The IDentif.AI-x pandemic readiness platform: Rapid prioritization of optimized COVID-19 combination therapy regimens.

IDentif.AI-x, a clinically actionable artificial intelligence platform, was used to rapidly pinpoint and prioritize optimal combination therapies against COVID-19 by pairing a prospective, experimental validation of multi-drug efficacy on a SARS-CoV-2 live virus and Vero E6 assay with a quadratic optimization workflow. A starting pool of 12 candidate drugs developed in collaboration with a community of infectious disease clinicians was first narrowed down to a six-drug pool and then interrogated in 50 combination regimens at three dosing levels per drug, representing 729 possible combinations. IDentif.AI-x revealed EIDD-1931 to be a strong candidate upon which multiple drug combinations can be derived, and pinpointed a number of clinically actionable drug interactions, which were further reconfirmed in SARS-CoV-2 variants B.1.351 (Beta) and B.1.617.2 (Delta). IDentif.AI-x prioritized promising drug combinations for clinical translation and can be immediately adjusted and re-executed with a new pool of promising therapies in an actionable path towards rapidly optimizing combination therapy following pandemic emergence.

Polymersomes as Stable Nanocarriers for a Highly Immunogenic and Durable SARS-CoV-2 Spike Protein Subunit Vaccine.

Multiple successful vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to address the ongoing coronavirus disease 2019 (Covid-19) pandemic. In the present work, we describe a subunit vaccine based on the SARS-CoV-2 spike protein coadministered with CpG adjuvant. To enhance the immunogenicity of our formulation, both antigen and adjuvant were encapsulated with our proprietary artificial cell membrane (ACM) polymersome technology. Structurally, ACM polymersomes are self-assembling nanoscale vesicles made up of an amphiphilic block copolymer comprising poly(butadiene)-b-poly(ethylene glycol) and a cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane. Functionally, ACM polymersomes serve as delivery vehicles that are efficiently taken up by dendritic cells (DC1 and DC2), which are key initiators of the adaptive immune response. Two doses of our formulation elicit robust neutralizing antibody titers in C57BL/6 mice that persist at least 40 days. Furthermore, we confirm the presence of functional memory CD4+ and CD8+ T cells that produce T helper type 1 cytokines. This study is an important step toward the development of an efficacious vaccine in humans.

JAK inhibition reduces SARS-CoV-2 liver infectivity and modulates inflammatory responses to reduce morbidity and mortality.

Using AI, we identified baricitinib as having antiviral and anticytokine efficacy. We now show a 71% (95% CI 0.15 to 0.58) mortality benefit in 83 patients with moderate-severe SARS-CoV-2 pneumonia with few drug-induced adverse events, including a large elderly cohort (median age, 81 years). An additional 48 cases with mild-moderate pneumonia recovered uneventfully. Using organotypic 3D cultures of primary human liver cells, we demonstrate that interferon-α2 increases ACE2 expression and SARS-CoV-2 infectivity in parenchymal cells by greater than fivefold. RNA-seq reveals gene response signatures associated with platelet activation, fully inhibited by baricitinib. Using viral load quantifications and superresolution microscopy, we found that baricitinib exerts activity rapidly through the inhibition of host proteins (numb-associated kinases), uniquely among antivirals. This reveals mechanistic actions of a Janus kinase-1/2 inhibitor targeting viral entry, replication, and the cytokine storm and is associated with beneficial outcomes including in severely ill elderly patients, data that incentivize further randomized controlled trials.

SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls.

Memory T cells induced by previous pathogens can shape susceptibility to, and the clinical severity of, subsequent infections1. Little is known about the presence in humans of pre-existing memory T cells that have the potential to recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we studied T cell responses against the structural (nucleocapsid (N) protein) and non-structural (NSP7 and NSP13 of ORF1) regions of SARS-CoV-2 in individuals convalescing from coronavirus disease 2019 (COVID-19) (n = 36). In all of these individuals, we found CD4 and CD8 T cells that recognized multiple regions of the N protein. Next, we showed that patients (n = 23) who recovered from SARS (the disease associated with SARS-CoV infection) possess long-lasting memory T cells that are reactive to the N protein of SARS-CoV 17 years after the outbreak of SARS in 2003; these T cells displayed robust cross-reactivity to the N protein of SARS-CoV-2. We also detected SARS-CoV-2-specific T cells in individuals with no history of SARS, COVID-19 or contact with individuals who had SARS and/or COVID-19 (n = 37). SARS-CoV-2-specific T cells in uninfected donors exhibited a different pattern of immunodominance, and frequently targeted NSP7 and NSP13 as well as the N protein. Epitope characterization of NSP7-specific T cells showed the recognition of protein fragments that are conserved among animal betacoronaviruses but have low homology to 'common cold' human-associated coronaviruses. Thus, infection with betacoronaviruses induces multi-specific and long-lasting T cell immunity against the structural N protein. Understanding how pre-existing N- and ORF1-specific T cells that are present in the general population affect the susceptibility to and pathogenesis of SARS-CoV-2 infection is important for the management of the current COVID-19 pandemic.

