Development of a Rapid Reverse Transcription-Recombinase Polymerase Amplification Couple Nucleic Acid Lateral Flow Method for Detecting Porcine Epidemic Diarrhoea Virus.

Porcine epidemic diarrhea virus (PEDV) infection is an important acute diarrheal disease of swine that results in economic and industrial losses worldwide. The clinical manifestations in infected piglets are severe diarrhea, dehydration with milk curd indigestion, leading to death. The diagnosis of PEDV is essential for monitoring and managing the disease. PEDV can be detected and identified by serology and the nucleic acid of the virus in clinical samples. Therefore, a novel isothermal amplification and detection technique, reverse transcription-recombinase polymerase amplification couple nucleic acid lateral flow (RT-RPA-NALF) was developed for the rapid detection of PEDV. Qualitative reverse transcription-polymerase chain reaction (RT-qPCR) was established as the gold standard assay to compare results. Specific primer pairs and probes were designed, and RT-RPA conditions were optimized to amplify the M gene of PEDV. The established RT-RPA-NALF assay could finish in 25 min at a temperature of 42 °C and the amplicon interpreted by visual detection. The developed RT-RPA-NALF assay was specific to the M gene of PEDV, did not detect other common swine diarrhea pathogens, and showed minimal detection at 102 TCID50/mL PEDV. The RT-RPA-NALF assay can detect PEDV in 5 simulated fecal samples. Furthermore, in 60 clinical fecal samples, the results of RT-RPA-NALF correlated with RT-qPCR assay, which provides sensitivity of 95.65% and specificity of 100%, with a coincident rate of 98.33%. The rapid RT-RPA-NALF is simple and rapid, increases high sensitivity, and can be used in the field.

Human Transbodies to Reverse Transcriptase Connection Subdomain of HIV-1 Gag-Pol Polyprotein Reduce Infectiousness of the Virus Progeny.

HIV-1 progeny are released from infected cells as immature particles that are unable to infect new cells. Gag-Pol polyprotein dimerization via the reverse transcriptase connection domain (RTCDs) is pivotal for proper activation of the virus protease (PR protein) in an early event of the progeny virus maturation process. Thus, the RTCD is a potential therapeutic target for a broadly effective anti-HIV agent through impediment of virus maturation. In this study, human single-chain antibodies (HuscFvs) that bound to HIV-1 RTCD were generated using phage display technology. Computerized simulation guided the selection of the transformed Escherichia coli-derived HuscFvs that bound to the RTCD dimer interface. The selected HuscFvs were linked molecularly to human-derived-cell-penetrating peptide (CPP) to make them cell-penetrable (i.e., become transbodies). The CPP-HuscFvs/transbodies produced by a selected transformed E. coli clone were tested for anti-HIV-1 activity. CPP-HuscFvs of transformed E. coli clone 11 (CPP-HuscFv11) that presumptively bound at the RTCD dimer interface effectively reduced reverse transcriptase activity in the newly released virus progeny. Infectiousness of the progeny viruses obtained from CPP-HuscFv11-treated cells were reduced by a similar magnitude to those obtained from protease/reverse transcriptase inhibitor-treated cells, indicating anti-HIV-1 activity of the transbodies. The CPP-HuscFv11/transbodies to HIV-1 RTCD could be an alternative, anti-retroviral agent for long-term HIV-1 treatment.

Engineered Human Monoclonal scFv to Receptor Binding Domain of Ebolavirus.

(1) Background: Ebolavirus (EBOV) poses as a significant threat for human health by frequently causing epidemics of the highly contagious Ebola virus disease (EVD). EBOV glycoprotein (GP), as a sole surface glycoprotein, needs to be cleaved in endosomes to fully expose a receptor-binding domain (RBD) containing a receptor-binding site (RBS) for receptor binding and genome entry into cytoplasm for replication. RBDs are highly conserved among EBOV species, so they are an attractive target for broadly effective anti-EBOV drug development. (2) Methods: Phage display technology was used as a tool to isolate human single-chain antibodies (HuscFv) that bind to recombinant RBDs from a human scFv (HuscFv) phage display library. The RBD-bound HuscFvs were fused with cell-penetrating peptide (CPP), and cell-penetrating antibodies (transbodies) were made, produced from the phage-infected E. coli clones and characterized. (3) Results: Among the HuscFvs obtained from phage-infected E. coli clones, HuscFvs of three clones, HuscFv4, HuscFv11, and HuscFv14, the non-cell-penetrable or cell-penetrable HuscFv4 effectively neutralized cellular entry of EBOV-like particles (VLPs). While all HuscFvs were found to bind cleaved GP (GPcl), their presumptive binding sites were markedly different, as determined by molecular docking. (4) Conclusions: The HuscFv4 could be a promising therapeutic agent against EBOV infection.

Human Antibodies to VP4 Inhibit Replication of Enteroviruses Across Subgenotypes and Serotypes, and Enhance Host Innate Immunity.

