Correction: Kaewchim et al. Neutralizing and Enhancing Epitopes of the SARS-CoV-2 Receptor-Binding Domain (RBD) Identified by Nanobodies. Viruses 2023, 15, 1252.

In the original publication [...].

Isolation and Characterization of scFv Antibody against Internal Ribosomal Entry Site of Enterovirus A71.

Enterovirus A71 (EV-A71) is one of the causative agents of hand-foot-mouth disease, which can be associated with neurocomplications of the central nervous system. A limited understanding of the virus's biology and pathogenesis has led to the unavailability of effective anti-viral treatments. The EV-A71 RNA genome carries type I internal ribosomal entry site (IRES) at 5' UTR that plays an essential role in the viral genomic translation. However, the detailed mechanism of IRES-mediated translation has not been elucidated. In this study, sequence analysis revealed that the domains IV, V, and VI of EV-A71 IRES contained the structurally conserved regions. The selected region was transcribed in vitro and labeled with biotin to use as an antigen for selecting the single-chain variable fragment (scFv) antibody from the naïve phage display library. The so-obtained scFv, namely, scFv #16-3, binds specifically to EV-A71 IRES. The molecular docking showed that the interaction between scFv #16-3 and EV-A71 IRES was mediated by the preferences of amino acid residues, including serine, tyrosine, glycine, lysine, and arginine on the antigen-binding sites contacted the nucleotides on the IRES domains IV and V. The so-produced scFv has the potential to develop as a structural biology tool to study the biology of the EV-A71 RNA genome.

Neutralizing and Enhancing Epitopes of the SARS-CoV-2 Receptor-Binding Domain (RBD) Identified by Nanobodies.

Engineered nanobodies (VHs) to the SARS-CoV-2 receptor-binding domain (RBD) were generated using phage display technology. A recombinant Wuhan RBD served as bait in phage panning to fish out nanobody-displaying phages from a VH/VHH phage display library. Sixteen phage-infected E. coli clones produced nanobodies with 81.79-98.96% framework similarity to human antibodies; thus, they may be regarded as human nanobodies. Nanobodies of E. coli clones 114 and 278 neutralized SARS-CoV-2 infectivity in a dose-dependent manner; nanobodies of clones 103 and 105 enhanced the virus's infectivity by increasing the cytopathic effect (CPE) in an infected Vero E6 monolayer. These four nanobodies also bound to recombinant Delta and Omicron RBDs and native SARS-CoV-2 spike proteins. The neutralizing VH114 epitope contains the previously reported VYAWN motif (Wuhan RBD residues 350-354). The linear epitope of neutralizing VH278 at Wuhan RBD 319RVQPTESIVRFPNITN334 is novel. In this study, for the first time, we report SARS-CoV-2 RBD-enhancing epitopes, i.e., a linear VH103 epitope at RBD residues 359NCVADVSVLYNSAPFFTFKCYG380, and the VH105 epitope, most likely conformational and formed by residues in three RBD regions that are spatially juxtaposed upon the protein folding. Data obtained in this way are useful for the rational design of subunit SARS-CoV-2 vaccines that should be devoid of enhancing epitopes. VH114 and VH278 should be tested further for clinical use against COVID-19.

Production and Immunological Characterization of scFv Specific to Epitope of Opisthorchis viverrini Rhophilin-Associated Tail Protein 1-like (OvROPN1L).

(1) Background: Opisthorchis viverrini is a significant health problem in the Mekong subregion of Southeast Asia, causing aggressive cholangiocarcinoma. Current diagnostic procedures do not cover early diagnosis and low infection. Hence, an effective diagnostic tool is still required. Immunodiagnosis seems promising, but attempts to generate monoclonal antibodies have not yet been successful. This study aims to develop a single-chain variable antibody fragment (scFv) against Rhophilin-associated tail protein 1-like (ROPN1L), the sperm-specific antigen of adult O. viverrini, which has not been reported elsewhere. (2) Methods: The target epitope for phage screening was L3-Q13 of OvROPN1L, which showed the highest antigenicity to human opisthorchiasis analyzed in a previous study. This peptide was commercially synthesized and used for phage library screening. The isolated phage was produced in a bacterial expression system and tested for specificity in vitro and in silico. (3) Results: One of fourteen phages, named scFv anti-OvROPN1L-CL19, significantly bound to rOvROPN1L compared with non-infected hamster fecal extracts. This phage clone was successfully produced and purified using Ni-NTA chromatography. Indirect ELISA demonstrated that scFv anti-OvROPN1L-CL19 has a high reactivity with O. viverrini-infected hamster fecal extracts (12 wpi, n = 6) in comparison with non-infected hamster fecal extracts (0 wpi, n = 6), while the polyclonal rOvROPN1L antibodies did not show such a difference. Molecular modeling and docking confirmed our in vitro findings. (4) Conclusion: scFv anti-OvROPN1L-CL19 could be used as an effective material for developing O. viverrini-immunodiagnostic procedures in the future.