Comparative Transcriptomic and Molecular Pathway Analyses of HL-CZ Human Pro-Monocytic Cells Expressing SARS-CoV-2 Spike S1, S2, NP, NSP15 and NSP16 Genes.

The ongoing COVID-19 pandemic is a clear and present threat to global public health. Research into how the causative SARS-CoV-2 virus together with its individual constituent genes and proteins interact with target host cells can facilitate the development of improved strategies to manage the acute and long-term complications of COVID-19. In this study, to better understand the biological roles of critical SARS-CoV-2 proteins, we determined and compared the host transcriptomic responses of the HL-CZ human pro-monocytic cell line upon transfection with key viral genes encoding the spike S1 subunit, S2 subunit, nucleocapsid protein (NP), NSP15 (endoribonuclease), and NSP16 (2'-O-ribose-methyltransferase). RNA sequencing followed by gene set enrichment analysis and other bioinformatics tools revealed that host genes associated with topologically incorrect protein, virus receptor activity, heat shock protein binding, endoplasmic reticulum stress, antigen processing and presentation were up-regulated in the presence of viral spike S1 expression. With spike S2 expression, pro-monocytic genes associated with the interferon-gamma-mediated signaling pathway, regulation of phosphatidylinositol 3-kinase activity, adipocytokine signaling pathway, and insulin signaling pathway were down-regulated, whereas those associated with cytokine-mediated signaling were up-regulated. The expression of NSP15 induced the up-regulation of genes associated with neutrophil degranulation, neutrophil-mediated immunity, oxidative phosphorylation, prion disease, and pathways of neurodegeneration. The expression of NSP16 resulted in the down-regulation of genes associated with S-adenosylmethionine-dependent methyltransferase activity. The expression of NP down-regulated genes associated with positive regulation of neurogenesis, nervous system development, and heart development. Taken together, the complex transcriptomic alterations arising from these viral-host gene interactions offer useful insights into host genes and their pathways that potentially contribute to SARS-CoV-2 pathogenesis.

Highly functional virus-specific cellular immune response in asymptomatic SARS-CoV-2 infection.

The efficacy of virus-specific T cells in clearing pathogens involves a fine balance between antiviral and inflammatory features. SARS-CoV-2-specific T cells in individuals who clear SARS-CoV-2 without symptoms could reveal nonpathological yet protective characteristics. We longitudinally studied SARS-CoV-2-specific T cells in a cohort of asymptomatic (n = 85) and symptomatic (n = 75) COVID-19 patients after seroconversion. We quantified T cells reactive to structural proteins (M, NP, and Spike) using ELISpot and cytokine secretion in whole blood. Frequencies of SARS-CoV-2-specific T cells were similar between asymptomatic and symptomatic individuals, but the former showed an increased IFN-γ and IL-2 production. This was associated with a proportional secretion of IL-10 and proinflammatory cytokines (IL-6, TNF-α, and IL-1ß) only in asymptomatic infection, while a disproportionate secretion of inflammatory cytokines was triggered by SARS-CoV-2-specific T cell activation in symptomatic individuals. Thus, asymptomatic SARS-CoV-2-infected individuals are not characterized by weak antiviral immunity; on the contrary, they mount a highly functional virus-specific cellular immune response.

Spike S2 Subunit: The Dark Horse in the Race for Prophylactic and Therapeutic Interventions against SARS-CoV-2.

In the midst of the unceasing COVID-19 pandemic, the identification of immunogenic epitopes in the SARS-CoV-2 spike (S) glycoprotein plays a vital role in the advancement and development of intervention strategies. S is expressed on the exterior of the SARS-CoV-2 virion and contains two subunits, namely the N-terminal S1 and C-terminal S2. It is the key element for mediating viral entry as well as a crucial antigenic determinant capable of stimulating protective immune response through elicitation of anti-SARS-CoV-2 antibodies and activation of CD4+ and CD8+ cells in COVID-19 patients. Given that S2 is highly conserved in comparison to the S1, here, we provide a review of the latest findings on the SARS-CoV-2 S2 subunit and further discuss its potential as an attractive and promising target for the development of prophylactic vaccines and therapeutic agents against COVID-19.

