Serological evidence of virus infection in Eidolon helvum fruit bats: implications for bushmeat consumption in Nigeria.

Introduction: The Eidolon helvum fruit bat is one of the most widely distributed fruit bats in Africa and known to be a reservoir for several pathogenic viruses that can cause disease in animals and humans. To assess the risk of zoonotic spillover, we conducted a serological survey of 304 serum samples from E. helvum bats that were captured for human consumption in Makurdi, Nigeria. Methods: Using pseudotyped viruses, we screened 304 serum samples for neutralizing antibodies against viruses from the Coronaviridae, Filoviridae, Orthomyxoviridae and Paramyxoviridae families. Results: We report the presence of neutralizing antibodies against henipavirus lineage GH-M74a virus (odds ratio 6.23; p < 0.001), Nipah virus (odds ratio 4.04; p = 0.00031), bat influenza H17N10 virus (odds ratio 7.25; p < 0.001) and no significant association with Ebola virus (odds ratio 0.56; p = 0.375) in this bat cohort. Conclusion: The data suggest a potential risk of zoonotic spillover including the possible circulation of highly pathogenic viruses in E. helvum populations. These findings highlight the importance of maintaining sero-surveillance of E. helvum, and the necessity for further, more comprehensive investigations to monitor changes in virus prevalence, distribution over time, and across different geographic locations.

Expanding the Repertoire of Low-Molecular-Weight Pentafluorosulfanyl-Substituted Scaffolds.

The pentafluorosulfanyl (-SF5 ) functional group is of increasing interest as a bioisostere in medicinal chemistry. A library of SF5 -containing compounds, including amide, isoxazole, and oxindole derivatives, was synthesised using a range of solution-based and solventless methods, including microwave and ball-mill techniques. The library was tested against targets including human dihydroorotate dehydrogenase (HDHODH). A subsequent focused approach led to synthesis of analogues of the clinically used disease modifying anti-rheumatic drugs (DMARDs), Teriflunomide and Leflunomide, considered for potential COVID-19 use, where SF5 bioisostere deployment led to improved inhibition of HDHODH compared with the parent drugs. The results demonstrate the utility of the SF5 group in medicinal chemistry.

Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes.

This protocol details a rapid and reliable method for the production and titration of high-titre viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G or other variants of concern, VOC) on a lentiviral vector core, and use for neutralisation assays in target cells expressing angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). It additionally provides detailed instructions on substituting in new spike variants via gene cloning, lyophilisation and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titres to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudotypes, neutralised by human convalescent plasma and monoclonal antibodies, and stored at a range of laboratory temperatures and lyophilised for distribution and subsequent application.

Cross-Neutralisation of Novel Bombali Virus by Ebola Virus Antibodies and Convalescent Plasma Using an Optimised Pseudotype-Based Neutralisation Assay.

Ebolaviruses continue to pose a significant outbreak threat, and while Ebola virus (EBOV)-specific vaccines and antivirals have been licensed, efforts to develop candidates offering broad species cross-protection are continuing. The use of pseudotyped virus in place of live virus is recognised as an alternative, safer, high-throughput platform to evaluate anti-ebolavirus antibodies towards their development, yet it requires optimisation. Here, we have shown that the target cell line impacts neutralisation assay results and cannot be selected purely based on permissiveness. In expanding the platform to incorporate each of the ebolavirus species envelope glycoprotein, allowing a comprehensive assessment of cross-neutralisation, we found that the recently discovered Bombali virus has a point mutation in the receptor-binding domain which prevents entry into a hamster cell line and, importantly, shows that this virus can be cross-neutralised by EBOV antibodies and convalescent plasma.

AP205 VLPs Based on Dimerized Capsid Proteins Accommodate RBM Domain of SARS-CoV-2 and Serve as an Attractive Vaccine Candidate.

COVID-19 is a novel disease caused by SARS-CoV-2 which has conquered the world rapidly resulting in a pandemic that massively impacts our health, social activities, and economy. It is likely that vaccination is the only way to form "herd immunity" and restore the world to normal. Here we developed a vaccine candidate for COVID-19 based on the virus-like particle AP205 displaying the spike receptor binding motif (RBM), which is the major target of neutralizing antibodies in convalescent patients. To this end, we genetically fused the RBM domain of SARS-CoV-2 to the C terminus of AP205 of dimerized capsid proteins. The fused VLPs were expressed in E. coli, which resulted in insoluble aggregates. These aggregates were denatured in 8 M urea followed by refolding, which reconstituted VLP formation as confirmed by electron microscopy analysis. Importantly, immunized mice were able to generate high levels of IgG antibodies recognizing eukaryotically expressed receptor binding domain (RBD) as well as spike protein of SARS-CoV-2. Furthermore, induced antibodies were able to neutralize SARS-CoV-2/ABS/NL20. Additionally, this vaccine candidate has the potential to be produced at large scale for immunization programs.

Intracellular immunization against HIV infection with an intracellular antibody that mimics HIV integrase binding to the cellular LEDGF protein.

