Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools.

In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua, and not the other standard nucleosides, inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphates (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTPs or NDPs modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes.

COVID-19 vaccine candidates based on modified vaccinia virus Ankara expressing the SARS-CoV-2 spike induce robust T- and B-cell immune responses and full efficacy in mice.

Vaccines against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are urgently needed. We developed two COVID-19 vaccines based on modified vaccinia virus Ankara (MVA) vectors expressing the entire SARS-CoV-2 spike (S) protein (MVA-CoV2-S); their immunogenicity was evaluated in mice using DNA/MVA or MVA/MVA prime/boost immunizations. Both vaccines induced robust, broad and polyfunctional S-specific CD4+ (mainly Th1) and CD8+ T-cell responses, with a T effector memory phenotype. DNA/MVA immunizations elicited higher T-cell responses. All vaccine regimens triggered high titers of IgG antibodies specific for the S, as well as for the receptor-binding domain; the predominance of the IgG2c isotype was indicative of Th1 immunity. Notably, serum samples from vaccinated mice neutralized SARS-CoV-2 in cell cultures, and those from MVA/MVA immunizations showed a higher neutralizing capacity. Remarkably, one or two doses of MVA-CoV2-S protect humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2. In addition, two doses of MVA-CoV2-S confer full inhibition of virus replication in the lungs. These results demonstrate the robust immunogenicity and full efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic.IMPORTANCE The continuous dissemination of the novel emerging SARS-CoV-2 virus, with more than 78 million infected cases worldwide and higher than 1,700,000 deaths as of December 23, 2020, highlights the urgent need for the development of novel vaccines against COVID-19. With this aim, we have developed novel vaccine candidates based on the poxvirus modified vaccinia virus Ankara (MVA) strain expressing the full-length SARS-CoV-2 spike (S) protein, and we have evaluated their immunogenicity in mice using DNA/MVA or MVA/MVA prime/boost immunization protocols. The results showed the induction of a potent S-specific T-cell response and high titers of neutralizing antibodies. Remarkably, humanized K18-hACE2 mice immunized with one or two doses of the MVA-based vaccine were 100% protected from SARS-CoV-2 lethality. Moreover, two doses of the vaccine prevented virus replication in lungs. Our findings prove the robust immunogenicity and efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic.

Effect of Clinically Used Microtubule Targeting Drugs on Viral Infection and Transport Function.

Microtubule targeting agents (MTAs) have been exploited mainly as anti-cancer drugs because of their impact on cellular division and angiogenesis. Additionally, microtubules (MTs) are key structures for intracellular transport, which is frequently hijacked during viral infection. We have analyzed the antiviral activity of clinically used MTAs in the infection of DNA and RNA viruses, including SARS-CoV-2, to find that MT destabilizer agents show a higher impact than stabilizers in the viral infections tested, and FDA-approved anti-helminthic benzimidazoles were among the most active compounds. In order to understand the reasons for the observed antiviral activity, we studied the impact of these compounds in motor proteins-mediated intracellular transport. To do so, we used labeled peptide tools, finding that clinically available MTAs impaired the movement linked to MT motors in living cells. However, their effect on viral infection lacked a clear correlation to their effect in motor-mediated transport, denoting the complex use of the cytoskeleton by viruses. Finally, we further delved into the molecular mechanism of action of Mebendazole by combining biochemical and structural studies to obtain crystallographic high-resolution information of the Mebendazole-tubulin complex, which provided insights into the mechanisms of differential toxicity between helminths and mammalians.

Host phosphatidic acid phosphatase lipin1 is rate limiting for functional hepatitis C virus replicase complex formation.

