Prediction of SARS-CoV-2 Omicron Variant Immunogenicity, Immune Escape and Pathogenicity, through Analysis of Spike Protein-specific Core Unique Peptides

The recently discovered Omicron variant of the SARS-CoV-2 corona virus has raised a new, global, awareness, since it is considered as a new variant of concern from all major health organizations, including WHO and ECDC. Omicron variant is characterized by 30 amino acid changes, three small deletions and one small insertion in the Spike protein. In this study, we have identified the Core Unique Peptides (CrUPs) that reside exclusively in the Omicron variant of Spike protein and are absent from the human proteome, thus creating a new dataset of peptides named as C/H-CrUPs. Furthermore, we have analyzed their protein locations and compared them with the respective ones of Alpha and Delta SARS-CoV-2 variants. In Omicron, 115 C/H-CrUPs were generated and 119 C/H-CrUPs were lost, almost four times as many compared to the other two variants. From position 440 to position 508, at the Receptor Binding Motif (RBM), 8 mutations were detected, resulting in the construction of 28 novel C/H-CrUPs. Most importantly, in Omicron variant, new C/H-CrUPs carrying two or three mutant amino acids were produced, as a consequence of the accumulation of multiple mutations in the RBM. Remarkably, these Omicron-derived C/H-CrUPs that bear several mutated amino acids could not be recognized in any other viral Spike variant. We suggest that virus binding to the ACE2 receptor is facilitated by the herein identified C/H-CrUPs in contact point mutations and Spike-cleavage sites, while the immunoregulatory NF9 peptide is not detectably affected. Taken together, our findings indicate that Omicron variant contains intrinsic abilities to escape immune-system attack, while its mutations can mediate strong viral binding to the ACE2 receptor, leading to highly efficient fusion of the virus to the target cell. However, the intact NF9 peptide suggests that Omicron exhibits reduced pathogenicity compared to Delta variant.

Evolutionary pathways to SARS-CoV-2 resistance are opened and closed by epistasis acting on ACE2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infects a broader range of mammalian species than previously predicted, binding a diversity of angiotensin converting enzyme 2 (ACE2) orthologs despite extensive sequence divergence. Within this sequence degeneracy, we identify a rare sequence combination capable of conferring SARS-CoV-2 resistance. We demonstrate that this sequence was likely unattainable during human evolution due to deleterious effects on ACE2 carboxypeptidase activity, which has vasodilatory and cardioprotective functions in vivo. Across the 25 ACE2 sites implicated in viral binding, we identify 6 amino acid substitutions unique to mouse—one of the only known mammalian species immune to SARS-CoV-2. Substituting human variants at these positions is sufficient to confer binding of the SARS-CoV-2 S protein to mouse ACE2, facilitating cellular infection. Conversely, substituting mouse variants into either human or dog ACE2 abolishes viral binding, diminishing cellular infection. However, these same substitutions decrease human ACE2 activity by 50% and are predicted as pathogenic, consistent with the extreme rarity of human polymorphisms at these sites. This trade-off can be avoided, however, depending on genetic background; if substituted simultaneously, these same mutations have no deleterious effect on dog ACE2 nor that of the rodent ancestor estimated to exist 70 million years ago. This genetic contingency (epistasis) may have therefore opened the road to resistance for some species, while making humans susceptible to viruses that use these ACE2 surfaces for binding, as does SARS-CoV-2.

12-month SARS-CoV-2 antibody persistency in a Tyrolean COVID-19 cohort

Summary Background Short-term antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been shown previously. The further development remains to be determined. Methods We prospectively followed 29 coronavirus disease 2019 cases, mean age 44 ± 13.2 years. Except for one participant in whom rheumatoid arthritis existed, all other cases were previously healthy. We determined anti-viral binding antibodies at 2–10 weeks, 3 months, 6 months, and 12 months after disease onset as well as neutralizing antibodies (NAb) against wild type at 6 and 12 months and the B.1.1.7 and B.1.351 variants at month 12. Three binding antibody assays were used, targeting the nucleocapsid protein (NCP), the S1 subunit of the spike protein, and the receptor binding domain (RBD). Results Antibodies to the RBD persisted for 12 months in all cases with increasing concentrations, whereas antibodies to S1 dropped below cut-off point in 7 participants and NCP antibodies were above cut-off point in only 5 subjects at month 12. The NAb against wild type were detected in all but 2 samples at 12 months of follow-up but clearly less frequently when targeting the variants. In 5 participants who were vaccinated against COVID-19 there was a strong increase of antibodies against S1 and RBD as well as an increase of NAb titres against wild type and the variants. Conclusion There was a persisting antibody response against SARS-CoV‑2 up to 12 months after COVID-19 with declining concentrations except for RBD and a strong increase of all antibody concentrations after vaccination.

