Agonistic Bivalent Human scFvs-Fcγ Fusion Antibodies to OX40 Ectodomain Enhance T Cell Activities against Cancer.

(1) Background: Understanding how advanced cancers evade host innate and adaptive immune opponents has led to cancer immunotherapy. Among several immunotherapeutic strategies, the reversal of immunosuppression mediated by regulatory T cells in the tumor microenvironment (TME) using blockers of immune-checkpoint signaling in effector T cells is the most successful treatment measure. Furthermore, agonists of T cell costimulatory molecules (CD40, 4-1BB, OX40) play an additional anti-cancer role to that of checkpoint blocking in combined therapy and serve also as adjuvant/neoadjuvant/induction therapy to conventional cancer treatments, such as tumor resection and radio- and chemo- therapies. (2) Methods and Results: In this study, novel agonistic antibodies to the OX40/CD134 ectodomain (EcOX40), i.e., fully human bivalent single-chain variable fragments (HuscFvs) linked to IgG Fc (bivalent HuscFv-Fcγ fusion antibodies) were generated by using phage-display technology and genetic engineering. The HuscFvs in the fusion antibodies bound to the cysteine-rich domain-2 of the EcOX40, which is known to be involved in OX40-OX40L signaling for NF-κB activation in T cells. The fusion antibodies caused proliferation, and increased the survival and cytokine production of CD3-CD28-activated human T cells. They showed enhancement trends for other effector T cell activities like granzyme B production and lysis of ovarian cancer cells when added to the activated T cells. (3) Conclusions: The novel OX40 agonistic fusion antibodies should be further tested step-by-step toward their safe use as an adjunctive non-immunogenic cancer immunotherapeutic agent.

Human super antibody to viral RNA-dependent RNA polymerase produced by a modified Sortase self-cleave-bacteria surface display system.

BACKGROUND: RNA-dependent RNA polymerase (RdRp) is a good target of anti-RNA virus agents; not only it is pivotal for the RNA virus replication cycle and highly conserved among RNA viruses across different families, but also lacks human homolog. Recently, human single-chain antibody (HuscFv) that bound to thumb domain of hepatitis C virus (HCV) RNA-dependent RNA polymerase (functionalized NS5B protein) was produced and engineered into cell-penetrating antibody (super antibody) in the form of cell-penetrating peptide (penetratin, PEN)-linked HuscFv (PEN-HuscFv34). The super antibody was produced and purified from inclusion body (IB) of a pen-huscfv34-vector-transformed Escherichia coli. The super antibody inhibited replication of alpha- and beta- coronaviruses, flaviviruses, and picornaviruses that were tested (broadly effective); thus, it has high potential for developing further towards a pan-anti-RNA virus agent. However, production, purification, and refolding of the super antibody molecules from the bacterial IB are laborious and hurdles to large-scale production. Therefore, in this study, Sortase-self-cleave method and bacteria surface display system were combined and modified for the super antibody production. METHODS AND RESULTS: BL21 (DE3) ΔA E. coli, a strain lacking predominant outer membrane protein (OmpA) and ion and OmpT proteases, that displayed a membrane-anchored fusion protein, i.e., chimeric lipoprotein (Lpp')-OmpA', SUMO, Sortase protease, Sortase cleavage site (LPET↓G) and PEN-HuscFv34-6× His was generated. The soluble PEN-HuscFv34-6× His with glycine at the N-terminus could be released from the E. coli surface, simply by incubating the bacterial cells in a Sortase-cleavage buffer. After centrifugation, the G-PEN-HuscFv34-6× His could be purified from the supernatant. The purified G-PEN-HuscFv34-6× retained original cell-penetrating ability (being super antibody) and the broadly effective anti-RNA virus activity of the original IB-derived-PEN-HuscFv34. CONCLUSION: The functionalized super antibody to RNA virus RdRp was successfully produced by using combined Sortase self-cleave and bacterial surface display systems with modification. The display system is suitable for downstream processing in a large-scale production of the super antibody. It is applicable also for production of other recombinant proteins in soluble free-folding form.