Hepatitis C virus (HCV) infection constitutes a significant health burden worldwide, because it is a major etiologic agent of chronic liver disease, cirrhosis and hepatocellular carcinoma. HCV replication cycle is closely tied to lipid metabolism and infection by this virus causes profound changes in host lipid homeostasis. We focused our attention on a phosphatidate phosphate (PAP) enzyme family (the lipin family), which mediate the conversion of phosphatidate to diacylglycerol in the cytoplasm, playing a key role in triglyceride biosynthesis and in phospholipid homeostasis. Lipins may also translocate to the nucleus to act as transcriptional regulators of genes involved in lipid metabolism. The best-characterized member of this family is lipin1, which cooperates with lipin2 to maintain glycerophospholipid homeostasis in the liver. Lipin1-deficient cell lines were generated by RNAi to study the role of this protein in different steps of HCV replication cycle. Using surrogate models that recapitulate different aspects of HCV infection, we concluded that lipin1 is rate limiting for the generation of functional replicase complexes, in a step downstream primary translation that leads to early HCV RNA replication. Infection studies in lipin1-deficient cells overexpressing wild type or phosphatase-defective lipin1 proteins suggest that lipin1 phosphatase activity is required to support HCV infection. Finally, ultrastructural and biochemical analyses in replication-independent models suggest that lipin1 may facilitate the generation of the membranous compartment that contains functional HCV replicase complexes.

Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment.

Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment.IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments.

Long noncoding RNA EGOT negatively affects the antiviral response and favors HCV replication.

The role of long noncoding RNAs (lncRNAs) in viral infection is poorly studied. We have identified hepatitis C virus (HCV)-Stimulated lncRNAs (CSRs) by transcriptome analysis. Interestingly, two of these CSRs (PVT1 and UCA1) play relevant roles in tumorigenesis, providing a novel link between HCV infection and development of liver tumors. Expression of some CSRs seems induced directly by HCV, while others are upregulated by the antiviral response against the virus. In fact, activation of pathogen sensors induces the expression of CSR32/EGOT RIG-I and the RNA-activated kinase PKR sense HCV RNA, activate NF-κB and upregulate EGOT EGOT is increased in the liver of patients infected with HCV and after infection with influenza or Semliki Forest virus (SFV). Genome-wide guilt-by-association studies predict that EGOT may function as a negative regulator of the antiviral pathway. Accordingly, EGOT depletion increases the expression of several interferon-stimulated genes and leads to decreased replication of HCV and SFV Our results suggest that EGOT is a lncRNA induced after infection that increases viral replication by antagonizing the antiviral response.

Polyanionic carbosilane dendrimers prevent hepatitis C virus infection in cell culture.

Hepatitis C virus (HCV) infection is a major biomedical problem worldwide. Although new direct antiviral agents (DAAs) have been developed for the treatment of chronic HCV infection, the potential emergence of resistant virus variants and the difficulties to implement their administration worldwide make the development of novel antiviral agents an urgent need. Moreover, no effective vaccine is available against HCV and transmission of the virus still occurs particularly when prophylactic measures are not taken. We used a cell-based system to screen a battery of polyanionic carbosilane dendrimers (PCDs) to identify compounds with antiviral activity against HCV and show that they inhibit effective virus adsorption of major HCV genotypes. Interestingly, one of the PCDs irreversibly destabilized infectious virions. This compound displays additive effect in combination with a clinically relevant DAA, sofosbuvir. Our results support further characterization of these molecules as nanotools for the control of hepatitis C virus spread.

Clathrin mediates infectious hepatitis C virus particle egress.