A Recombinant Adenoviral Vector With a Specific Tropism to CD4-positive Cells: a New Tool for HIV-1 Inhibition

Gene therapy can be an option to overcoming the side effects of chemotherapy and preventing the development of drug-resistant HIV viruses in HIV-infected patients. The need to develop a safe and efficient vector for gene transfer is always necessary and an appropriate option might be adenovirus (Ad). the use of Ad vectors in the gene delivery applications is limited due to the semi-specific tropism. A strategy to overcome this tropism limitation may be the modification of fiber protein domain involved in the viral binding to cells. Therefore, we designed an Ad5 vector with a specific tropism to CD4+ cells containing an expression system limited to HIV-infected cells. We replaced the knob region of Ad5 fiber protein with the extracellular region of HIV-1 envelope. We also used a specific Tat-inducible promoter to express two anti-HIV-1 shRNAs. Tropism of recombinant Ad5 was assayed by comparison of shRNA expression level in CEM and PBMC cells (as CD4+ cells) and HEK293 cells (as CD4- cells). HIV-1 inhibition was assayed by determination of p24 antigen in the HIV-infected CEM cells transduced with the recombinant Ad5 vector. Our results showed that shRNA expression was significantly higher in CEM and PBMC cells than HEK293 cells when were transduced with recombinant Ad5 vector. This new Ad5 vector also inhibited HIV-1 proliferation in a Tat-inducible manner. Our new recombinant Ad5 vector has a specific tropism to CD4-positive cells that can effectively suppress the HIV-1 replication.

Mutating novel interaction sites in NRP1 reduces SARS-CoV-2 spike protein internalization

The global pandemic of Coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 has become a severe global health problem because of its rapid spread. Both angiotensin-converting enzyme 2 and neuropilin 1 provide initial viral binding sites for SARS-CoV-2. Here, we show that three cysteine residues located in a1/a2 and b1 domains of neuropilin 1 are necessary for SARS-CoV-2 spike protein internalization in human cells. Mutating cysteines C82, C104, and C147 altered neuropilin 1 stability and binding ability as well as cellular internalization and lysosomal translocation of the spike protein. This resulted in up to 4 times reduction in spike protein load in cells for the original, alpha, and delta SARS-CoV-2 variants even in the presence of the endogenous angiotensin-converting enzyme 2 receptors. Transcriptome analysis of cells transfected with mutated NRP1 revealed significantly reduced expression of genes involved in viral infection and replication, including eight members of the ribosomal protein L, ten members of ribosomal protein S, and five members of the proteasome β subunit family proteins. We also observed higher expression of genes involved in the suppression of inflammation and endoplasmic reticulum-associated degradation. These observations suggest that these cysteines offer viable targets for therapies against COVID-19.

Preliminary Evidence for IL-10-Induced ACE2 mRNA Expression in Lung-Derived and Endothelial Cells: Implications for SARS-Cov-2 ARDS Pathogenesis