Targeted Nanoparticles for the Binding of Injured Vascular Endothelium after Percutaneous Coronary Intervention.

Percutaneous coronary intervention (PCI) is a common procedure for the management of coronary artery obstruction. However, it usually causes vascular wall injury leading to restenosis that limits the long-term success of the PCI endeavor. The ultimate objective of this study was to develop the targeting nanoparticles (NPs) that were destined for the injured subendothelium and attract endothelial progenitor cells (EPCs) to the damaged location for endothelium regeneration. Biodegradable poly(lactic-co-glycolic acid) (PLGA) NPs were conjugated with double targeting moieties, which are glycoprotein Ib alpha chain (GPIbα) and human single-chain antibody variable fragment (HuscFv) specific to the cluster of differentiation 34 (CD34). GPIb is a platelet receptor that interacts with the von Willebrand factor (vWF), highly deposited on the damaged subendothelial surface, while CD34 is a surface marker of EPCs. A candidate anti-CD34 HuscFv was successfully constructed using a phage display biopanning technique. The HuscFv could be purified and showed binding affinity to the CD34-positive cells. The GPIb-conjugated NPs (GPIb-NPs) could target vWF and prevent platelet adherence to vWF in vitro. Furthermore, the HuscFv-conjugated NPs (HuscFv-NPs) could capture CD34-positive cells. The bispecific NPs have high potential to locate at the damaged subendothelial surface and capture EPCs for accelerating the vessel repair.

Anaplasma marginale: Molecular discrimination, recombinant expression and characterization of major surface protein 2.

A. marginale's major surface protein 2 (MSP2) is an immunodominant protein that is encoded by a multigene family. Phylogenetic analysis revealed that the msp2 sequence Thailand strain was clustered in third clade, with similarity values between 90.4 and 100%. The haplotype diversity showed 10 haplotypes of the msp2 genes. The entropy analysis of the nucleic and amino sequences revealed 289 and 117 high entropy peaks, respectively. Interestingly, one predicted allele belonging to MHC-II represented the hypervariable region (HVR) of MSP2. A. marginale's recombinant MSP2 (rAmMSP2), which has a molecular weight of 42 kDa, was examined in SDS-PAGE. Antigenicity of rAmMSP2 (42 kDa) and AmMSP2 (36 kDa) showed the conserved epitopes. The distribution of AmMSP2 on infected erythrocytes' membrane and outside was demonstrated by immunofluorescence detection. Therefore, the rMSP2 could be utilized in the establishment of immunodiagnostic tools and vaccine approaches for the monitoring of anaplasmosis.

Targeting Emerging RNA Viruses by Engineered Human Superantibody to Hepatitis C Virus RNA-Dependent RNA Polymerase.

RNA-dependent RNA polymerase (RdRp) is a unique and highly conserved enzyme across all members of the RNA virus superfamilies. Besides, humans do not have a homolog of this protein. Therefore, the RdRp is an attractive target for a broadly effective therapeutic agent against RNA viruses. In this study, a formerly generated cell-penetrating human single-chain antibody variable fragment (superantibody) to a conformational epitope of hepatitis C virus (HCV) RdRp, which inhibited the polymerase activity leading to the HCV replication inhibition and the host innate immunity restoration, was tested against emerging/reemerging RNA viruses. The superantibody could inhibit the replication of the other members of the Flaviviridae (DENV serotypes 1-4, ZIKV, and JEV), Picornaviridae (genus Enterovirus: EV71, CVA16), and Coronaviridae (genus Alphacoronavirus: PEDV, and genus Betacoronavirus: SARS-CoV-2 (Wuhan wild-type and the variants of concern), in a dose-dependent manner, as demonstrated by the reduction of intracellular viral RNAs and numbers of the released infectious particles. Computerized simulation indicated that the superantibody formed contact interfaces with many residues at the back of the thumb domain (thumb II site, T2) of DENV, ZIKV, JEV, EV71, and CVA16 and fingers and thumb domains of the HCV and coronaviruses (PEDV and SARS-CoV-2). The superantibody binding may cause allosteric change in the spatial conformation of the enzyme and disrupt the catalytic activity, leading to replication inhibition. Although the speculated molecular mechanism of the superantibody needs experimental support, existing data indicate that the superantibody has high potential as a non-chemical broadly effective anti-positive sense-RNA virus agent.

