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This study aims to characterize the intermediates, and the final product (FP) obtained during the production of human intramuscular hyperimmune gamma globulin anti-SARS-CoV-2 (hIHGG anti-SARS-CoV-2) and to determine its stability. Material and methods : hIHGG anti-SARS-CoV-2 was fractionated from 270 convalescent plasma donations with the Cohn method. Prior to fractionation, the plasma was inactivated (Theraflex MB Plasma). Samples were defined using enzyme immunoassays (EIA) for anti-S1, anti-RBD S1, and anti-N antibodies, and neutralization assays with SARS-CoV-2 (VN) and pseudoviruses (PVN, decorated with SARS-CoV-2 S protein). Results were expressed as a titer (EIA) or 50% of the neutralization titer (IC 50 ) estimated in a four-parameter nonlinear regression model. Results: Concentration of anti-S1 antibodies in plasma was similar before and after inactivation. Following fractionation, the anti-S1, anti-RBD, and anti-N (total tests) titers in FP were concentrated approximately 15-fold from 1:4 to 1:63 (1800 BAU/mL), 7-fold from 1:111 to 1:802 and from 1:13 to 1:88, respectively. During production, the IgA (anti-S1) antibody titer was reduced to an undetectable level and the IgM (anti-RBD) titer from 1:115 to 1:24. The neutralizing antibodies (nAb) titer increased in both VN (from 1:40 to 1:160) and PVN (IC 50 from 63 to 313). The concentration of specific IgG in the FP did not change significantly for 14 months. Conclusions: The hIHGG anti-SARS-CoV-2 was stable, with concentration up to approximately 15-fold nAb compared to the source plasma pool.

SARS-CoV-2 genome annotation revealed the presence of 10 open reading frames (ORFs), of which the last one (ORF10) is positioned downstream of the N gene. It is a hypothetical gene, which was speculated to encode a 38 aa protein. This hypothetical protein does not share sequence similarity with any other known protein and cannot be associated with a function. While the role of this ORF10 was proposed, there is growing evidence showing that the ORF10 is not a coding region. Here, we identified SARS-CoV-2 variants in which the ORF10 gene was prematurely terminated. The disease was not attenuated, and the transmissibility between humans was maintained. Also, in vitro, the strains replicated similarly to the related viruses with the intact ORF10. Altogether, based on clinical observation and laboratory analyses, it appears that the ORF10 protein is not essential in humans. This observation further proves that the ORF10 should not be treated as the protein-coding gene, and the genome annotations should be amended., Competing Interests: The authors declare no competing interests.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a major epidemic threat since the beginning of 2020. Efforts to combat the virus and the associated coronavirus disease 2019 (COVID-19) disease are being undertaken worldwide. To facilitate the research on the virus itself, a number of surrogate systems have been developed. Here, we report the efficient production of SARS-CoV-2 virus-like particles (VLPs) in insect cells. Contrary to widely used pseudovirus particles, where only one coronaviral protein is displayed within a heterologous scaffold, developed VLPs are structurally similar to the native virus and allow for more throughput studies on the biology of the infection. On the other hand, being devoid of the viral genome, VLPs are unable to replicate and thus safe to work with. Importantly, this is the first report showing that SARS-CoV-2 VLPs can be efficiently produced in insect cells and purified using scalable affinity chromatography. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks. [Display omitted] • Acriflavine (ACF) is a potent inhibitor of SARS-CoV-2 papain-like protease PLpro • A co-crystal structure shows how ACF blocks the active site with a unique binding mode • ACF has low-nanomolar IC 50 in vitro , ex vivo , and in animal models • ACF is a registered drug and a promising candidate for clinical studies Napolitano et al. discovered acriflavine (ACF), a clinically approved drug, as an effective inhibitor of SARS-CoV-2 papain-like protease (PLpro). ACF inhibits viral replication at nanomolar concentrations in vitro and ex vivo , as well as in vivo. These findings open a promising therapeutic approach against COVID-19 and other betacoronaviruses. [ABSTRACT FROM AUTHOR]

Currently, there are four seasonal coronaviruses associated with relatively mild respiratory tract disease in humans. However, there is also a plethora of animal coronaviruses which have the potential to cross the species border. This regularly results in the emergence of new viruses in humans. In 2002, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged and rapidly disappeared in May 2003. In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a possible threat to humans, but its pandemic potential so far is minimal, as human-to-human transmission is ineffective. The end of 2019 brought us information about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emergence, and the virus rapidly spread in 2020, causing an unprecedented pandemic. At present, studies on the virus are carried out using a surrogate system based on the immortalized simian Vero E6 cell line. This model is convenient for diagnostics, but it has serious limitations and does not allow for understanding of the biology and evolution of the virus. Here, we show that fully differentiated human airway epithelium cultures constitute an excellent model to study infection with the novel human coronavirus SARS-CoV-2. We observed efficient replication of the virus in the tissue, with maximal replication at 2 days postinfection. The virus replicated in ciliated cells and was released apically. [ABSTRACT FROM AUTHOR]