Virtual screening and in vitro validation of natural compound inhibitors against SARS-CoV-2 spike protein.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has led to a major public health burden and has resulted in millions of deaths worldwide. As effective treatments are limited, there is a significant requirement for high-throughput, low resource methods for the discovery of novel antivirals. The SARS-CoV-2 spike protein plays a key role in viral entry and has been identified as a therapeutic target. Using the available spike crystal structure, we performed a virtual screen with a library of 527 209 natural compounds against the receptor binding domain of this protein. Top hits from this screen were subjected to a second, more comprehensive molecular docking experiment and filtered for favourable ADMET properties. The in vitro activity of 10 highly ranked compounds was assessed using a virus neutralisation assay designed to facilitate viral entry in a physiologically relevant manner via the plasma membrane route. Subsequently, four compounds ZINC02111387, ZINC02122196, SN00074072 and ZINC04090608 were identified to possess antiviral activity in the µM range. These findings validate the virtual screening method as a tool for identifying novel antivirals and provide a basis for future drug development against SARS-CoV-2.

Refinement and Neutralization Evaluation of the F(ab')2 Type of Antivenom against the Deadly Jellyfish Nemopilema nomurai Toxins.

Jellyfish stings threaten people's health and even life in coastal areas worldwide. Nemopilema nomurai is one of the most dangerous jellyfish in the East Asian Marginal Seas, which not only stings hundreds of thousands of people every year but also is assumed to be responsible for most deaths by jellyfish stings in China. However, there is no effective first-aid drug, such as antivenoms, for the treatment of severe stings by N. nomurai to date. In this study, we prepared a N. nomurai antiserum from rabbits using inactivated N. nomurai toxins (NnTXs) and isolated the IgG type of antivenom (IgG-AntiNnTXs) from the antiserum. Subsequently, IgG-AntiNnTXs were refined with multiple optimizations to remove Fc fragments. Finally, the F(ab')2 type of antivenom (F(ab')2-AntiNnTXs) was purified using Superdex 200 and protein A columns. The neutralization efficacy of both types of antivenom was analyzed in vitro and in vivo, and the results showed that both IgG and F(ab')2 types of antivenom have some neutralization effect on the metalloproteinase activity of NnTXs in vitro and could also decrease the mortality of mice in the first 4 h after injection. This study provides some useful information for the development of an effective antivenom for N. nomurai stings in the future.

Analysis of the molecular mechanism of SARS-CoV-2 antibodies.

A newly-emergent beta-coronavirus, SARS-CoV-2, rapidly has become a pandemic since 2020. It is a serious respiratory disease and caused more than 100 million of deaths in the world. WHO named it COVIA-19 and there is no effective targeted drug for it. The main treatment strategies include chemical medicine, traditional Chinese medicine (TCM) and biologics. Due to SARS-CoV-2 uses the spike proteins (S proteins) on its envelope to infect human cells, monoclonal antibodies that neutralize the S protein have become one of the hot research areas in the current research and treatment of SARS-CoV-2. In this study, we reviewed the antibodies that have been reported to have neutralizing activity against the SARS-CoV-2 infection. According to their different binding epitope regions in RBD or NTD, they are classified, and the mechanism of the representative antibodies in each category is discussed in depth, which provides potential foundation for future antibody and vaccine therapy and the development of antibody cocktails against SARS-CoV-2 mutants.

Evaluation of antibody response of sheep to foot-and-mouth disease vaccine prepared by using different MontanideTM oil adjuvants.