UNLABELLED: Although it is well established that hepatitis C virus (HCV) entry into hepatocytes depends on clathrin-mediated endocytosis, the possible roles of clathrin in other steps of the viral cycle remain unexplored. Thus, we studied whether cell culture-derived HCV (HCVcc) exocytosis was altered after clathrin interference. Knockdown of clathrin or the clathrin adaptor AP-1 in HCVcc-infected human hepatoma cell cultures impaired viral secretion without altering intracellular HCVcc levels or apolipoprotein B (apoB) and apoE exocytosis. Similar reductions in HCVcc secretion were observed after treatment with specific clathrin and dynamin inhibitors. Furthermore, detergent-free immunoprecipitation assays, neutralization experiments, and immunofluorescence analyses suggested that whereas apoE associated with infectious intracellular HCV precursors in endoplasmic reticulum (ER)-related structures, AP-1 participated in HCVcc egress in a post-ER compartment. Finally, we observed that clathrin and AP-1 knockdown altered the endosomal distribution of HCV core, reducing and increasing its colocalization with early endosome and lysosome markers, respectively. Our data support a model in which nascent HCV particles associate with apoE in the ER and exit cells following a clathrin-dependent transendosomal secretory route. IMPORTANCE: HCV entry into hepatocytes depends on clathrin-mediated endocytosis. Here we demonstrate for the first time that clathrin also participates in HCV exit from infected cells. Our data uncover important features of HCV egress, which may lead to the development of new therapeutic interventions. Interestingly, we show that secretion of the very-low-density lipoprotein (VLDL) components apoB and apoE is not impaired after clathrin interference. This is a significant finding, since, to date, it has been proposed that HCV and VLDL follow similar exocytic routes. Given that lipid metabolism recently emerged as a potential target for therapies against HCV infection, our data may help in the design of new strategies to interfere specifically with HCV exocytosis without perturbing cellular lipid homeostasis, with the aim of achieving more efficient, selective, and safe antivirals.

Dysregulation of interferon regulatory factors impairs the expression of immunostimulatory molecules in hepatitis C virus genotype 1-infected hepatocytes.

BACKGROUND: IL-7 and IL-15 are produced by hepatocytes and are critical for the expansion and function of CD8 T cells. IL-15 needs to be presented by IL-15Rα for efficient stimulation of CD8 T cells. METHODS: We analysed the hepatic levels of IL-7, IL-15, IL-15Rα and interferon regulatory factors (IRF) in patients with chronic hepatitis C (CHC) (78% genotype 1) and the role of IRF1 and IRF2 on IL-7 and IL-15Rα expression in Huh7 cells with or without hepatitis C virus (HCV) replicon. RESULTS: Hepatic expression of both IL-7 and IL-15Rα, but not of IL-15, was reduced in CHC. These patients exhibited decreased hepatic IRF2 messenger RNA levels and diminished IRF2 staining in hepatocyte nuclei. We found that IRF2 controls basal expression of both IL-7 and IL-15Rα in Huh7 cells. IRF2, but not IRF1, is downregulated in cells with HCV genotype 1b replicon and this was accompanied by decreased expression of IL-7 and IL-15Rα, a defect reversed by overexpressing IRF2. Treating Huh7 cells with IFNα plus oncostatin M increased IL-7 and IL-15Rα mRNA more intensely than either cytokine alone. This effect was mediated by strong upregulation of IRF1 triggered by the combined treatment. Induction of IRF1, IL-7 and IL-15Rα by IFNα plus oncostatin M was dampened in replicon cells but the combination was more effective than either cytokine alone. CONCLUSIONS: HCV genotype 1 infection downregulates IRF2 in hepatocytes attenuating hepatocellular expression of IL-7 and IL-15Rα. Our data reveal a new mechanism by which HCV abrogates specific T-cell responses and point to a novel therapeutic approach to stimulate anti-HCV immunity.

Selective inhibition of hepatitis C virus infection by hydroxyzine and benztropine.

Hepatitis C virus (HCV) infection is a major biomedical problem worldwide as it causes severe liver disease in millions of humans around the world. Despite the recent approval of specific drugs targeting HCV replication to be used in combination with alpha interferon (IFN-α) and ribavirin, there is still an urgent need for pangenotypic, interferon-free therapies to fight this genetically diverse group of viruses. In this study, we used an unbiased screening cell culture assay to interrogate a chemical library of compounds approved for clinical use in humans. This system enables identifying nontoxic antiviral compounds targeting every aspect of the viral life cycle, be the target viral or cellular. The aim of this study was to identify drugs approved for other therapeutic applications in humans that could be effective components of combination therapies against HCV. As a result of this analysis, we identified 12 compounds with antiviral activity in cell culture, some of which had previously been identified as HCV inhibitors with antiviral activity in cell culture and had been shown to be effective in patients. We selected two novel HCV antivirals, hydroxyzine and benztropine, to characterize them by determining their specificity and genotype spectrum as well as by defining the step of the replication cycle targeted by these compounds. We found that both compounds effectively inhibited viral entry at a postbinding step of genotypes 1, 2, 3, and 4 without affecting entry of other viruses.

