Search Results (31)

Hepatitis E virus (HEV) causes significant morbidity and mortality globally, particularly affecting vulnerable populations such as pregnant women. HEV239 (Hecolin^®), a recombinant vaccine containing the immunodominant protruding (E2) domain of the HEV capsid protein, has demonstrated effectiveness, yet detailed human cellular immune responses remain understudied. This study characterized humoral and cellular immune responses following vaccination with HEV239 or natural HEV infection in healthy Bangladeshi women aged 16–39 years. Using dual IFNγ and IL-4 ELISpot assays, we found robust, predominantly Th1-mediated cellular responses at 30 days after the third vaccine dose, comparable to responses during acute infection. Longitudinal antibody assessments confirmed sustained antibody production, primarily against the E2 domain of genotypes 1 and 3, persisting up to two years post-vaccination. Despite limitations related to sample size and assay sensitivity, our findings underscore the immunogenic potential of HEV239 and support a broader use in HEV-endemic regions. Full article

The emergence of coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has become a global issue since 2019. The prominent characteristic of SARS-CoV-2 is the presence of the spike (S) protein protruding from the virus particle envelope. The S protein is a major drug and vaccine target because it initiates the key step in infection. Medicinal herbs are a potential treatment option to enhance immunity to fight viral infections. Caesalpinia sappan L. has been reported to display promising anti-viral activities. Specifically, brazilin (BRA), a major bioactive compound in C. sappan, was reported to play a role in inhibiting viral infection. Thus, the ability of BRA as a COVID-19 treatment was tested. The S protein was used as the BRA target of this work. Understanding the binding mechanism of BRA to the S protein is crucial for future utilisation of C. sappan as a COVID-19 treatment or other coronavirus-caused pandemics. Here, we performed molecular docking of BRA onto the S protein receptor binding domain (RBD) and multimerisation (MM) pockets. Molecular dynamics (MD) simulations were conducted to study the stability of binding to glycosylated and non-glycosylated S protein constructs. BRA can bind to the Receptor-binding motif (RBM) on an RBD surface stably; however, it is too large to fit into the MM pocket, resulting in dissociation. Nonetheless, BRA is bound by residues near the S1/S2 interface. We found that glycosylation has no effect on BRA binding, as the proposed binding site is far from any glycans. Our results thus indicate that C. sappan may act as a promising preventive and therapeutic alternative for COVID-19 treatment. Full article

Background: Noroviruses, which cause epidemic acute gastroenteritis, and Plasmodium parasites, which lead to malaria, are two infectious pathogens that pose threats to public health. The protruding (P) domain of norovirus VP1 and the αTSR domain of the circumsporozoite protein (CSP) of Plasmodium sporozoite are the glycan receptor-binding domains of the two pathogens for host cell attachment, making them excellent targets for vaccine development. Modified norovirus P domains self-assemble into a 24-meric octahedral P nanoparticle (P₂₄ NP). Methods: We generated a unique P₂₄-αTSR NP by inserting the αTSR domain into a surface loop of the P domain. The P-αTSR fusion proteins were produced in the Escherichia coli expression system and the fusion protein self-assembled into the P₂₄-αTSR NP. Results: The formation of the P₂₄-αTSR NP was demonstrated through gel filtration, electron microscopy, and dynamic light scattering. A 3D structural model of the P₂₄-αTSR NP was constructed, using the known cryo-EM structure of the previously developed P₂₄ NP and P₂₄-VP8* NP as templates. Each P₂₄-αTSR NP consists of a P₂₄ NP core, with 24 surface-exposed αTSR domains that have retained their general conformations and binding function to heparan sulfate proteoglycans. The P₂₄-αTSR NP is immunogenic, eliciting strong antibody responses in mice toward both the norovirus P domain and the αTSR domain of Plasmodium CSP. Notably, sera from mice immunized with the P₂₄-αTSR NP bound strongly to Plasmodium sporozoites and blocked norovirus VLP attachment to their glycan receptors. Conclusion: These data suggest that the P₂₄-αTSR NP may serve as a combination vaccine against both norovirus and Plasmodium parasites. Full article

Caliciviruses are a diverse group of non-enveloped, positive-sense RNA viruses with a wide range of hosts and transmission routes. Norovirus is the most well-known member of the Caliciviridae; the acute gastroenteritis caused by human norovirus (HuNoV), for example, frequently results in closures of hospital wards and schools during the winter months. One area of calicivirus biology that has gained increasing attention over the past decade is the conformational flexibility exhibited by the protruding (P) domains of the major capsid protein VP1. This was observed in structure analyses of capsids encoded by many species and is often a consequence of environmental cues such as metal ions, changes to pH, or receptor/co-factor engagement. This review summarises the current understanding of P-domain flexibility, discussing the role this region plays in caliciviral infection and immune evasion, and highlighting potential avenues for further investigation. Full article