Enhancing epitope of PEDV spike protein.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of a highly contagious enteric disease of pigs characterized by diarrhea, vomiting, and severe dehydration. PEDV infects pigs of all ages, but neonatal pigs during the first week of life are highly susceptible; the mortality rates among newborn piglets may reach 80-100%. Thus, PEDV is regarded as one of the most devastating pig viruses that cause huge economic damage to pig industries worldwide. Vaccination of sows and gilts at the pre-fertilization or pre-farrowing stage is a good strategy for the protection of suckling piglets against PEDV through the acquisition of the lactating immunity. However, vaccination of the mother pigs for inducing a high level of virus-neutralizing antibodies is complicated with unstandardized immunization protocol and unreliable outcomes. Besides, the vaccine may also induce enhancing antibodies that promote virus entry and replication, so-called antibody-dependent enhancement (ADE), which aggravates the disease upon new virus exposure. Recognition of the virus epitope that induces the production of the enhancing antibodies is an existential necessity for safe and effective PEDV vaccine design. In this study, the enhancing epitope of the PEDV spike (S) protein was revealed for the first time, by using phage display technology and mouse monoclonal antibody (mAbG3) that bound to the PEDV S1 subunit of the S protein and enhanced PEDV entry into permissive Vero cells that lack Fc receptor. The phages displaying mAbG3-bound peptides derived from the phage library by panning with the mAbG3 matched with several regions in the S1-0 sub-domain of the PEDV S1 subunit, indicating that the epitope is discontinuous (conformational). The mAbG3-bound phage sequence also matched with a linear sequence of the S1-BCD sub-domains. Immunological assays verified the phage mimotope results. Although the molecular mechanism of ADE caused by the mAbG3 via binding to the newly identified S1 enhancing epitope awaits investigation, the data obtained from this study are helpful and useful in designing a safe and effective PEDV protein subunit/DNA vaccine devoid of the enhancing epitope.

Production of human embryonic kidney 293T cells stably expressing C-X-C chemokine receptor type 4 (CXCR4) as a screening tool for anticancer lead compound targeting CXCR4.

AIM: The C-X-C chemokine-receptor type 4 (CXCR4) is an emerging target for cancer drug discovery due to its high expression in cancer cells. The present study aimed to produce CXCR4 overexpressing HEK293T cells for a non-radioactive binding assay as a platform to identify drug candidates targeting CXCR4. MAIN METHODS: HEK293T cells stably expressing human CXCR4 were constructed by transfection of CXCR4 plasmids from the human CXCR4 gene. The CXCR4 overexpressing HEK293T cells were obtained by fluorescence-activated sorting and verified by conducting the competition binding assay of a known CXCR4 inhibitor, AMD3100 (plerixafor), to determine the IC50 value against monoclonal anti-human CD184 (hCD184) antibody tagged with fluorescence probe, phycoerythrin (PE). The non-radioactive binding assay using CXCR4 overexpressing HEK293T cells and PE-anti hCD184 was applied as a platform for identifying the target of natural compounds that exhibited cytotoxicity against cancer cell lines. KEY FINDINGS: The CXCR4 overexpressing HEK293T cells were produced with high expression (99.8%). The IC50 value of plerixafor determined by fluorescence tagged antibody-based competition assay using our developed cells agree with previously reported values using a radioligand binding assay. We observed no significant displacement of bound PE-anti-hCD184 by the test natural compounds which could be due to non-specific binding to other functional targets or organelles, low potency of the natural compounds, or binding to CXCR4 at deeper pockets. SIGNIFICANCE: The verified non-radioactive binding assay can serve as an alternative screening tool for anticancer lead compounds targeting CXCR4 and an essential tool for proof of mechanism study in the drug discovery.