Despite the widespread use of the conventional inactivated foot-and-mouth disease (FMD) vaccine, its immunogenicity is poor and the duration of its protection is short. In this study, humoral response to commercial ready-to-use MontanideTM ISA 201 VG and MontanideTM ISA 61 VG oil adjuvants and a common adjuvant MontanideTM ISA 206 VG developed by Seppic Inc., were evaluated for FMD antigens in sheep and double oil emulsion (w/o/w) formulations of MontanideTM ISA 201 and 206 and single oil emulsion (w/o) of MontanideTM ISA 61 have been prepared by using current FMDV antigens (O/TUR/07, A/ASIA/G-VII, A/TUR/16 and ASIA/ TUR/15). The animals (n=48) were vaccinated subcutaneously with formulations and five sheep were maintained as an unvaccinated control group. Blood samples were taken at day 0, 7, 14, 21, 28, 60, 90, 120 and 150. Virus neutralization and liquid phase blocking ELISA tests were used to compare antibody response to vaccines prepared by using different MontanideTM mineral oils. The results showed that vaccines prepared by using MontanideTM ISA 61 and 201 gave better antibody response to FMD antigens than MontanideTM ISA 206 formulation, although results were not statistically significant for certain days of sampling. Moreover, the overall type O antibody response of MontanideTM ISA 201 was found to be superior to MontanideTM ISA 61.

Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2.

Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.

A Novel Apilic Antivenom to Treat Massive, Africanized Honeybee Attacks: A Preclinical Study from the Lethality to Some Biochemical and Pharmacological Activities Neutralization.

Massive, Africanized honeybee attacks have increased in Brazil over the years. Humans and animals present local and systemic effects after envenomation, and there is no specific treatment for this potentially lethal event. This study evaluated the ability of a new Apilic antivenom, which is composed of F(ab')2 fraction of specific immunoglobulins in heterologous and hyperimmune equine serum, to neutralize A. mellifera venom and melittin, in vitro and in vivo, in mice. Animal experiments were performed in according with local ethics committee license (UFRJ protocol no. DFBCICB072-04/16). Venom dose-dependent lethality was diminished with 0.25-0.5 µL of intravenous Apilic antivenom/µg honeybee venom. In vivo injection of 0.1-1 µg/g bee venom induced myotoxicity, hemoconcentration, paw edema, and increase of vascular permeability which were antagonized by Apilic antivenom. Cytotoxicity, assessed in renal LLC-PK1 cells and challenged with 10 µg/mL honeybee venom or melittin, was neutralized by preincubation with Apilic antivenom, as well the hemolytic activity. Apilic antivenom inhibited phospholipase and hyaluronidase enzymatic activities. In flow cytometry experiments, Apilic antivenom neutralized reduction of cell viability due to necrosis by honeybee venom or melittin. These results showed that this antivenom is effective inhibitor of honeybee venom actions. Thus, this next generation of Apilic antivenom emerges as a new promising immunobiological product for the treatment of massive, Africanized honeybee attacks.

Generation of recombinant rotaviruses encoding a split NanoLuc peptide tag.

Recombinant viruses expressing fluorescent or luminescent reporter proteins are used to quantitate and visualize viral replication and transmission. Here, we used a split NanoLuc luciferase (NLuc) system comprising large LgBiT and small HiBiT peptide fragments to generate stable reporter rotaviruses (RVs). Reporter RVs expressing NSP1-HiBiT fusion protein were generated by placing an 11 amino acid HiBiT peptide tag at the C-terminus of the intact simian RV NSP1 open reading frame or truncated human RV NSP1 open reading frame. Virus-infected cell lysates exhibited NLuc activity that paralleled virus replication. The antiviral activity of neutralizing antibodies and antiviral reagents against the recombinant HiBiT reporter viruses were monitored by measuring reductions in NLuc expression. These findings demonstrate that the HiBiT reporter RV systems are powerful tools for studying the viral life cycle and pathogenesis, and a robust platform for developing novel antiviral drugs.

A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite.

Snakebite is a medical emergency causing high mortality and morbidity in rural tropical communities that typically experience delayed access to unaffordable therapeutics. Viperid snakes are responsible for the majority of envenomings, but extensive interspecific variation in venom composition dictates that different antivenom treatments are used in different parts of the world, resulting in clinical and financial snakebite management challenges. Here, we show that a number of repurposed Phase 2-approved small molecules are capable of broadly neutralizing distinct viper venom bioactivities in vitro by inhibiting different enzymatic toxin families. Furthermore, using murine in vivo models of envenoming, we demonstrate that a single dose of a rationally-selected dual inhibitor combination consisting of marimastat and varespladib prevents murine lethality caused by venom from the most medically-important vipers of Africa, South Asia and Central America. Our findings support the translation of combinations of repurposed small molecule-based toxin inhibitors as broad-spectrum therapeutics for snakebite.