Sigma-1 receptor regulates early steps of viral RNA replication at the onset of hepatitis C virus infection.

Hepatitis C virus (HCV) genome replication is thought to occur in a membranous cellular compartment derived from the endoplasmic reticulum (ER). The molecular mechanisms by which these membrane-associated replication complexes are formed during HCV infection are only starting to be unraveled, and both viral and cellular factors contribute to their formation. In this study, we describe the discovery of nonopioid sigma-1 receptor (S1R) as a cellular factor that mediates the early steps of viral RNA replication. S1R is a cholesterol-binding protein that resides in lipid-rich areas of the ER and in mitochondrion-associated ER membranes (MAMs). Several functions have been ascribed to this ER-resident chaperone, many of which are related to Ca(2+) signaling at the MAMs and lipid storage and trafficking. Downregulation of S1R expression by RNA interference (RNAi) in Huh-7 cells leads to a proportional decrease in susceptibility to HCV infection, as shown by reduced HCV RNA accumulation and intra- and extracellular infectivity in single-cycle infection experiments. Similar RNAi studies in persistently infected cells indicate that S1R expression is not rate limiting for persistent HCV RNA replication, as marked reduction in S1R in these cells does not lead to any decrease in HCV RNA or viral protein expression. However, subgenomic replicon transfection experiments indicate that S1R expression is rate limiting for HCV RNA replication without impairing primary translation. Overall, our data indicate that the initial steps of HCV infection are regulated by S1R, a key component of MAMs, suggesting that these structures could serve as platforms for initial RNA replication during HCV infection.

SARS-CoV-2 membrane protein-specific antibodies from critically ill SARS-CoV-2-infected individuals interact with Fc receptor-expressing cells but do not neutralize the virus.

The membrane (M) glycoprotein of SARS-CoV-2 is one of the key viral proteins regulating virion assembly and morphogenesis. Immunologically, the M protein is a major source of peptide antigens driving T cell responses, and most individuals who have been infected with SARS-CoV-2 make antibodies to the N-terminal, surface-exposed peptide of the M protein. We now report that although the M protein is abundant in the viral particle, antibodies to the surface-exposed N-terminal epitope of M do not appear to neutralize the virus. M protein-specific antibodies do, however, activate antibody-dependent cell-mediated cytotoxicity and cytokine secretion by primary human natural killer cells. Interestingly, while patients with severe or mild disease make comparable levels of M antigen-binding antibodies, M-specific antibodies from the serum of critically ill patients are significantly more potent activators of antibody-dependent cell-mediated cytotoxicity than antibodies found in individuals with mild or asymptomatic infection.

Unbiased probing of the entire hepatitis C virus life cycle identifies clinical compounds that target multiple aspects of the infection.

Over 170 million people are chronically infected by the hepatitis C virus (HCV) and at risk for dying from liver cirrhosis and hepatocellular carcinoma. Current therapy is expensive, associated with significant side effects, and often ineffective. Discovery of antiviral compounds against HCV traditionally involves a priori target identification followed by biochemical screening and confirmation in cell-based replicon assays. Typically, this results in the discovery of compounds that address a few predetermined targets and are prone to select for escape variants. To attempt to identify antiviral compounds with broad target specificity, we developed an unbiased cell-based screening system involving multiple rounds of infection in a 96-well format. Analysis of a publicly available library of 446 clinically approved drugs identified 33 compounds that targeted both known and previously unexplored aspects of HCV infection, including entry, replication, and assembly. Discovery of novel viral and cellular targets in this manner will broaden the therapeutic armamentarium against this virus, allowing for the development of drug mixtures that should reduce the likelihood of mutational escape.

Plasmacytoid dendritic cells sense hepatitis C virus-infected cells, produce interferon, and inhibit infection.

