Kittirat Glab-Ampai

Humans get SARS-CoV-2 infection mainly through inhalation; thus, vaccine that induces protective immunity at the virus entry site is important for early control of the infection. In this study, two anionic liposome (L)-adjuvanted VLP vaccines against SARS-CoV-2 were formulated. Baculovirus-Sf21 insect cell system was used for production of VLPs made of full-length S, M and E proteins. S protein of one vaccine (L-SME-VLPs) contained furin cleavage site at the S1/S2 junction, while that of another vaccine (L-S′ME-VLPs) did not. Both vaccines were innocuous and immunogenic when administered IP and IN to mice. Mice immunized IP with L-SME-VLPs/L-S′ME-VLPs (three doses, two-weeks intervals) had serum virus neutralizing (VN) antibodies (in falling order of isotype frequency): IgG3, IgA and IgG2a/IgG3, IgA and IgM, respectively. The L-S′ME VLPs vaccine induced significantly higher serum VN antibody titers than the L-SME-VLPs vaccine. All mice immunized IN with both vaccines had significant rise of VN antibodies in their bronchoalveolar lavage fluids (BALF). The VN antibodies in 67% of immunized mice were Th1- isotypes (IgG2a and/or IgG2b); the immunized mice had also other antibody isotypes in BALF. The intranasal L-S′ME-VLPs should be tested further step-by-step towards the clinical use as effective and safe vaccine against SARS-CoV-2.

Background: Reduced or absent H antigens on red cells with the (para‐)Bombay phenotype can arise from FUT1 gene mutations, impacting the structure and function of 1,2‐L‐fucosyltransferase 1 (1,2‐L‐FucT1). Here, we identified the novel mutations in one patient displaying the para‐Bombay phenotype and examined the potential molecular mechanisms underlying this phenotype.Materials and Methods: ABH antigens and antibodies were detected in patient's blood and saliva using serological methods. The genotypes of ABO, FUT1, and FUT2 were imputed using the genetic variations discovered in the whole exome sequencing data. Three‐dimensional (3D) models of FUT1 variants were built using Deepmind's AlphaFold2 and HDOCK, and the possible effects of the variants were predicted to evaluate using DynaMut2 and Polyphen‐2.Results: Serological analysis confirmed the para‐Bombay B phenotype producing anti‐HI and exhibiting normal genotypes ABO*B.01/O.01.02 and FUT2*01.09/01.09. Remarkably, the phenotype is caused by a compound heterozygous genotype: one allele containing the novel c.–35A>T mutation and the known c.725T>G mutation (p.Leu242Arg) of FUT1, and the other allele containing the c.–35A>T mutation. From the computerized stimulation analysis, p.Arg242 of the FUT1 variant may be detrimental, destabilizing, and probably damaging to 1,2‐L‐FucT1, although not altering the 3D structure of the entire enzyme. The c.–35A>T promoter DNA left at the binding interface of both ZID and c‐Rel transcription factors may enable regulation of 1,2‐L‐FucT1 function at gene promoters.Conclusion: We identified the two novel variants, c.–35A>T and c.[−35A>T, 725T>G], in the FUT1 causing the para‐Bombay phenotype. This finding may clarify the molecular mechanisms and enhance blood transfusion safety.

(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.

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.

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/V_HH 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.

(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.

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.

Broadly effective and safe anti-coronavirus agent is existentially needed. Major protease (3CL^pro) 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 3CL^pro were generated using phage display technology. The 3CL^pro-bound phages were used to infect Escherichia coli host for the production the 3CL^pro-bound HuscFvs. Computerized simulation was used to guide the selection of the phage infected-E. coli clones that produced HuscFvs with the 3CL^pro inhibitory potential. HuscFvs of three phage infected-E. coli clones were predicted to form contact interface with residues for 3CL^pro 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 3CL^pro activity in vitro, were not toxic to human cells, traversed across membrane of 3CL^pro-expressing cells to co-localize with the intracellular 3CL^pro and most of all, they inhibited replication of authentic SARS-CoV-2 Wuhan wild type and α, β, 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.