A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition.

An In Vitro Microneutralization Assay for SARS-CoV-2 Serology and Drug Screening.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the city of Wuhan, Hubei Province, China, in late 2019. Since then, the virus has spread globally and caused a pandemic. Assays that can measure the antiviral activity of antibodies or antiviral compounds are needed for SARS-CoV-2 vaccine and drug development. Here, we describe in detail a microneutralization assay, which can be used to assess in a quantitative manner if antibodies or drugs can block entry and/or replication of SARS-CoV-2 in vitro. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Microneutralization assay to test inhibition of virus by antibodies (purified antibodies or serum/plasma) Basic Protocol 2: Screening of anti-SARS-CoV-2 compounds in vitro Support Protocol: SARS-CoV-2 propagation.

Phytochemical screening of Alstonia scholaris leaf and bark extracts and their antimicrobial activities.

Alstonia sholaris is an evergreen tree commonly found in South East Asia. In traditional medicine pharmacological activities are attributed to the leaves and bark of this plant. The aim of this study is characterizing the chemicals present in A. sholaris leaves and bark extracts and study their antimicrobial activities. Solvent extractions with Soxhlet apparatus of leaves and bark were obtained using hexane, benzene, isopropanol, methanol, and water. The crude extracts were concentrated and screened for qualitative phytochemical analysis and thin layer chromatography, and the antibacterial, antifungal an antiviral activity of crude extracts were measured by in vitro methods. Isopropanol and methanol extracts showed significant antibacterial activity and it was more pronounced against Gram positive than against Gram negative bacteria. Hexane, benzene, isopropanol and methanol fractions of A. scholaris bark and leaf showed activity against Enterobacter cloacae. Isopropanol extract showed maximum activity against selected human pathogenic fungus. In conclusion, the leaves and bark of A. scholaris are rich in phytochemicals with antimicrobial activities against human pathogens, being the isopropanol fraction the one with the highest antibacterial, antifungal, antiviral and anti-mycobacterial activities.

Revealing new tick-borne encephalitis virus foci by screening antibodies in sheep milk.

BACKGROUND: Tick distribution in Sweden has increased in recent years, with the prevalence of ticks predicted to spread towards the northern parts of the country, thus increasing the risk of tick-borne zoonoses in new regions. Tick-borne encephalitis (TBE) is the most significant viral tick-borne zoonotic disease in Europe. The disease is caused by TBE virus (TBEV) infection which often leads to severe encephalitis and myelitis in humans. TBEV is usually transmitted to humans via tick bites; however, the virus can also be excreted in the milk of goats, sheep and cattle and infection may then occur via consumption of unpasteurised dairy products. Virus prevalence in questing ticks is an unreliable indicator of TBE infection risk as viral RNA is rarely detected even in large sample sizes collected at TBE-endemic areas. Hence, there is a need for robust surveillance techniques to identify emerging TBEV risk areas at early stages. METHODS: Milk and colostrum samples were collected from sheep and goats in Örebro County, Sweden. The milk samples were analysed for the presence of TBEV antibodies by ELISA and validated by western blot in which milk samples were used to detect over-expressed TBEV E-protein in crude cell extracts. Neutralising titers were determined by focus reduction neutralisation test (FRNT). The stability of TBEV in milk and colostrum was studied at different temperatures. RESULTS: In this study we have developed a novel strategy to identify new TBEV foci. By monitoring TBEV antibodies in milk, we have identified three previously unknown foci in Örebro County which also overlap with areas of TBE infection reported during 2009-2018. In addition, our data indicates that keeping unpasteurised milk at 4 °C will preserve the infectivity of TBEV for several days. CONCLUSIONS: Altogether, we report a non-invasive surveillance technique for revealing risk areas for TBE in Sweden, by detecting TBEV antibodies in sheep milk. This approach is robust and reliable and can accordingly be used to map TBEV "hotspots". TBEV infectivity in refrigerated milk was preserved, emphasising the importance of pasteurisation (i.e. 72 °C for 15 s) prior to consumption.

