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.

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.

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: 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.