Tularemia - a re-emerging disease with growing concern.

Tularemia caused by Gram-negative, coccobacillus bacterium, Francisella tularensis, is a highly infectious zoonotic disease. Human cases have been reported mainly from the United States, Nordic countries like Sweden and Finland, and some European and Asian countries. Naturally, the disease occurs in several vertebrates, particularly lagomorphs. Type A (subspecies tularensis) is more virulent and causes disease mainly in North America; type B (subspecies holarctica) is widespread, while subspecies mediasiatica is present in central Asia. F. tularensis is a possible bioweapon due to its lethality, low infectious dosage, and aerosol transmission. Small mammals like rabbits, hares, and muskrats are primary sources of human infections, but true reservoir of F. tularensis is unknown. Vector-borne tularemia primarily involves ticks and mosquitoes. The bacterial subspecies involved and mode of transmission determine the clinical picture. Early signs are flu-like illnesses that may evolve into different clinical forms of tularemia that may or may not include lymphadenopathy. Ulcero-glandular and glandular forms are acquired by arthropod bite or handling of infected animals, oculo-glandular form as a result of conjunctival infection, and oro-pharyngeal form by intake of contaminated food or water. Pulmonary form appears after inhalation of bacteria. Typhoidal form may occur after infection via different routes. Human-to-human transmission has not been known. Diagnosis can be achieved by serology, bacterial culture, and molecular methods. Treatment for tularemia typically entails use of quinolones, tetracyclines, or aminoglycosides. Preventive measures are necessary to avoid infection although difficult to implement. Research is underway for the development of effective live attenuated and subunit vaccines.

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 [...].

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.

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.

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.

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.

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.

Peptide of Trichinella spiralis Infective Larval Extract That Harnesses Growth of Human Hepatoma Cells.

Trichinella spiralis, a tissue-dwelling helminth, causes human trichinellosis through ingestion of undercooked meat containing the parasite's infective larvae. However, benefits from T. spiralis infection have been documented: reduction of allergic diseases, inhibition of collagen-induced arthritis, delay of type 1 diabetes progression, and suppression of cancer cell proliferation. Since conventional cancer treatments have limited and unreliable efficacies with adverse side effects, novel adjunctive therapeutic agents and strategies are needed to enhance the overall treatment outcomes. This study aimed to validate the antitumor activity of T. spiralis infective larval extract (LE) and extricate the parasite-derived antitumor peptide. Extracts of T. spiralis infective larvae harvested from striated muscles of infected mice were prepared and tested for antitumor activity against three types of carcinoma cells: hepatocellular carcinoma HepG2, ovarian cancer SK-OV-3, and lung adenocarcinoma A549. The results showed that LE exerted the greatest antitumor effect on HepG2 cells. Proteomic analysis of the LE revealed 270 proteins. They were classified as cellular components, proteins involved in metabolic processes, and proteins with diverse biological functions. STRING analysis showed that most LE proteins were interconnected and played pivotal roles in various metabolic processes. In silico analysis of anticancer peptides identified three candidates. Antitumor peptide 2 matched the hypothetical protein T01_4238 of T. spiralis and showed a dose-dependent anti-HepG2 effect, not by causing apoptosis or necrosis but by inducing ROS accumulation, leading to inhibition of cell proliferation. The data indicate the potential application of LE-derived antitumor peptide as a complementary agent for human hepatoma treatment.

Zoonotic diseases of fish and their prevention and control.