Three-finger proteins (TFPs), or Ly6/uPAR proteins, are characterized by the beta-structural LU domain containing three protruding “fingers” and stabilized by four conserved disulfide bonds. TFPs were initially characterized as snake alpha-neurotoxins, but later many studies showed their regulatory roles in different organisms. Despite a known expression of TFPs in vertebrates, they are poorly studied in other taxa. The presence of TFPs in starfish was previously shown, but their targets and functional role still remain unknown. Here, we analyzed expression, target, and possible function of the Lystar5 protein from the Asterias rubens starfish using bioinformatics, qPCR, and immunoassay. First, the presence of Lystar5 homologues in all classes of echinoderms was demonstrated. qPCR revealed that mRNA of Lystar5 and LyAr2 are expressed mainly in coelomocytes and coelomic epithelium of Asterias, while mRNA of other TFPs, LyAr3, LyAr4, and LyAr5, were also found in a starfish body wall. Using anti-Lystar5 serum from mice immunized by a recombinant Lystar5, we confirmed that this protein is expressed on the surface of coelomocytes and coelomic epithelium cells. According to ELISA, a recombinant analogue of Lystar5 bound to the membrane fraction of coelomocytes and coelomic epithelium but not to the body wall or starfish arm tip. Analysis by LC-MALDI MS/MS suggested integrin α-8-like protein expressed in the coelomocytes and coelomic epithelium as a target of Lystar5. Thus, our insights propose the important role of TFPs in regulation of starfish physiology and show prospects for their further research. Full article

The spectrums of one type of object under different conditions have the same features (up, down, protruding, concave) at the same spectral positions, which can be used as primary parameters to evaluate the difference among remotely sensed pixels. The wavelet-feature correlation ratio Markov clustering algorithm (WFCRMCA) for remotely sensed data is proposed based on an accurate description of abrupt spectral features and an optimized Markov clustering in the wavelet feather space. The peak points can be captured and identified by applying a wavelet transform to spectral data. The correlation ratio between two samples is a statistical calculation of the matched peak point positions on the wavelet feature within an adjustable spectrum domain or a range of wavelet scales. The evenly sampled data can be used to create class centers, depending on the correlation ratio threshold at each Markov step, accelerating the clustering speed by avoiding the computation of Euclidean distance for traditional clustering algorithms, such as K-means and ISODATA. Markov clustering applies several strategies, such as a simulated annealing method and gradually shrinking the clustering size, to control the clustering convergence. It can quickly obtain the best class centers at each clustering temperature. The experimental results of the Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) and Thermal Mapping (TM) data have verified its acceptable clustering accuracy and high convergence velocity. Full article

To understand the evolution of GII.P6-GII.6 and GII.P7-GII.6 strains, the prevalent human norovirus genotypes, we analysed both the RdRp region and VP1 gene in globally collected strains using authentic bioinformatics technologies. A common ancestor of the P6- and P7-type RdRp region emerged approximately 50 years ago and a common ancestor of the P6- and P7-type VP1 gene emerged approximately 110 years ago. Subsequently, the RdRp region and VP1 gene evolved. Moreover, the evolutionary rates were significantly faster for the P6-type RdRp region and VP1 gene than for the P7-type RdRp region and VP1 genes. Large genetic divergence was observed in the P7-type RdRp region and VP1 gene compared with the P6-type RdRp region and VP1 gene. The phylodynamics of the RdRp region and VP1 gene fluctuated after the year 2000. Positive selection sites in VP1 proteins were located in the antigenicity-related protruding 2 domain, and these sites overlapped with conformational epitopes. These results suggest that the GII.6 VP1 gene and VP1 proteins evolved uniquely due to recombination between the P6- and P7-type RdRp regions in the HuNoV GII.P6-GII.6 and GII.P7-GII.6 virus strains. Full article

Integrating point absorber wave energy converters (PAWECs) and an offshore floating wind platform provide a cost-effective way of joint wind and wave energy exploitation. However, the coupled dynamics of the complicated hybrid system and its influence on power performance are not well understood. Here, a frequency-domain-coupled hydrodynamics, considering the constraints and the power output through the relative motion between the PAWECs and the semi-submersible platform, is introduced to optimize the size, power take-off damping, and layout of the PAWECs. Results show that the annual wave power generation of a PAWEC can be improved by 30% using a 90° conical or a hemispherical bottom instead of a flat bottom. Additionally, while letting the PAWECs protrude out the sides of the triangular frame of the platform by a distance of 1.5 times the PAWEC radius, the total power generation can be improved by up to 18.2% without increasing the motion response of the platform. The PAWECs can reduce the resonant heave motion of the platform due to the power take-off damping force. This study provides a reference for the synergistic use of wave and wind energy. Full article

