ACE2-Fc and DPP4-Fc decoy receptors against SARS-CoV-2 and MERS-CoV variants: a quick therapeutic option for current and future coronaviruses outbreaks.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the Middle East respiratory syndrome coronavirus (MERS-CoV) are highly pathogenic human coronaviruses (CoVs). Anti-CoVs mAbs and vaccines may be effective, but the emergence of neutralization escape variants is inevitable. Angiotensin-converting enzyme 2 and dipeptidyl peptidase 4 enzyme are the getaway receptors for SARS-CoV-2 and MERS-CoV, respectively. Thus, we reformatted these receptors as Fc-fusion decoy receptors. Then, we tested them in parallel with anti-SARS-CoV (ab1-IgG) and anti-MERS-CoV (M336-IgG) mAbs against several variants using pseudovirus neutralization assay. The generated Fc-based decoy receptors exhibited a strong inhibitory effect against all pseudotyped CoVs. Results showed that although mAbs can be effective antiviral drugs, they might rapidly lose their efficacy against highly mutated viruses. We suggest that receptor traps can be engineered as Fc-fusion proteins for highly mutating viruses with known entry receptors, for a faster and effective therapeutic response even against virus harboring antibodies escape mutations.

Construction of VSVΔ51M oncolytic virus expressing human interleukin-12.

The use of oncolytic viruses (OVs) in combination with cytokines, such as IL-12, is a promising approach for cancer treatment that addresses the limitations of current standard treatments and traditional cancer immunotherapies. IL-12, a proinflammatory cytokine, triggers intracellular signaling pathways that lead to increased apoptosis of tumor cells and enhanced antitumor activity of immune cells via IFN-γ induction, making this cytokine a promising candidate for cancer therapy. Targeted expression of IL-12 within tumors has been shown to play a crucial role in tumor eradication. The recent development of oncolytic viruses enables targeted delivery and expression of IL-12 at the tumor site, thereby addressing the systemic toxicities associated with traditional cancer therapy. In this study, we constructed an oncolytic virus, VSVΔ51M, based on the commercially available VSV wild-type backbone and further modified it to express human IL-12. Our preclinical data confirmed the safety and limited toxicity of the modified virus, VSV-Δ51M-hIL-12, supporting its potential use for clinical development.

Early Humoral Response Correlates with Disease Severity and Outcomes in COVID-19 Patients.

The Coronavirus Disease 2019 (COVID-19), caused by SARS-CoV-2, continues to spread globally with significantly high morbidity and mortality rates. Antigen-specific responses are of unquestionable value for clinical management of COVID-19 patients. Here, we investigated the kinetics of IgM, IgG against the spike (S) and nucleoproteins (N) proteins and their neutralizing capabilities in hospitalized COVID-19 patients with different disease presentations (i.e., mild, moderate or severe), need for intensive care units (ICU) admission or outcomes (i.e., survival vs death). We show that SARS-CoV-2 specific IgG, IgM and neutralizing antibodies (nAbs) were readily detectable in almost all COVID-19 patients with various clinical presentations. Interestingly, significantly higher levels of nAbs as well as anti-S1 and -N IgG and IgM antibodies were found in patients with more severe symptoms, patients requiring admission to ICU or those with fatal outcomes. More importantly, early after symptoms onset, we found that the levels of anti-N antibodies correlated strongly with disease severity. Collectively, these findings provide new insights into the kinetics of antibody responses in COVID-19 patients with different disease severity.

Evaluation of Neutralizing Antibodies Against Highly Pathogenic Coronaviruses: A Detailed Protocol for a Rapid Evaluation of Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudovirus-Based Assay.

Emerging highly pathogenic human coronaviruses (CoVs) represent a serious ongoing threat to the public health worldwide. The spike (S) proteins of CoVs are surface glycoproteins that facilitate viral entry into host cells via attachment to their respective cellular receptors. The S protein is believed to be a major immunogenic component of CoVs and a target for neutralizing antibodies (nAbs) and most candidate vaccines. Development of a safe and convenient assay is thus urgently needed to determine the prevalence of CoVs nAbs in the population, to study immune response in infected individuals, and to aid in vaccines and viral entry inhibitor evaluation. While live virus-based neutralization assays are used as gold standard serological methods to detect and measure nAbs, handling of highly pathogenic live CoVs requires strict bio-containment conditions in biosafety level-3 (BSL-3) laboratories. On the other hand, use of replication-incompetent pseudoviruses bearing CoVs S proteins could represent a safe and useful method to detect nAbs in serum samples under biosafety level-2 (BSL-2) conditions. Here, we describe a detailed protocol of a safe and convenient assay to generate vesicular stomatitis virus (VSV)-based pseudoviruses to evaluate and measure nAbs against highly pathogenic CoVs. The protocol covers methods to produce VSV pseudovirus bearing the S protein of the Middle East respiratory syndrome-CoV (MERS-CoV) and the severe acute respiratory syndrome-CoV-2 (SARS-CoV-2), pseudovirus titration, and pseudovirus neutralization assay. Such assay could be adapted by different laboratories and researchers working on highly pathogenic CoVs without the need to handle live viruses in the BSL-3 environment.

SARS-CoV-2 S1 and N-based serological assays reveal rapid seroconversion and induction of specific antibody response in COVID-19 patients.