Subclade 2.2.1-Specific Human Monoclonal Antibodies That Recognize an Epitope in Antigenic Site A of Influenza A(H5) Virus HA Detected between 2015 and 2018.

Highly pathogenic avian H5 influenza viruses persist among poultry and wild birds throughout the world. They sometimes cause interspecies transmission between avian and mammalian hosts. H5 viruses possessing the HA of subclade 2.3.4.4, 2.3.2.1, 2.2.1, or 7.2 were detected between 2015 and 2018. To understand the neutralizing epitopes of H5-HA, we characterized 15 human monoclonal antibodies (mAbs) against the HA of H5 viruses, which were obtained from volunteers who received the H5N1 vaccine that contains a subclade 2.2.1 or 2.1.3.2 virus as an antigen. Twelve mAbs were specific for the HA of subclade 2.2.1, two mAbs were specific for the HA of subclade 2.1.3.2, and one mAb was specific for the HA of both. Of the 15 mAbs analyzed, nine, which were specific for the HA of subclade 2.2.1, and shared the VH and VL genes, possessed hemagglutination inhibition and neutralizing activities, whereas the others did not. A single amino acid substitution or insertion at positions 144-147 in antigenic site A conferred resistance against these nine mAbs to the subclade 2.2.1 viruses. The amino acids at positions 144-147 are highly conserved among subclade 2.2.1, but differ from those of other subclades. These results show that the neutralizing epitope including amino acids at positions 144-147 is targeted by human antibodies, and plays a role in the antigenic difference between subclade 2.2.1 and other subclades.

Antibody Responses to Bovine Alphaherpesvirus 1 (BoHV-1) in Passively Immunized Calves.

To date, in countries where infectious bovine rhinotracheitis (IBR) is widespread, its control is associated with deleted marker vaccines. These products lack one or more genes responsible for the synthesis of glycoproteins or enzymes. In Europe, the most widely used marker vaccine is one in which glycoprotein E (gE-) is deleted, and it is marketed in a killed or modified-live form. Using this type of immunization, it is possible to differentiate vaccinated animals (gE-) from those infected or injected with non-deleted (gE+) products using diagnostic tests specific for gE. The disadvantage of using modified-live gE-products is that they may remain latent in immunized animals and be reactivated or excreted following an immunosuppressive stimulus. For this reason, in the last few years, a new marker vaccine became commercially available containing a double deletion related to genes coding for gE and the synthesis of the thymidine-kinase (tk) enzyme, the latter being associated with the reduction of the neurotropism, latency, and reactivation of the vaccine virus. Intramuscularly and intranasally administered marker products induce a humoral immune response; however, the mother-to-calf antibody kinetics after vaccination with marker vaccines is poorly understood. This review discusses several published articles on this topic.

Rational design of a triple-type human papillomavirus vaccine by compromising viral-type specificity.

Sequence variability in surface-antigenic sites of pathogenic proteins is an important obstacle in vaccine development. Over 200 distinct genomic sequences have been identified for human papillomavirus (HPV), of which more than 18 are associated with cervical cancer. Here, based on the high structural similarity of L1 surface loops within a group of phylogenetically close HPV types, we design a triple-type chimera of HPV33/58/52 using loop swapping. The chimeric VLPs elicit neutralization titers comparable with a mix of the three wild-type VLPs both in mice and non-human primates. This engineered region of the chimeric protein recapitulates the conformational contours of the antigenic surfaces of the parental-type proteins, offering a basis for this high immunity. Our stratagem is equally successful in developing other triplet-type chimeras (HPV16/35/31, HPV56/66/53, HPV39/68/70, HPV18/45/59), paving the way for the development of an improved HPV prophylactic vaccine against all carcinogenic HPV strains. This technique may also be extrapolated to other microbes.

Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays.

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000⁻300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies.

