Objectives A major goal of curative hepatitis B virus (HBV) treatments is the reduction or inactivation of intrahepatic viral covalently closed circular DNA (cccDNA). Hence, precise cccDNA quantification is essential in preclinical and clinical studies. Southern blot (SB) permits cccDNA visualisation but lacks sensitivity and is very laborious. Quantitative PCR (qPCR) has no such limitations but inaccurate quantification due to codetection of viral replicative intermediates (RI) can occur. The use of different samples, preservation conditions, DNA extraction, nuclease digestion methods and qPCR strategies has hindered standardisation. Within the ICE-HBV consortium, available and novel protocols for cccDNA isolation and qPCR quantification in liver tissues and cell cultures were compared in six laboratories to develop evidence-based guidance for best practices. Design Reference material (HBV-infected humanised mouse livers and HepG2-NTCP cells) was exchanged for cross-validation. Each group compared different DNA extraction methods (Hirt extraction, total DNA extraction with or without proteinase K treatment (+PK/−PK)) and nuclease digestion protocols (plasmid-safe ATP-dependent DNase (PSD), T5 exonuclease, exonucleases I/III). Samples were analysed by qPCR and SB. Results Hirt and −PK extraction reduced coexisting RI forms. However, both cccDNA and the protein-free relaxed circular HBV DNA (pf-rcDNA) form were detected by qPCR. T5 and Exo I/III nucleases efficiently removed all RI forms. In contrast, PSD did not digest pf-rcDNA, but was less prone to induce cccDNA overdigestion. In stabilised tissues (eg, Allprotect), nucleases had detrimental effects on cccDNA. Conclusions We present here a comprehensive evidence-based guidance for optimising, controlling and validating cccDNA measurements using available qPCR assays.

Hepatitis B virus (HBV) is a major causative agent of human hepatitis. Its viral genome comprises partially double-stranded DNA, which is complexed with viral polymerase within an icosahedral capsid consisting of a dimeric core protein. Here, we describe the effects of capsid assembly modulators (CAMs) on the geometric or kinetic disruption of capsid construction and the virus life cycle. We highlight classical, early-generation CAMs such as heteroaryldihydropyrimidines, phenylpropenamides or sulfamoylbenzamides, and focus on the chemical structure and antiviral efficacy of recently identified non-classical CAMs, which consist of carboxamides, aryl ureas, bithiazoles, hydrazones, benzylpyridazinones, pyrimidines, quinolines, dyes, and antimicrobial compounds. We summarize the therapeutic efficacy of four representative classical compounds with data from clinical phase 1 studies in chronic HBV patients. Most of these compounds are in phase 2 trials, either as monotherapy or in combination with approved nucleos(t)ides drugs or other immunostimulatory molecules. As followers of the early CAMs, the therapeutic efficacy of several non-classical CAMs has been evaluated in humanized mouse models of HBV infection. It is expected that these next-generation HBV CAMs will be promising candidates for a series of extended human clinical trials.

Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in hospitalized and non-hospitalized patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 SIMPLE clinical studies evaluating RDV in hospitalized participants with severe or moderate COVID-19 disease. The severe and moderate studies enrolled participants with radiologic evidence of pneumonia and a room-air oxygen saturation of ≤94% or >94%, respectively. Virology sample collection was optional in the study protocols. Sequencing and related viral load data were obtained retrospectively from participants at a subset of study sites with local sequencing capabilities (10 of 183 sites) at timepoints with detectable viral load. Among participants with both baseline and post-baseline sequencing data treated with RDV, emergent Nsp12 substitutions were observed in 4 of 19 (21%) participants in the severe study and none of the 2 participants in the moderate study. The following 5 substitutions emerged: T76I, A526V, A554V, E665K, and C697F. The substitutions T76I, A526V, A554V, and C697F had an EC₅₀ fold change of ≤1.5 relative to the wildtype reference using a SARS-CoV-2 subgenomic replicon system, indicating no significant change in the susceptibility to RDV. The phenotyping of E665K could not be determined due to a lack of replication. These data reveal no evidence of relevant resistance emergence and further confirm the established efficacy profile of RDV with a high resistance barrier in COVID-19 patients.

Influenza A virus continuously infects humans and the antigenic shifts of this respiratory virus enable it to cross the species barrier, threatening public health with the risk of pandemics. Broadly neutralizing antibodies (bnAbs) that target the antigenic surface glycoprotein, hemagglutinin (HA), of influenza A virus protect against various subtypes of the virus. Here, we screened a human scFv library, through phage display and panning against recombinant HA proteins, to discover human monoclonal antibodies (mAbs) that are broadly active. Consequently, two human mAbs, named G1 and G2, were identified, which target the HA proteins of the H1N1 and H3N2 subtypes, respectively. G1 was shown to have broad binding ability to different HA subtypes of group 1. By contrast, G2 had higher binding affinity but sensed exclusively H3 subtype-derived HAs. In a cell culture-based virus-neutralizing assay, both G1 and G2 efficiently suppressed infection of the parental influenza A viruses of H1N1 and H3N2 subtypes. Mode-of-action studies showed that the G1 antibody blocked HA2-mediated membrane fusion. Meanwhile, G2 inhibited HA1-mediated viral attachment to host cells. It is noteworthy that both antibodies elicited antibody-dependent cellular cytotoxicity (ADCC) activities by recruiting FcγRIIIA-expressing effector cells. In mouse challenge models, single-shot, intraperitoneal administration of chimeric G1 and G2 antibodies with the mouse IgG constant region completely protected mice from viral infections at doses above 10 and 1 mg/kg, respectively. The newly identified bnAbs, G1 and G2, could provide insight into the development of broad-spectrum antivirals against future pandemic influenza A virus involving group 1- or H3-subtyped strains.