A Therapeutically Active Minibody Exhibits an Antiviral Activity in Oseltamivir-Resistant Influenza-Infected Mice via Direct Hydrolysis of Viral RNAs

Abstract

Emerging Oseltamivir-resistant influenza strains pose a critical public health threat due to antigenic shifts and drifts. We report an innovative strategy for controlling influenza A infections by use of a novel minibody of the 3D8 single chain variable fragment (scFv) showing intrinsic viral RNA hydrolyzing activity, cell penetration activity, and epidermal cell penetration ability. In this study, we examined 3D8 scFv’s antiviral activity in vitro on three different H1N1 influenza strains, one Oseltamivir-resistant (A/Korea/2785/2009pdm) strain, and two Oseltamivir-sensitive (A/PuertoRico/8/1934 and A/X-31) strains. Interestingly, the 3D8 scFv directly digested viral RNAs in the ribonucleoprotein complex. scFv’s reduction of influenza viral RNA including viral genomic RNA, complementary RNA, and messenger RNA during influenza A infection cycles indicated that this minibody targets all types of viral RNAs during the early, intermediate, and late stages of the virus’s life cycle. Moreover, we further addressed the antiviral effects of 3D8 scFv to investigate in vivo clinical outcomes of influenza-infected mice. Using both prophylactic and therapeutic treatments of intranasal administered 3D8 scFv, we found that Oseltamivir-resistant H1N1-infected mice showed 90% (prophylactic effects) and 40% (therapeutic effects) increased survival rates, respectively, compared to the control group. The pathological signs of influenza A in the lung tissues, and quantitative analyses of the virus proliferations supported the antiviral activity of the 3D8 single chain variable fragment. Taken together, these results demonstrate that 3D8 scFv has antiviral therapeutic potentials against a wide range of influenza A viruses via the direct viral RNA hydrolyzing activity.

A synergistic therapy against influenza virus A/H1N1/PR8 by a HA1 specific neutralizing single-domain VL and an RNA hydrolyzing scFv

“…depending on the IAV strain, NVLH8 demonstrated the specific neutralization activity (H1N1/pdm, H3N2/Brisbane, H3N2/ Switzerland), which supported that they are specific to H1N1/PR8 (Supplementary Figure S3B). In a previous study, a 3D8 scFv was able to hydrolyze RNA in RNP form, resulting in an antiviral activity against influenza viruses (Lee et al, 2022). Here, we strongly agree that the 3D8 scFv digested RNA in RNP form, particularly to H1N1/PR8 strain.…”

“…Our previous study reported the antiviral activity of 3D8 scFv against influenza H1N1/H275Y virus through hydrolyzing viral RNA in mRNA and vRNPs and cRNPs form (Lee et al, 2022). Here, we strongly agreed that 3D8 scFv targeted not only mRNA (naked RNA) but also vRNA and cRNA in RNP forms (vRNPs or cRNPs) of H1N1/PR8.…”

“…3D8 scFv with the ability to non-specifically hydrolyze both DNA and RNA and enter into the cytoplasm of the cell via caveolae/lipid raft endocytosis demonstrated a broadly antiviral effect against different viruses (Kim et al, 2006;Jun et al, 2010;Lee et al, 2014Lee et al, , 2021Cho et al, 2015;Park et al, 2017;). An earlier report indicated that 3D8 scFv can hydrolyze messenger RNA (mRNA), viral genomic RNA (vRNA), and complementary RNA (cRNA) of influenza A viruses, resulting in a therapeutic effect in vivo (Lee et al, 2022).…”

Targeting the Human Influenza a Virus: The Methods, Limitations, and Pitfalls of Virtual Screening for Drug-like Candidates Including Scaffold Hopping and Compound Profiling

“…Moreover, vaccine production time is a bottleneck, so vaccines are not readily available during the initial spread of a pandemic. As such, alternative treatments such as novel mini-antibodies have been proposed [ 1 ]. Human influenza A vaccines constitute the cornerstone of flu prevention while drugs have been developed for monotherapeutic purposes in the early stages of infection or as a co-medication, such as orally inhaled zanamivir (Relenza™) or orally administered oseltamivir (Tamiflu™).…”