Human Superantibodies to 3CLpro Inhibit Replication of SARS-CoV-2 across Variants.

Broadly effective and safe anti-coronavirus agent is existentially needed. Major protease (3CLpro) is a highly conserved enzyme of betacoronaviruses. The enzyme plays pivotal role in the virus replication cycle. Thus, it is a good target of a broadly effective anti-Betacoronavirus agent. In this study, human single-chain antibodies (HuscFvs) of the SARS-CoV-2 3CLpro were generated using phage display technology. The 3CLpro-bound phages were used to infect Escherichia coli host for the production the 3CLpro-bound HuscFvs. Computerized simulation was used to guide the selection of the phage infected-E. coli clones that produced HuscFvs with the 3CLpro inhibitory potential. HuscFvs of three phage infected-E. coli clones were predicted to form contact interface with residues for 3CLpro catalytic activity, substrate binding, and homodimerization. These HuscFvs were linked to a cell-penetrating peptide to make them cell-penetrable, i.e., became superantibodies. The superantibodies blocked the 3CLpro activity in vitro, were not toxic to human cells, traversed across membrane of 3CLpro-expressing cells to co-localize with the intracellular 3CLpro and most of all, they inhibited replication of authentic SARS-CoV-2 Wuhan wild type and α, ß, δ, and Omicron variants that were tested. The superantibodies should be investigated further towards clinical application as a safe and broadly effective anti-Betacoronavirus agent.

Novel Neutralizing Epitope of PEDV S1 Protein Identified by IgM Monoclonal Antibody.

Porcine epidemic diarrhea virus (PEDV) causes devastating enteric disease that inflicts huge economic damage on the swine industry worldwide. A safe and highly effective PEDV vaccine that contains only the virus-neutralizing epitopes (not enhancing epitope), as well as a ready-to-use PEDV neutralizing antibody for the passive immunization of PEDV vulnerable piglets (during the first week of life) are needed, particularly for PEDV-endemic farms. In this study, we generated monoclonal antibodies (mAbs) to the recombinant S1 domain of PEDV spike (S) protein and tested their PEDV neutralizing activity by CPE-reduction assay. The mAb secreted by one hybrodoma clone (A3), that also bound to the native S1 counterpart from PEDV-infected cells (tested by combined co-immunoprecipitation and Western blotting), neutralized PEDV infectivity. Epitope of the neutralizing mAb (mAbA3) locates in the S1A subdomain of the spike protein, as identified by phage mimotope search and multiple sequence alignment, and peptide binding-ELISA. The newly identified epitope is shared by PEDV G1 and G2 strains and other alphacoronaviruses. In summary, mAbA3 may be useful as a ready-to-use antibody for passive immunization of PEDV-susceptible piglets, while the novel neutralizing epitope, together with other, previously known protective epitopes, have potential as an immunogenic cocktail for a safe, next-generation PEDV vaccine.

Engineered Fully Human Single-Chain Monoclonal Antibodies to PIM2 Kinase.

Proviral integration site of Moloney virus-2 (PIM2) is overexpressed in multiple human cancer cells and high level is related to poor prognosis; thus, PIM2 kinase is a rational target of anti-cancer therapeutics. Several chemical inhibitors targeting PIMs/PIM2 or their downstream signaling molecules have been developed for treatment of different cancers. However, their off-target toxicity is common in clinical trials, so they could not be advanced to official approval for clinical application. Here, we produced human single-chain antibody fragments (HuscFvs) to PIM2 by using phage display library, which was constructed in a way that a portion of phages in the library carried HuscFvs against human own proteins on their surface with the respective antibody genes in the phage genome. Bacterial derived-recombinant PIM2 (rPIM2) was used as an antigenic bait to fish out the rPIM2-bound phages from the library. Three E. coli clones transfected with the HuscFv genes derived from the rPIM2-bound phages expressed HuscFvs that bound also to native PIM2 from cancer cells. The HuscFvs presumptively interact with the PIM2 at the ATP binding pocket and kinase active loop. They were as effective as small chemical drug inhibitor (AZD1208, which is an ATP competitive inhibitor of all PIM isoforms for ex vivo use) in inhibiting PIM kinase activity. The HuscFvs should be engineered into a cell-penetrating format and tested further towards clinical application as a novel and safe pan-anti-cancer therapeutics.