Fish and aquatic-derived zoonotic diseases have caused considerable problems in the aquaculture industry and fishery worldwide. In particular, zoonotic diseases can pose widespread threats to humans. With the world's growing population and potential global trade of aquaculture and fish, the risk of environmental contamination and development of fish and aquatic-derived zoonoses in humans are increasing. The important causes of zoonoses include bacteria, parasites, viruses, and fungi. The zoonotic bacterial agents are divided into two main groups: Gram-positive (Mycobacteriaceae, Streptococcaceae, Erysipelothricaceae families) and Gram-negative (Aeromonadaceae, Vibrionaceae, Pseudomondaceae, Enterobacteriaceae, and Hafniaceae families). The premier parasitic agents include cestodes (tapeworm; e.g. Diphyllobothrium spp.), trematodes (fluke; e.g. Opisthorchis spp.), and nematodes (round worm; e.g. Anisakis spp.). In addition, protozoan organisms such as Cryptosporidium spp. are also considered fish-derived zoonotic pathogens. Two groups of fish-associated fungi causing basidiobolomycosis and sporotrichosis also pose a zoonotic risk for humans. The majority of the fish-derived zoonotic diseases are transmitted to humans mainly via the consumption of improperly cooked or raw fish or fish products. Therefore, the incidence of zoonotic diseases can be reduced by properly processing fish and fish products, e.g. by thermal (heat/freezing) treatment. The prevalence of zoonotic agents in fishes varies seasonally and should be regularly monitored to evaluate the prevalence of pathogens in both wild and cultured fish populations. This review focuses on the fish zoonotic agents/diseases and their control and prevention.

Swine coronaviruses (SCoVs) and their emerging threats to swine population, inter-species transmission, exploring the susceptibility of pigs for SARS-CoV-2 and zoonotic concerns.

Swine coronaviruses (SCoVs) are one of the most devastating pathogens affecting the livelihoods of farmers and swine industry across the world. These include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and porcine delta coronavirus (PDCoV). Coronaviruses infect a wide variety of animal species and humans because these are having single stranded-RNA that accounts for high mutation rates and thus could break the species barrier. The gastrointestinal, cardiovascular, and nervous systems are the primary organ systems affected by SCoVs. Infection is very common in piglets compared to adult swine causing high mortality in the former. Bat is implicated to be the origin of all CoVs affecting animals and humans. Since pig is the only domestic animal in which CoVs cause a wide range of diseases; new coronaviruses with high zoonotic potential could likely emerge in the future as observed in the past. The recently emerged severe acute respiratory syndrome coronavirus virus-2 (SARS-CoV-2), causing COVID-19 pandemic in humans, has been implicated to have animal origin, also reported from few animal species, though its zoonotic concerns are still under investigation. This review discusses SCoVs and their epidemiology, virology, evolution, pathology, wildlife reservoirs, interspecies transmission, spill-over events and highlighting their emerging threats to swine population. The role of pigs amid ongoing SARS-CoV-2 pandemic will also be discussed. A thorough investigation should be conducted to rule out zoonotic potential of SCoVs and to design appropriate strategies for their prevention and control.

Antimicrobial Resistance and Virulence of Non-Typhoidal Salmonella from Retail Foods Marketed in Bangkok, Thailand.

Nontyphoidal-Salmonella bacteria cause foodborne gastroenteritis that may lead to fatal bacteremia, osteomyelitis, and meningitis if not treated properly. The emergence of multidrug-resistant Salmonella strains is a global public health threat. Regular monitoring of genotypes and phenotypes of Salmonella isolated from humans, animals, foods, and environments is mandatory for effective reduction and control of this food-borne pathogen. In this study, antimicrobial-resistant and virulent genotypes and phenotypes of Salmonella isolated from retail food samples in Bangkok, Thailand, were investigated. From 252 raw food samples, 58 Salmonella strains that belonged only to serotype Enteritidis were isolated. Disc diffusion method showed that all isolates were still sensitive to amikacin and carbapenems. More than 30% of the isolates were resistant to ampicillin, tetracycline, and ciprofloxacin. Twenty isolates resist at least three antibiotic classes. Minimum inhibitory concentration tests showed that 12.07% of the isolates produced extended-spectrum ß-Lactamase. Polymerase chain reaction indicated that 32.76, 81.03, 39.66, and 5.17% of the isolates carried blaTEM-1, tetA, sul2, and dfrA7, respectively. All isolates were positive for invasion-associated genes. Effective prevention and control of Salmonella (as well as other food-borne pathogens) is possible by increasing public awareness and applying food hygienic practices. Active and well harmonised "One Health" co-operation is required to effectively control food-borne zoonosis.

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.