Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals

Abstract

Neutralizing monoclonal antibodies are being found to be increasingly useful in viral infections. In hepatitis B infection, antibodies are proven to be useful for passive prophylaxis. The preS1 region (21–47a.a.) of HBV contains the viral hepatocyte-binding domain crucial for its attachment and infection of hepatocytes. Antibodies against this region are neutralizing and are best suited for immune-based neutralization of HBV, especially in view of their not recognizing decoy particles. Anti-preS1 (21–47a.a.) antibodies are present in serum of spontaneously recovered individuals. We generated a phage-displayed scFv library using circulating lymphocytes from these individuals and selected four preS1-peptide specific scFvs with markedly distinct sequences from this library. All the antibodies recognized the blood-derived and recombinant preS1 containing antigens. Each scFv showed a discrete binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker for neutralizing capacity. These antibodies inhibited preS1-hepatocyte interaction individually and even better in combination. Such a combination of potentially neutralizing recombinant antibodies with defined specificities could be used for preventing/managing HBV infections, including those by possible escape mutants.

Cancer cells govern miR‐2909 exosomal recruitment through its 3′‐end post‐transcriptional modification

“…The small noncoding RNAs (sncRNAs) were separated from the cancer cells and their secreted exosomes by employing NucleoSpin ® miRNA (MACHEREY NAGEL) Isolation Kit as per the manufacturer's instructions. These sncRNAs were further processed for cDNA synthesis employing miScript Reverse Transcriptase Kit as per supplier's protocol . Subsequently, the intrinsic expression of miR‐2909 within the cancer cells as well as the amount of miR‐2909 recruited to their secreted exosomes was quantified by using quantitative reverse transcription polymerase chain reaction (RT‐PCR) with gene‐specific primers.…”

“…These sncRNAs were further processed for cDNA synthesis employing miScript Reverse Transcriptase Kit as per supplier's protocol. 17 Subsequently, the intrinsic expression of miR-2909 within the cancer cells as well as the amount of miR-2909 recruited to their secreted exosomes was quantified by using quantitative reverse transcription polymerase chain reaction (RT-PCR) with gene-specific primers. The primer sequence is as follows (shown 5′ to 3′): miRNA-2909 (Forward Primer), GTTAGGGCCAACATCTCTT.…”

Tumorigenic PVT‐1 gene locus is governed by miR‐2909 RNomics

“…The isolated RNA from each culture well was subjected to expression analysis of genes coding for miR‐2909, KLF4, c‐Myc, AATF, STAT3, PVT‐1, Deptor, mTORC1, and PVT‐1 encoded microRNAs using gene specific primers and Real‐Time PCR method as described earlier . U6 and GAPDH were used as invariant controls for the expression of microRNAs and other genes, respectively . The isolated proteins from each culture well were subjected to SDS‐PAGE followed by Western immuno‐detection using specific antibodies against KLF4, c‐Myc, STAT3, p53, and β‐actin (used as an invariant control) and employing standard methodology described earlier …”

“…[6][7][8] U6 and GAPDH were used as invariant controls for the expression of microRNAs and other genes, respectively. 11 The isolated proteins from each culture well were subjected to SDS-PAGE followed by Western immuno-detection using specific antibodies against KLF4, c-Myc, STAT3, p53, and β-actin (used as an invariant control) and employing standard methodology described earlier. [6][7][8]…”

Disease Pathways and Mechanisms of Potential Drug Targets

“…The NTCP receptor confers the species specificity to HBV . Understanding this process has led to the development of several classes of specific and nonspecific inhibitors of viral entry (see Table ) . Entry inhibitors may be useful by preventing de novo infection of uninfected hepatocytes.…”