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.

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.

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.

The Bacterial Mucosal Immunotherapy MV130 Protects Against SARS-CoV-2 Infection and Improves COVID-19 Vaccines Immunogenicity.

COVID-19-specific vaccines are efficient prophylactic weapons against SARS-CoV-2 virus. However, boosting innate responses may represent an innovative way to immediately fight future emerging viral infections or boost vaccines. MV130 is a mucosal immunotherapy, based on a mixture of whole heat-inactivated bacteria, that has shown clinical efficacy against recurrent viral respiratory infections. Herein, we show that the prophylactic intranasal administration of this immunotherapy confers heterologous protection against SARS-CoV-2 infection in susceptible K18-hACE2 mice. Furthermore, in C57BL/6 mice, prophylactic administration of MV130 improves the immunogenicity of two different COVID-19 vaccine formulations targeting the SARS-CoV-2 spike (S) protein, inoculated either intramuscularly or intranasally. Independently of the vaccine candidate and vaccination route used, intranasal prophylaxis with MV130 boosted S-specific responses, including CD8+-T cell activation and the production of S-specific mucosal IgA antibodies. Therefore, the bacterial mucosal immunotherapy MV130 protects against SARS-CoV-2 infection and improves COVID-19 vaccines immunogenicity.

Imaging of Virus-Infected Cells with Soft X-ray Tomography.

Viruses are obligate parasites that depend on a host cell for replication and survival. Consequently, to fully understand the viral processes involved in infection and replication, it is fundamental to study them in the cellular context. Often, viral infections induce significant changes in the subcellular organization of the host cell due to the formation of viral factories, alteration of cell cytoskeleton and/or budding of newly formed particles. Accurate 3D mapping of organelle reorganization in infected cells can thus provide valuable information for both basic virus research and antiviral drug development. Among the available techniques for 3D cell imaging, cryo-soft X-ray tomography stands out for its large depth of view (allowing for 10 µm thick biological samples to be imaged without further thinning), its resolution (about 50 nm for tomographies, sufficient to detect viral particles), the minimal requirements for sample manipulation (can be used on frozen, unfixed and unstained whole cells) and the potential to be combined with other techniques (i.e., correlative fluorescence microscopy). In this review we describe the fundamentals of cryo-soft X-ray tomography, its sample requirements, its advantages and its limitations. To highlight the potential of this technique, examples of virus research performed at BL09-MISTRAL beamline in ALBA synchrotron are also presented.

SARS-CoV-2 Fears Green: The Chlorophyll Catabolite Pheophorbide A Is a Potent Antiviral.

SARS-CoV-2 pandemic is having devastating consequences worldwide. Although vaccination advances at good pace, effectiveness against emerging variants is unpredictable. The virus has displayed a remarkable resistance to treatments and no drugs have been proved fully effective against COVID-19. Thus, despite the international efforts, there is still an urgent need for new potent and safe antivirals against SARS-CoV-2. Here, we exploited the enormous potential of plant metabolism using the bryophyte Marchantia polymorpha L. and identified a potent SARS-CoV-2 antiviral, following a bioactivity-guided fractionation and mass-spectrometry approach. We found that the chlorophyll derivative Pheophorbide a (PheoA), a porphyrin compound similar to animal Protoporphyrin IX, has an extraordinary antiviral activity against SARS-CoV-2, preventing infection of cultured monkey and human cells, without noticeable cytotoxicity. We also show that PheoA targets the viral particle, interfering with its infectivity in a dose- and time-dependent manner. Besides SARS-CoV-2, PheoA also displayed a broad-spectrum antiviral activity against enveloped RNA viral pathogens such as HCV, West Nile, and other coronaviruses. Our results indicate that PheoA displays a remarkable potency and a satisfactory therapeutic index, which together with its previous use in photoactivable cancer therapy in humans, suggest that it may be considered as a potential candidate for antiviral therapy against SARS-CoV-2.

Identification of Niemann-Pick C1 protein as a potential novel SARS-CoV-2 intracellular target.

Niemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol traffic. This protein has been shown to play an important role for several viruses. It has been reported that SARS-CoV-2 enters the cell through plasma membrane fusion and/or endosomal entry upon availability of proteases. However, the whole process is not fully understood yet and additional viral/host factors might be required for viral fusion and subsequent viral replication. Here, we report a novel interaction between the SARS-CoV-2 nucleoprotein (N) and the cholesterol transporter NPC1. Furthermore, we have found that some compounds reported to interact with NPC1, carbazole SC816 and sulfides SC198 and SC073, were able to reduce SARS-CoV-2 viral infection with a good selectivity index in human cell infection models. These findings suggest the importance of NPC1 for SARS-CoV-2 viral infection and a new possible potential therapeutic target to fight against COVID-19.

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.