Preventing the protein-protein interaction of the cellular chromatin binding protein Lens Epithelium-Derived Growth Factor (LEDGF) and human immunodeficiency virus (HIV) integrase is an important possible strategy for anti-viral treatment for AIDS. We have used Intracellular Antibody Capture technology to isolate a single VH antibody domain that binds to LEDGF. The crystal structure of the LEDGF-VH complex reveals that the single domain antibody mimics the effect of binding of HIV integrase to LEDGF which is crucial for HIV propagation. CD4-expressing T cell lines were constructed to constitutively express the LEDGF-binding VH and these cells showed interference with HIV viral replication, assayed by virus capsid protein p24 production. Therefore, pre-conditioning cells to express antibody fragments confers effective intracellular immunization for preventing chronic viral replication and can be a way to prevent HIV spread in infected patients. This raises the prospect that intracellular immunization strategies that focus on cellular components of viral integrase protein interactions can be used to combat the problems associated with latent HIV virus re-emergence in patients. New genome editing development, such as using CRISPR/cas9, offer the prospect intracellularly immunized T cells in HIV+ patients.

Survivability of immunoassay reagents exposed to the space radiation environment on board the ESA BIOPAN-6 platform as a prelude to performing immunoassays on Mars.

The Life Marker Chip (LMC) instrument is an immunoassay-based sensor that will attempt to detect signatures of life in the subsurface of Mars. The molecular reagents at the core of the LMC have no heritage of interplanetary mission use; therefore, the design of such an instrument must take into account a number of risk factors, including the radiation environment that will be encountered during a mission to Mars. To study the effects of space radiation on immunoassay reagents, primarily antibodies, a space study was performed on the European Space Agency's 2007 BIOPAN-6 low-Earth orbit (LEO) space exposure platform to complement a set of ground-based radiation studies. Two antibodies were used in the study, which were lyophilized and packaged in the intended LMC format and loaded into a custom-made sample holder unit that was mounted on the BIOPAN-6 platform. The BIOPAN mission went into LEO for 12 days, after which all samples were recovered and the antibody binding performance was measured via enzyme-linked immunosorbent assays (ELISA). The factors expected to affect antibody performance were the physical conditions of a space mission and the exposure to space conditions, primarily the radiation environment in LEO. Both antibodies survived inactivation by these factors, as concluded from the comparison between the flight samples and a number of shipping and storage controls. This work, in combination with the ground-based radiation tests on representative LMC antibodies, has helped to reduce the risk of using antibodies in a planetary exploration mission context.

Effects of simulated space radiation on immunoassay components for life-detection experiments in planetary exploration missions.

The Life Marker Chip (LMC) instrument is part of the proposed payload on the ESA ExoMars rover that is scheduled for launch in 2018. The LMC will use antibody-based assays to detect molecular signatures of life in samples obtained from the shallow subsurface of Mars. For the LMC antibodies, the ability to resist inactivation due to space particle radiation (both in transit and on the surface of Mars) will therefore be a prerequisite. The proton and neutron components of the mission radiation environment are those that are expected to have the dominant effect on the operation of the LMC. Modeling of the radiation environment for a mission to Mars led to the calculation of nominal mission fluences for proton and neutron radiation. Various combinations and multiples of these values were used to demonstrate the effects of radiation on antibody activity, primarily at the radiation levels envisaged for the ExoMars mission as well as at much higher levels. Five antibodies were freeze-dried in a variety of protective molecular matrices and were exposed to various radiation conditions generated at a cyclotron facility. After exposure, the antibodies' ability to bind to their respective antigens was assessed and found to be unaffected by ExoMars mission level radiation doses. These experiments indicated that the expected radiation environment of a Mars mission does not pose a significant risk to antibodies packaged in the form anticipated for the LMC instrument.

Synthesis and characterisation of immunogens for the production of antibodies against small hydrophobic molecules with biosignature properties.

In the present study, five different classes of small hydrophobic molecular targets, atypical for antibody generation, were structurally modified in order to introduce suitable reactive functionalities and/or spacers which allow covalent coupling to a carrier protein resulting in a stable carrier-hapten complex. These targets were chosen to serve as markers of extant and/or extinct life in the context of the development of the Life Marker Chip (LMC), an antibody-based instrument, which is being developed by a UK-led international consortium for flight to Mars on board the joint ESA/NASA Mars exploration ExoMars mission. The hapten-protein conjugates were designed to be used as immunogens for antibody generation and immunoassay reagents in subsequent stages of the LMC development. The extent of protein modification due to covalent attachment of hapten was determined by two independent methods, i.e. trinitrobenzenesulfonic acid (TNBSA) titrations of remaining protein reactive groups and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of the resultant hapten-protein conjugates. In a further quality validation step, the conjugates were presented to an animal's immune system and polyclonal antibody titres with moderate specificity were obtained. These results suggest that conjugates synthesized as described herein can successfully be used in the generation of antibodies targeting small hydrophobic molecules.

Use of polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline for the preparation of a hapten-protein conjugate for antibody development.

Polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline (PS-IIDQ), a polymer-supported covalent coupling reagent, was successfully employed for the first time in the bioconjugation of an example hapten (phytanic acid derivative) to a carrier protein (bovine serum albumin (BSA)) within the context of immunogen preparation for antibody development. The ability of the prepared example phytanic acid derivative-BSA conjugate to bind an anti-phytanic acid antibody was confirmed using an enzyme-linked immunosorbent assay (ELISA).