Hepatitis C virus (HCV), a member of the Flaviviridae family, is a single-stranded positive-sense RNA virus that infects >170 million people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite its ability to block the innate host response in infected hepatocyte cell lines in vitro, HCV induces a strong type 1 interferon (IFN) response in the infected liver. The source of IFN in vivo and how it is induced are currently undefined. Here we report that HCV-infected cells trigger a robust IFN response in plasmacytoid dendritic cells (pDCs) by a mechanism that requires active viral replication, direct cell-cell contact, and Toll-like receptor 7 signaling, and we show that the activated pDC supernatant inhibits HCV infection in an IFN receptor-dependent manner. Importantly, the same events are triggered by HCV subgenomic replicon cells but not by free virus particles, suggesting the existence of a novel cell-cell RNA transfer process whereby HCV-infected cells can activate pDCs to produce IFN without infecting them. These results may explain how HCV induces IFN production in the liver, and they reveal a heretofore unsuspected aspect of the innate host response to viruses that can subvert the classical sensing machinery in the cells they infect, and do not infect or directly activate pDCs.

[Meeting Report: 20 years after the First International Symposium on hepatitis C virus and related viruses]. / Resumen de congreso: 20 años después del primer Simposium Internacional sobre el virus de la hepatitis C y otros virus relacionados.

The hepatitis C virus (HCV) was discovered by the team of Michael Houghton at Chiron Corporation in 1989 and the first symposium on HCV and related viruses was held in Venice, Italy, shortly after, in 1992. This conference was organized to advance knowledge on what then was a mysterious virus responsible for most cases of «non-A, non-B¼ hepatitis. During the 20 years since the first conference, the scientific quality of presentations has steadily increased, together with the tremendous advances in basic and clinical research and epidemiology. What started as a small conference on a new virus, about which there were very few data, has today become a first-in-class congress: a meeting place for basic researchers, clinicians, epidemiologists, public health experts, and industry members to present the most important advances and their application to HCV treatment and control. The nineteenth HCV symposium was held in September 2012, once again in Venice.

Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography.

Plus-strand RNA viruses are proficient at remodeling host cell membranes for optimal viral genome replication and the production of infectious progeny. These ultrastructural alterations result in the formation of viral membranous organelles and may be observed by different imaging techniques, providing nanometric resolution. Guided by confocal and electron microscopy, this study describes the generation of wide-field volumes using cryogenic soft-X-ray tomography (cryo-SXT) on SARS-CoV-2-infected human lung adenocarcinoma cells. Confocal microscopy showed accumulation of double-stranded RNA (dsRNA) and nucleocapsid (N) protein in compact perinuclear structures, preferentially found around centrosomes at late stages of the infection. Transmission electron microscopy (TEM) showed accumulation of membranous structures in the vicinity of the infected cell nucleus, forming a viral replication organelle containing characteristic double-membrane vesicles and virus-like particles within larger vesicular structures. Cryo-SXT revealed viral replication organelles very similar to those observed by TEM but indicated that the vesicular organelle observed in TEM sections is indeed a vesiculo-tubular network that is enlarged and elongated at late stages of the infection. Overall, our data provide additional insight into the molecular architecture of the SARS-CoV-2 replication organelle.

Antiviral stilbene 1,2-diamines prevent initiation of hepatitis C virus RNA replication at the outset of infection.

The recent development of a cell culture model of hepatitis C virus (HCV) infection based on the JFH-1 molecular clone has enabled discovery of new antiviral agents. Using a cell-based colorimetric screening assay to interrogate a 1,200-compound chemical library for anti-HCV activity, we identified a family of 1,2-diamines derived from trans-stilbene oxide that prevent HCV infection at nontoxic, low micromolar concentrations in cell culture. Structure-activity relationship analysis of ~ 300 derivatives synthesized using click chemistry yielded compounds with greatly enhanced low nanomolar potency and a > 1,000:1 therapeutic ratio. Using surrogate models of HCV infection, we showed that the compounds selectively block the initiation of replication of incoming HCV RNA but have no impact on viral entry, primary translation, or ongoing HCV RNA replication, nor do they suppress persistent HCV infection. Selection of an escape variant revealed that NS5A is directly or indirectly targeted by this compound. In summary, we have identified a family of HCV inhibitors that target a critical step in the establishment of HCV infection in which NS5A translated de novo from an incoming genomic HCV RNA template is required to initiate the replication of this important human pathogen.