Many efforts in norovirus vaccine development have focused on subunit or recombinant protein vaccines, such as subviral P particles formed by the protruding (P) domain of VP1. P particles are immunogenic and have a region with a human histo-blood group antigen binding site, an interaction critical for infecting the host. In the past, only intracellular NoV P proteins expressed in Escherichia coli and Pichia pastoris were reported, and the low yield and difficulty in purification limited their applications. In this study, the Taiwan-native NoV P domain was successfully expressed and secreted by P. pastoris. The secretion efficiency was greatly enhanced by integrating oligosaccharyl transferase (Ost1) into the α-factor signal peptide and coexpressing Hac1. The production of NoV P in fermentation cultures reached 345 mg/L, and the purity and recovery were 94.8% and 66.9%, respectively, after only ion-exchange chromatography. Transmission electron microscopy analysis showed that the small P particles were mostly ring-, square-, and triangle-shaped, with diameters of 10-15 nm. The biological activity of NoV P was confirmed by saliva-binding assay using human histo-blood group antigen. This study describes the secretion and characterization of the Taiwan-native norovirus P domain in P. pastoris. Particles formed from the P domain were similar in size, morphology, and binding ability to those expressed intracellularly. The strategy described in this study provides great potential in scale-up production and antiviral vaccine development. Full article

Since the outbreak of the coronavirus disease 2019 (COVID-19), various vaccines have been developed for emergency use. The efficacy of the initial vaccines based on the ancestral strain of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has become a point of contention due to the emergence of new variants of concern (VOCs). Therefore, continuous innovation of new vaccines is required to target upcoming VOCs. The receptor binding domain (RBD) of the virus spike (S) glycoprotein has been extensively used in vaccine development due to its role in host cell attachment and penetration. In this study, the RBDs of the Beta (β) and Delta (δ) variants were fused to the truncated Macrobrachium rosenbergii nodavirus capsid protein without the protruding domain (CΔ116-MrNV-CP). Immunization of BALB/c mice with the virus-like particles (VLPs) self-assembled from the recombinant CP showed that, with AddaVax as an adjuvant, a significantly high level of humoral response was elicited. Specifically, mice injected with equimolar of adjuvanted CΔ116-MrNV-CP fused with the RBD of the β- and δ-variants increased T helper (Th) cell production with a CD8⁺/CD4⁺ ratio of 0.42. This formulation also induced proliferation of macrophages and lymphocytes. Overall, this study demonstrated that the nodavirus truncated CP fused with the SARS-CoV-2 RBD has potential to be developed as a VLP-based COVID-19 vaccine. Full article

Ly6/uPAR proteins regulate many essential functions in the nervous and immune systems and epithelium. Most of these proteins contain single β-structural LU domains with three protruding loops and are glycosylphosphatidylinositol (GPI)-anchored to a membrane. The GPI-anchor role is currently poorly studied. Here, we investigated the positional and orientational preferences of six GPI-anchored proteins in the receptor-unbound state by molecular dynamics simulations. Regardless of the linker length between the LU domain and GPI-anchor, the proteins interacted with the membrane by polypeptide parts and N-/O-glycans. Lynx1, Lynx2, Lypd6B, and Ly6H contacted the membrane by the loop regions responsible for interactions with nicotinic acetylcholine receptors, while Lypd6 and CD59 demonstrated unique orientations with accessible receptor-binding sites. Thus, GPI-anchoring does not guarantee an optimal ‘pre-orientation’ of the LU domain for the receptor interaction. Full article

A sodium channel blocker mexiletine (MEX) is used to treat chronic pain, myotonia and some arrhythmias. Mutations in the pore domain (PD) of voltage-gated sodium channels differently affect tonic block (TB) and use-dependent block (UDB) by MEX. Previous studies identified several MEX-sensing residues in the hNav1.5 channel and demonstrated that the channel block by MEX increases with activation of the voltage-sensing domain III (VSD_III), whereas MEX stabilizes the activated state of VSD_III. Structural rationales for these observations are unclear. Here, Monte Carlo (MC) energy minimizations were used to dock MEX and its more potent analog, Thio-Me2, into the hNav1.5 cryo-EM structure with activated VSDs and presumably inactivated PD. Computations yielded two ensembles of ligand binding poses in close contacts with known MEX-sensing residues in helices S6_III, S6_IV and P1_IV. In both ensembles, the ligand NH₃ group approached the cation-attractive site between backbone carbonyls at the outer-pore bottom, while the aromatic ring protruded ether into the inner pore (putative UDB pose) or into the III/IV fenestration (putative TB pose). In silico deactivation of VSD_III shifted helices S4–S5_III, S5_III, S6_III and S6_IV and tightened the TB site. In a model with activated VSD_III and three resting VSDs, MC-minimized energy profile of MEX pulled from the TB site towards lipids shows a deep local minimum due to interactions with 11 residues in S5_III, P1_III, S6_III and S6_IV. The minimum may correspond to an interim binding site for MEX in the hydrophobic path to the TB site along the lipid-exposed sides of repeats III and IV where 15 polar and aromatic residues would attract cationic blockers. The study explains numerous experimental data and suggests the mechanism of allosteric modification of the MEX binding site by VSD_III. Full article