The black mamba (Dendroaspis polylepis) is one of the most feared snake species of the African savanna. It has a potent, fast-acting neurotoxic venom comprised of dendrotoxins and α-neurotoxins associated with high fatality in untreated victims. Current antivenoms are both scarce on the African continent and present a number of drawbacks as they are derived from the plasma of hyper-immunized large mammals. Here, we describe the development of an experimental recombinant antivenom by a combined toxicovenomics and phage display approach. The recombinant antivenom is based on a cocktail of fully human immunoglobulin G (IgG) monoclonal antibodies capable of neutralizing dendrotoxin-mediated neurotoxicity of black mamba whole venom in a rodent model. Our results show the potential use of fully human monoclonal IgGs against animal toxins and the first use of oligoclonal human IgG mixtures against experimental snakebite envenoming.

Golden-star nanoparticles as adjuvant effectively promotes immune response to foot-and-mouth disease virus-like particles vaccine.

Virus-like particles (VLPs) have become a hot topic in modern vaccine research because of its safety, facile production, and immune properties. To further enhance the immune effect of VLPs, we synthesized and used gold-star nanoparticles (AuSNs) as adjuvant for vaccine. Foot-and-mouth disease (FMD) VLPs as target antigen were combined with AuSNs. The FMD VLPs-AuSNs complex was characterized through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, ultraviolet light absorption, and morphological measurement analyses. Result indicated that the FMD VLPs-AuSNs complex is non-toxic in different cell lines. AuSNs can effectively promote the entry of FMD VLPs into cells and improve macrophages activation when combined with FMD VLPs compared with FMD VLPs alone. Further animal vaccination and challenge tests revealed that the specific immune response and protection rate of AuSNs adjuvant group is higher than that of conventional mineral oil (ISA206) adjuvant group. AuSNs can effectively improve the immune protection effects of FMD VLPs vaccines, and exhibit potential as a new adjuvant for other vaccines.

Immunogenicity and Protection After Vaccination With a Modified Vaccinia Virus Ankara-Vectored Yellow Fever Vaccine in the Hamster Model.

The highly efficacious live-attenuated 17D yellow fever (YF) vaccine is occasionally associated with rare life-threatening adverse events. Modified vaccinia virus Ankara (MVA), a non-replicating poxvirus, has been used as a vaccine platform to safely deliver various antigens. A MVA-based YF vaccine (MVA-BN-YF) was tested with and without a non-mineral oil adjuvant in a hamster model of lethal YF disease and protective efficacy of this vaccine was compared with the 17D vaccine. The vaccine candidate MVA-BN-YF generated a protective response in hamsters infected with YFV that was comparable to protection by the live 17D vaccine. Similar levels of neutralizing antibody were observed in animals vaccinated with either vaccine alone or vaccine with adjuvant. Significant improvement in survival, weight change, and serum alanine aminotransferase levels were observed in vaccinated hamsters when administered 42 and 14 days prior to challenge with Jimenez YF virus (YFV). Neutralizing antibodies induced by MVA-BN-YF were transferred to naïve hamsters prior to virus challenge. Passive administration of neutralizing antibody 24 h prior to virus infection resulted in significantly improved survival and weight change. A trend toward reduced liver enzyme levels was also observed. MVA-BN-YF, therefore, represents a safe alternative to vaccination with live-attenuated YFV.

Colostral transmission of BTV-8 antibodies from dairy cows six years after vaccination.

Bluetongue virus (BTV) antibodies were analysed in 27 Swiss calves born in 2016 at the age of 16-19 days using competitive enzyme-linked-immunosorbent-assay (cELISA) and virus neutralization test (VNT) (animal trial permission number: 75684). Obligatory documentation proved that 15 of 27 dams were BTV-8 vaccinated once or three times in 2008-2010. The offsprings of the non-vaccinated dams were seronegative. Two of three calves and 11 of 12 calves descending from dams who had been vaccinated one or three times, respectively, had BTV specific serum antibodies. As Switzerland is considered BTV-free from 2010 to 2016, it is likely that BTV-8 antibodies were transferred via colostrum. Furthermore, we confirmed neutralizing cross-reactivity of BTV-8 with BTV-4 antibodies as 5 samples positive for BTV-8 were also reactive with BTV-4 antibodies.