A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction.

A robust serological test to detect neutralizing antibodies to SARS-CoV-2 is urgently needed to determine not only the infection rate, herd immunity and predicted humoral protection, but also vaccine efficacy during clinical trials and after large-scale vaccination. The current gold standard is the conventional virus neutralization test requiring live pathogen and a biosafety level 3 laboratory. Here, we report a SARS-CoV-2 surrogate virus neutralization test that detects total immunodominant neutralizing antibodies targeting the viral spike (S) protein receptor-binding domain in an isotype- and species-independent manner. Our simple and rapid test is based on antibody-mediated blockage of the interaction between the angiotensin-converting enzyme 2 (ACE2) receptor protein and the receptor-binding domain. The test, which has been validated with two cohorts of patients with COVID-19 in two different countries, achieves 99.93% specificity and 95-100% sensitivity, and differentiates antibody responses to several human coronaviruses. The surrogate virus neutralization test does not require biosafety level 3 containment, making it broadly accessible to the wider community for both research and clinical applications.

Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2.

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.

Molecular and Biochemical Characterization of the SARS-CoV Accessory Proteins ORF8a, ORF8b and ORF8ab

A novel coronavirus was identified as the aetiological agent for the global outbreak of severe acute respiratory syndrome (SARS) at the beginning of the twenty-first century. The SARS coronavirus genome encodes for proteins that are common to all members of the coronavirus, i.e. replicase polyproteins (pp1a and pp1b) and structural proteins (spike, membrane, nucleocapsid and envelope), as well as eight accessory proteins. The accessory proteins have been designated as open reading frames (ORF) 3a, 3b, 6, 7a, 7b, 8a, 8b and 9b, and they do not show significant homology to viral proteins of other known coronaviruses. Epidemiological studies have revealed that the part of the viral genome that encodes for ORF8a and ORF8b showed major variations and the animal isolates contain an additional 29-nucleotide sequence which is absent in most of the human isolates. As a result, ORF8a and ORF8b in the human isolates become one ORF, termed ORF8ab. In this chapter, we will discuss the genetic variation in the ORF8 region, expression of ORF8a, ORF8b and ORF8ab during infection, cellular localization and posttranslational modification of ORF8a, ORF8b and ORF8ab, participation of ORF8a, ORF8b and ORF8ab in viral–viral interactions, their effects on other viral proteins and impact on viral replication and/or pathogenesis. FAU - Keng, Choong-Tat

Serological differentiation between COVID-19 and SARS infections.

In response to the coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, multiple diagnostic tests are required for acute disease diagnosis, contact tracing, monitoring asymptomatic infection rates and assessing herd immunity. While PCR remains the frontline test of choice in the acute diagnostic setting, serological tests are urgently needed. Unlike PCR tests which are highly specific, cross-reactivity is a major challenge for COVID-19 antibody tests considering there are six other coronaviruses known to infect humans. SARS-CoV is genetically related to SARS-CoV-2 sharing approximately 80% sequence identity and both belong to the species SARS related coronavirus in the genus Betacoronavirus of family Coronaviridae. We developed and compared the performance of four different serological tests to comprehensively assess the cross-reactivity between COVID-19 and SARS patient sera. There is significant cross-reactivity when N protein of either virus is used. The S1 or RBD regions from the spike (S) protein offers better specificity. Amongst the different platforms, capture ELISA performed best. We found that SARS survivors all have significant levels of antibodies remaining in their blood 17 years after infection. Anti-N antibodies waned more than anti-RBD antibodies, and the latter is known to play a more important role in providing protective immunity.

Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients.

Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID-19 infection via proposed anti-cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID-19 infection. We validated the AI-predicted biochemical inhibitory effects of baricitinib on human numb-associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID-19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS-CoV-2 viral load, inflammatory markers, and IL-6 levels. Collectively, these data support further evaluation of the anti-cytokine and anti-viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID-19 patients.

Lack of cross-neutralization by SARS patient sera towards SARS-CoV-2.

Despite initial findings indicating that SARS-CoV and SARS-CoV-2 are genetically related belonging to the same virus species and that the two viruses used the same entry receptor, angiotensin-converting enzyme 2 (ACE2), our data demonstrated that there is no detectable cross-neutralization by SARS patient sera against SARS-CoV-2. We also found that there are significant levels of neutralizing antibodies in recovered SARS patients 9-17 years after initial infection. These findings will be of significant use in guiding the development of serologic tests, formulating convalescent plasma therapy strategies, and assessing the longevity of protective immunity for SARS-related coronaviruses in general as well as vaccine efficacy.