Capsid protein of Hepatitis E virus (HEV) is capable of self-assembly into virus-like particles (VLPs) when expressed in Nicotiana benthamiana plants. Such VLPs could be used as carriers of antigens for vaccine development. In this study, we obtained VLPs based on truncated coat protein of HEV bearing the M2e peptide of Influenza A virus or receptor-binding domain of SARS-CoV-2 spike glycoprotein (RBD). We optimized the immunogenic epitopes’ presentation by inserting them into the protruding domain of HEV ORF2 at position Tyr485. The fusion proteins were expressed in Nicotiana benthamiana plants using self-replicating potato virus X (PVX)-based vector. The fusion protein HEV/M2, targeted to the cytosol, was expressed at the level of about 300–400 μg per gram of fresh leaf tissue and appeared to be soluble. The fusion protein was purified using metal affinity chromatography under native conditions with the final yield about 200 μg per gram of fresh leaf tissue. The fusion protein HEV/RBD, targeted to the endoplasmic reticulum, was expressed at about 80–100 μg per gram of fresh leaf tissue; the yield after purification was up to 20 μg per gram of fresh leaf tissue. The recombinant proteins HEV/M2 and HEV/RBD formed nanosized virus-like particles that could be recognized by antibodies against inserted epitopes. The ELISA assay showed that antibodies of COVID-19 patients can bind plant-produced HEV/RBD virus-like particles. This study shows that HEV capsid protein is a promising carrier for presentation of foreign antigen. Full article

Intracellular trafficking of human papillomavirus (HPV) during virus entry requires γ-secretase, a cellular protease consisting of a complex of four cellular transmembrane (TM) proteins. γ-secretase typically cleaves substrate proteins but it plays a non-canonical role during HPV entry. γ-secretase binds to the HPV minor capsid protein L2 and facilitates its insertion into the endosomal membrane. After insertion, L2 protrudes into the cytoplasm, which allows HPV to bind other cellular factors required for proper virus trafficking into the retrograde transport pathway. Here, we further characterize the interaction between γ-secretase and HPV L2. We show that γ-secretase is required for cytoplasmic protrusion of L2 and that L2 associates strongly with the PS1 catalytic subunit of γ-secretase and stabilizes the γ-secretase complex. Mutational studies revealed that a putative TM domain in HPV16 L2 cannot be replaced by a foreign TM domain, that infectivity of HPV TM mutants is tightly correlated with γ-secretase binding and stabilization, and that the L2 TM domain is required for protrusion of the L2 protein into the cytoplasm. These results provide new insight into the interaction between γ-secretase and L2 and highlight the importance of the native HPV L2 TM domain for proper virus trafficking during entry. Full article

Galectins are multi-purpose effectors acting via interactions with distinct counterreceptors based on protein-glycan/protein recognition. These processes are emerging to involve several regions on the protein so that the availability of a detailed structural characterization of a full-length galectin is essential. We report here the first crystallographic information on the N-terminal extension of the carbohydrate recognition domain of rat galectin-5, which is precisely described as an N-tailed proto-type-like galectin. In the ligand-free protein, the three amino-acid stretch from Ser2 to Ser5 is revealed to form an extra β-strand (F0), and the residues from Thr6 to Asn12 are part of a loop protruding from strands S1 and F0. In the ligand-bound structure, amino acids Ser2–Tyr10 switch position and are aligned to the edge of the β-sandwich. Interestingly, the signal profile in our glycan array screening shows the sugar-binding site to preferentially accommodate the histo-blood-group B (type 2) tetrasaccharide and N-acetyllactosamine-based di- and oligomers. The crystal structures revealed the characteristically preformed structural organization around the central Trp77 of the CRD with involvement of the sequence signature’s amino acids in binding. Ligand binding was also characterized calorimetrically. The presented data shows that the N-terminal extension can adopt an ordered structure and shapes the hypothesis that a ligand-induced shift in the equilibrium between flexible and ordered conformers potentially acts as a molecular switch, enabling new contacts in this region. Full article