Publisher Correction: Human single chain-transbodies that bound to domain-I of non-structural protein 5A (NS5A) of hepatitis C virus.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Identification and production of mouse scFv to specific epitope of enterovirus-71 virion protein-2 (VP2).

Enterovirus-71 (EV71) and coxsackievirus-A16 (CA16) frequently cause hand-foot-mouth disease (HFMD) epidemics among infants and young children. CA16 infections are usually mild, while EV71 disease may be fatal due to neurologic complications. As such, the ability to rapidly and specifically recognize EV71 is needed to facilitate proper case management and epidemic control. Accordingly, the aim of this study was to generate antibodies to EV71-virion protein-2 (VP2) by phage display technology for further use in specific detection of EV71. A recombinant peptide sequence of EV71-VP2, carrying a predicted conserved B cell epitope fused to glutathione-S-transferase (GST) (designated GST-EV71-VP2/131-160), was produced. The fusion protein was used as bait in in-solution biopanning to separate protein-bound phages from a murine scFv (MuscFv) phage display library constructed from an immunoglobulin gene repertoire from naïve ICR mice. Three phage-transformed E. coli clones (clones 63, 82, and 83) produced MuscFvs that bound to the GST-EV71-VP2/131-160 peptide. The MuscFv of clone 83 (MuscFv83), which produced the highest ELISA signal to the target antigen, was further tested. MuscFv83 also bound to full-length EV71-VP2 and EV71 particles, but did not bind to GST, full-length EV71-VP1, or the antigenically related CA16. MuscFv83 could be a suitable reagent for rapid antigen-based immunoassay, such as immunochromatography (ICT), for the specific detection and/or diagnosis of EV71 infection as well as epidemic surveillance.

Human single chain-transbodies that bound to domain-I of non-structural protein 5A (NS5A) of hepatitis C virus.

A safe and broadly effective direct acting anti-hepatitis C virus (HCV) agent that can withstand the viral mutation is needed. In this study, human single chain antibody variable fragments (HuscFvs) to conserved non-structural protein-5A (NS5A) of HCV were produced by phage display technology. Recombinant NS5A was used as bait for fishing-out the protein bound-phages from the HuscFv-phage display library. NS5A-bound HuscFvs produced by five phage transfected-E. coli clones were linked molecularly to nonaarginine (R9) for making them cell penetrable (become transbodies). The human monoclonal transbodies inhibited HCV replication in the HCVcc infected human hepatic cells and also rescued the cellular antiviral immune response from the viral suppression. Computerized simulation verified by immunoassays indicated that the transbodies used several residues in their multiple complementarity determining regions (CDRs) to form contact interface with many residues of the NS5A domain-I which is important for HCV replication complex formation and RNA binding as well as for interacting with several host proteins for viral immune evasion and regulation of cellular physiology. The human monoclonal transbodies have high potential for testing further as a new ramification of direct acting anti-HCV agent, either alone or in combination with their cognates that target other HCV proteins.

Inhibition of HCV replication by humanized-single domain transbodies to NS4B.

NS4B of hepatitis C virus (HCV) initiates membrane web formation, binds RNA and other HCV proteins for viral replication complex (RC) formation, hydrolyses NTP, and inhibits innate anti-viral immunity. Thus, NS4B is an attractive target of a novel anti-HCV agent. In this study, humanized-nanobodies (VHs/VHHs) that bound to recombinant NS4B were produced by means of phage display technology. The nanobodies were linked molecularly to a cell penetrating peptide, penetratin (PEN), for making them cell penetrable (become transbodies). Human hepatic (Huh7) cells transfected with HCV JFH1-RNA that were treated with transbodies from four Escherichia coli clones (PEN-VHH7, PEN-VHH9, PEN-VH33, and PEN-VH43) had significant reduction of HCV RNA amounts in their culture fluids and intracellularly when compared to the transfected cells treated with control transbody and medium alone. The results were supported by the HCV foci assay. The transbody treated-transfected cells also had upregulation of the studied innate cytokine genes, IRF3, IFNß and IL-28b. The transbodies have high potential for testing further as a novel anti-HCV agent, either alone, adjunct of existing anti-HCV agents/remedies, or in combination with their cognates specific to other HCV enzymes/proteins.