As the Coronavirus Disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses (CoVs). The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

Seroprevalence of MERS-CoV in healthy adults in western Saudi Arabia, 2011-2016.

BACKGROUND: The Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly recognized zoonotic coronavirus. Current evidence confirms the role of dromedaries in primary human infections but does not explain the sporadic community cases. However, asymptomatic or subclinical cases could represent a possible source of infection in the community. METHODS: Archived human sera (7461) collected between 2011 and 2016 from healthy adult blood donors from 50 different nationalities in the western part of Saudi Arabia were obtained for MERS-CoV seroprevalence investigation. Samples were tested for MERS-CoV S1-specific antibodies (Abs) by ELISA and confirmed by testing for neutralizing Abs (nAbs) using both pseudotyped and live virus neutralization assays. RESULTS: Out of 7461 samples, 174 sera from individuals with 18 different nationalities were ELISA positive (2.3%, 95% CI 2.0-2.7). Presence of nAbs was confirmed in 17 samples (0.23%, 95% CI 0.1-0.4) of which one sample exhibited positivity in both neutralization assays. Confirmed seropositivity was identified in young (15-44 years) men and women from Saudi Arabia, Egypt, Yemen, Pakistan, Palestine, Sudan, and India without significant preference. CONCLUSIONS: An increasing trend of MERS-CoV seroprevalence was observed in the general population in western Saudi Arabia, suggesting that asymptomatic or mild infections might exist and act as an unrecognized source of infection. Seropositivity of individuals from different nationalities underscores the potential MERS exportation outside of the Arabian Peninsula. Thus, enhanced and continuous surveillance is highly warranted.

Unnatural amino acids: production and biotechnological potential.

Unnatural amino acids (UAAs) are valuable building blocks in the manufacture of a wide range of pharmaceuticals. UAAs exhibit biological activity as free acids and they can be incorporated into linear or cyclic peptides with biological activity. However, the scope of biotechnological application of UAAs goes beyond this, as they can be used to investigate the structure and dynamics of proteins, to study protein interactions, or to modulate the activity of proteins in living cells. The means to expand nature's repertoire of amino acids include chemical and biological routes. An UAA can be made through chemical modifications of natural amino acids, or related compounds. These modifications typically rely on utilisation of ligands and palladium catalysts. Employing biocatalysts in the synthesis of UAAs can also afford novel molecules with different physical and chemical properties. A number of transaminases for example have been identified and employed in the production of UAAs. This review will compare the chemical and biological routes for the synthesis of UAAs and provide an overview of their applications.

Single point mutations reveal amino acid residues important for Chromobacterium violaceum transaminase activity in the production of unnatural amino acids.

Unnatural amino acids (UAAs) are chiral amines with high application potential in drug discovery and synthesis of other valuable chemicals. Biocatalysis offers the possibility to synthesise novel optically pure UAAs with different physical and chemical properties. While the biocatalytic potential of transaminases in the synthesis of UAAs has been demonstrated, there is still a need to improve the activity with non-native substrates and to understand which amino acids residues are important for activity with these UAAs. Using a rational design approach, six variants of Chromobacterium violaceum DSM30191 transaminase (CV_TA) carrying a single and one variant carrying two substitutions were generated. Among the variants with a single substitution, CV_Y168F showed a 2 to 2.6-fold increased affinity for 2-oxooctanoic acid (2-OOA) and 3-oxobutyric acid (3-OBA) methyl ester used to synthesise an α- and ß-UAA. Analysis of the first half of the transaminase reaction showed no change in the activity with the donor (S)-1-phenylethylamine. The combination of W60C and Y168F substitutions improved the CV_TA affinity for 2-OOA 10-fold compared to the wild type. Other substitutions showed no change, or reduced activity with the tested substrates. Our findings provide structural information on CV_TA and demonstrate the potential of rational design for biosynthesis of UAAs.

Biosynthesis of 2-aminooctanoic acid and its use to terminally modify a lactoferricin B peptide derivative for improved antimicrobial activity.

Terminal modification of peptides is frequently used to improve their hydrophobicity. While N-terminal modification with fatty acids (lipidation) has been reported previously, C-terminal lipidation is limited as it requires the use of linkers. Here we report the use of a biocatalyst for the production of an unnatural fatty amino acid, (S)-2-aminooctanoic acid (2-AOA) with enantiomeric excess > 98% ee and the subsequent use of 2-AOA to modify and improve the activity of an antimicrobial peptide. A transaminase originating from Chromobacterium violaceum was employed with a conversion efficiency 52-80% depending on the ratio of amino group donor to acceptor. 2-AOA is a fatty acid with amino functionality, which allowed direct C- and N-terminal conjugation respectively to an antimicrobial peptide (AMP) derived from lactoferricin B. The antibacterial activity of the modified peptides was improved by up to 16-fold. Furthermore, minimal inhibitory concentrations (MIC) of C-terminally modified peptide were always lower than N-terminally conjugated peptides. The C-terminally modified peptide exhibited MIC values of 25 µg/ml for Escherichia coli, 50 µg/ml for Bacillus subtilis, 100 µg/ml for Salmonella typhimurium, 200 µg/ml for Pseudomonas aeruginosa and 400 µg/ml for Staphylococcus aureus. The C-terminally modified peptide was the only peptide tested that showed complete inhibition of growth of S. aureus.