Hand, foot, and mouth disease (HFMD) is a highly contagious disease that usually affects infants and young children (<5 years). HFMD outbreaks occur frequently in the Asia-Pacific region, and these outbreaks are associated with enormous healthcare and socioeconomic burden. There is currently no specific antiviral agent to treat HFMD and/or the severe complications that are frequently associated with the enterovirus of serotype EV71. Therefore, the development of a broadly effective and safe anti-enterovirus agent is an existential necessity. In this study, human single-chain antibodies (HuscFvs) specific to the EV71-internal capsid protein (VP4) were generated using phage display technology. VP4 specific-HuscFvs were linked to cell penetrating peptides to make them cell penetrable HuscFvs (transbodies), and readily accessible to the intracellular target. The transbodies, as well as the original HuscFvs that were tested, entered the enterovirus-infected cells, bound to intracellular VP4, and inhibited replication of EV71 across subgenotypes A, B, and C, and coxsackieviruses CVA16 and CVA6. The antibodies also enhanced the antiviral response of the virus-infected cells. Computerized simulation, indirect and competitive ELISAs, and experiments on cells infected with EV71 particles to which the VP4 and VP1-N-terminus were surface-exposed (i.e., A-particles that don't require receptor binding for infection) indicated that the VP4 specific-antibodies inhibit virus replication by interfering with the VP4-N-terminus, which is important for membrane pore formation and virus genome release leading to less production of virus proteins, less infectious virions, and restoration of host innate immunity. The antibodies may inhibit polyprotein/intermediate protein processing and cause sterically strained configurations of the capsid pentamers, which impairs virus morphogenesis. These antibodies should be further investigated for application as a safe and broadly effective HFMD therapy.

Cell-penetrable nanobodies (transbodies) that inhibit the tyrosine kinase activity of EGFR leading to the impediment of human lung adenocarcinoma cell motility and survival.

Most patients suffering from non-small cell lung cancer (NSCLC) have epidermal growth factor receptor (EGFR) overexpression. Currently, EGFR tyrosine kinase inhibitors (TKIs) that act as the ATP-analogs and monoclonal antibodies (MAbs) to EGFR-ectodomain that block intracellular signaling are used for the treatment of advanced NSCLC. Unfortunately, adverse effects due to the TKI off-target and drug resistance occur in a significant number of the treated patients while some NSCLC genotypes do not respond to the therapeutic MAbs. Thus, a more effective remedy for the treatment of EGFR-overexpressed cancers is deemed necessary. In this study, VH/VH H displayed-phage clones that are bound to recombinant EGFR-TK were fished-out from a humanized-camel VH/VH H phage display library. VH/VH H of three phage-infected Escherichia coli clones (VH18, VH H35, and VH36) were linked molecularly to nonaarginine (R9) for making them cell penetrable. R9-VH18, R9-VH H35, and R9-VH36 were cytotoxic to human adenocarcinomic alveolar basal epithelial cells (A549) at the fifty percent inhibitory concentration (IC50 ) 0.181 ± 0.132, 0.00961 ± 0.00516, and 0.00996 ± 0.00752 µM, respectively, which were approximately 1000-fold more effective than small molecular TKIs. R9-VH18 and R9-VH36 also delayed cancer cell migration in a scratch-wound assay. Computerized homology modeling and intermolecular docking revealed that VH18 and VH H35 used CDR3 to interact with EGFR-TK residues close to the catalytic site, which might sterically hinder the ATP-binding of the TK; VH36 used CDR2 to bind at the asymmetric dimerization surface, which might disrupt EGFR dimerization leading to inhibition of intracellular signaling. The humanized-cell penetrable nanobodies have a high potential for developing further towards a clinical application.

Human Monoclonal scFvs that Neutralize Fribrinogenolytic Activity of Kaouthiagin, a Zinc-Metalloproteinase in Cobra (Naja kaouthia) Venom.

Snake venom-metalloproteinases (SVMPs) are the primary factors that disturb hemostasis and cause hemorrhage in the venomous snake bitten subjects. Kaouthiagin is a unique SVMP that binds and cleaves von Willebrand factor (vWF) at a specific peptide bond leading to inhibition of platelet aggregation, which enhances the hemorrhage. Kaouthiagin is a low abundant venom component of Thai cobra (Naja kaouthia); thus, most horse-derived antivenins used for cobra bite treatment do not contain adequate anti-kaouthiagin. This study aimed to produce human single-chain antibody variable fragments (HuscFvs) that bind to and interfere with kaouthiagin activity for further clinical use. Kaouthiagin was purified from N. kaouthia-holovenom by a single-step gel-filtration chromatography. The purified venom component was used in phage-biopanning to select the kaouthiagin-bound HuscFv-displayed-phage clones from a HuscFv-phage display library. The selected phages were used to infect Escherichia coli bacteria. Soluble HuscFvs expressed by three phage-transformed-E. coli clones interfered with cobra kaouthiagin binding to human vWF. Computerized simulation indicated that HuscFv of two phage-transformed E. coli clones formed contact interface with kaouthiagin residues at or near catalytic site and effectively inhibited fibrinogenolytic activity of the kaouthiagin. The HuscFvs have therapeutic potential as an adjunct of antivenins in treatment of bleeding caused by venomous snakebites.

Anemia in patients with coinherited thalassemia and glucose-6-phosphate dehydrogenase deficiency.

Thalassemia and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency are genetic disorders that cause hemolytic anemia. In areas with high frequencies of both hematological disorders, coinheritance of G-6-PD deficiency with thalassemia can be found. Whether G-6-PD deficiency, coinherited with thalassemia, enhances severe anemia is still unclear. Hematological parameters between thalassemia carriers with G-6-PD deficiency and those without G-6-PD deficiency were compared. The G-6-PD deficiency was diagnosed in 410 blood samples from thalassemia patients using a fluorescent spot test. The levels of hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV) and Hb A2/Hb E [ß26(B8)Glu→Lys; HBB: c.79G>A] were measured using an automated blood counter and high performance liquid chromatography (HPLC), respectively. The G-6-PD deficiency was found in 37 samples (9.02%). Mean levels of Hb, PCV, MCV and Hb A2/E were similar between the two groups. Thus, G-6-PD deficiency did not enhance red blood cell pathology or induce more anemic severity in thalassemia patients.