Angiotensin-converting enzyme 2 (ACE2) is a receptor for the spike protein of SARS-COV-2 that allows viral binding and entry and is expressed on the surface of several pulmonary and non-pulmonary cell types, with induction of a “cytokine storm” upon binding. Other cell types present the receptor and can be infected, including cardiac, renal, intestinal, and endothelial cells. High ACE2 levels protect from inflammation. Despite the relevance of ACE2 levels in COVID-19 pathogenesis, experimental studies to comprehensively address the question of ACE2 regulations are still limited. A relevant observation from the clinic is that, besides the pro-inflammatory cytokines, such as IL-6 and IL-1β, the anti-inflammatory cytokine IL-10 is also elevated in worse prognosis patients. This could represent somehow a “danger signal”, an alarmin from the host organism, given the immuno-regulatory properties of the cytokine. Here, we investigated whether IL-10 could increase ACE2 expression in the lung-derived Calu-3 cell line. We provided preliminary evidence of ACE2 mRNA increase in cells of lung origin in vitro, following IL-10 treatment. Endothelial cell infection by SARS-COV-2 is associated with vasculitis, thromboembolism, and disseminated intravascular coagulation. We confirmed ACE2 expression enhancement by IL-10 treatment also on endothelial cells. The sartans (olmesartan and losartan) showed non-statistically significant ACE2 modulation in Calu-3 and endothelial cells, as compared to untreated control cells. We observed that the antidiabetic biguanide metformin, a putative anti-inflammatory agent, also upregulates ACE2 expression in Calu-3 and endothelial cells. We hypothesized that IL-10 could be a danger signal, and its elevation could possibly represent a feedback mechanism fighting inflammation. Although further confirmatory studies are required, inducing IL-10 upregulation could be clinically relevant in COVID-19-associated acute respiratory distress syndrome (ARDS) and vasculitis, by reinforcing ACE2 levels.

A novel hamster model of SARS-CoV-2 respiratory infection using a pseudotyped virus

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a biosafety level (BSL)-3 pathogen; therefore, its research environment is strictly limited. Pseudotyped viruses that mimic SARS-CoV-2 have been widely used for in vitro evaluation because they are available in BSL-2 containment laboratories; however, in vivo application is inadequate. Therefore, animal models that can be instigated with animal BSL-2 will increase opportunities for in vivo evaluations. Methods: Hamsters (6- to 10-week-old males) were intratracheally inoculated with luciferase-expressing vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudotyped virus. The lungs were harvested 24 h after inoculation, and luminescence was measured using an in vivo imaging system. Results: Lung luminescence after inoculation with the SARS-CoV-2 pseudotyped virus increased in a dose-dependent manner. VSV-G (envelope [G]) pseudotyped virus also induced luminescence; however, a 100-fold concentration was required to reach a level similar to that of the SARS-CoV-2 pseudotyped virus. Conclusions: The SARS-CoV-2 pseudotyped virus is applicable to SARS-CoV-2 respiratory infections in a hamster model. Because of the single-round infectious virus, the model can be used to study the steps from viral binding to entry, which will be useful for future research regarding SARS-CoV-2 entry without using live SARS-CoV-2 or transgenic animals.

Development of a novel, pan-variant aerosol intervention for COVID-19

To develop a universal strategy to block SARS-CoV-2 cellular entry and infection represents a central aim for effective COVID-19 therapy. The growing impact of emerging variants of concern increases the urgency for development of effective interventions. Since ACE2 is the critical SARS-CoV-2 receptor and all tested variants bind to ACE2, some even at much increased affinity (see accompanying paper), we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here we show that intranasal administration of APN01 in a mouse model of SARS-CoV-2 infection dramatically reduced weight loss and prevented animal death. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers can now be initiated, with subsequent Phase II testing in individuals with SARS-CoV-2 infection. This strategy could be used to develop a viable and rapidly actionable therapy to prevent and treat COVID-19, against all current and future SARS-CoV-2 variants.

O-Glycosylation Landscapes of SARS-CoV-2 Spike Proteins

The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized through high-resolution mass spectrometry. Following digestion with trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the GalNAc-type O-glycosylation pattern of S proteins, including O-glycosites and the six most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 255 intact O-glycopeptides composed of 50 peptides sequences and 43 O-glycosites were discovered by higher energy collision-induced dissociation (HCD), and three O-glycosites were confidently identified by electron transfer/higher energy collision-induced dissociation (EThcD) in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex (1). In contrast, in the human cell-expressed S protein S1 subunit, 407 intact O-glycopeptides composed of 34 peptides sequences and 30 O-glycosites were discovered by HCD, and 11 O-glycosites were unambiguously assigned by EThcD. However, the measurement of O-glycosylation occupancy hasn’t been made. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex (1)NeuAc (1) and HexNAc(1)Hex (1)NeuAc (2). Our results reveal that the SARS-CoV-2 S protein is an O-glycoprotein; the O-glycosites and O-glycan compositions vary with the host cell type. These comprehensive O-glycosylation landscapes of the S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.