The autophagy machinery is required to initiate hepatitis C virus replication.

In addition to its cellular homeostasis function, autophagy is emerging as a central component of antimicrobial host defense against diverse infections. To counteract this mechanism, many pathogens have evolved to evade, subvert, or exploit autophagy. Here, we report that autophagy proteins (i.e., Beclin-1, Atg4B, Atg5, and Atg12) are proviral factors required for translation of incoming hepatitis C virus (HCV) RNA and, thereby, for initiation of HCV replication, but they are not required once infection is established. These results illustrate a previously unappreciated role for autophagy in the establishment of a viral infection and they suggest that different host factors regulate the translation of incoming viral genome and translation of progeny HCV RNA once replication is established.

Structure and inhibition of SARS-CoV-1 and SARS-CoV-2 main proteases by oral antiviral compound AG7404.

Severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) pose a threat to global public health. The 3C-like main protease (Mpro), which presents structural similarity with the active site domain of enterovirus 3C protease, is one of the best-characterized drug targets of these viruses. Here we studied the antiviral activity of the orally bioavailable enterovirus protease inhibitor AG7404 against SARS-CoV-1 and SARS-CoV-2 from a structural, biochemical, and cellular perspective, comparing it with the related molecule rupintrivir (AG7800). Crystallographic structures of AG7404 in complex with SARS-CoV-1 Mpro and SARS-CoV-2 Mpro and of rupintrivir in complex with SARS-CoV-2 Mpro were solved, revealing that all protein residues interacting with the inhibitors are conserved between the two proteins. A detailed analysis of protein-inhibitor interactions indicates that AG7404 has a better fit to the active site of the target protease than rupintrivir. This observation was further confirmed by biochemical FRET assays showing IC50 values of 47 µM and 101 µM for AG7404 and rupintrivir, respectively, in the case of SARS-CoV-2 Mpro. Equivalent IC50 values for SARS-CoV-1 also revealed greater inhibitory capacity of AG7404, with a value of 29 µM vs. 66 µM for rupintrivir. Finally, the antiviral activity of the two inhibitors against SARS-CoV-2 was confirmed in a human cell culture model of SARS-CoV-2 infection, although rupintrivir showed a higher potency and selectivity index in this assay.

Nanobodies Protecting From Lethal SARS-CoV-2 Infection Target Receptor Binding Epitopes Preserved in Virus Variants Other Than Omicron.

The emergence of SARS-CoV-2 variants that escape from immune neutralization are challenging vaccines and antibodies developed to stop the COVID-19 pandemic. Thus, it is important to establish therapeutics directed toward multiple or specific SARS-CoV-2 variants. The envelope spike (S) glycoprotein of SARS-CoV-2 is the key target of neutralizing antibodies (Abs). We selected a panel of nine nanobodies (Nbs) from dromedary camels immunized with the receptor-binding domain (RBD) of the S, and engineered Nb fusions as humanized heavy chain Abs (hcAbs). Nbs and derived hcAbs bound with subnanomolar or picomolar affinities to the S and its RBD, and S-binding cross-competition clustered them in two different groups. Most of the hcAbs hindered RBD binding to its human ACE2 (hACE2) receptor, blocked cell entry of viruses pseudotyped with the S protein and neutralized SARS-CoV-2 infection in cell cultures. Four potent neutralizing hcAbs prevented the progression to lethal SARS-CoV-2 infection in hACE2-transgenic mice, demonstrating their therapeutic potential. Cryo-electron microscopy identified Nb binding epitopes in and out the receptor binding motif (RBM), and showed different ways to prevent virus binding to its cell entry receptor. The Nb binding modes were consistent with its recognition of SARS-CoV-2 RBD variants; mono and bispecific hcAbs efficiently bound all variants of concern except omicron, which emphasized the immune escape capacity of this latest variant.