Characterization of SARS-CoV-2 Entry Factors' Expression in Corneal and Limbal Tissues of Adult Human Donors Aged from 58 to 85.

Purpose: Recent studies have shown the presence of SARS-CoV-2 entry factors on the ocular surface, identifying the eye as an additional entry route for the virus. Moreover, the coexpression of angiotensin-converting enzyme 2 (ACE2) with other SARS-CoV-2 entry factors [transmembrane protease serine 2 (TMPRSS2), transmembrane protease serine 4 (TMPRSS4), and dipeptidyl peptidase-4 (DPP4)] facilitates the virus infection. Methods: Here, we performed a study over 10 adult corneal and limbal tissues from human donors, both male and female between 58 and 85 years of age. Some of the main virus entry factors were analyzed and their expression was quantified and correlated with the age and sex of the donors through western blot. The receptors' localization was investigated through immunofluorescence. Results: Immunofluorescence confirmed the localization of ACE2 and TMPRSS2 on the ocular surface and showed, for the first time, the localization of TMPRSS4 and DPP4 in limbal and corneal epithelial superficial cells. The quantitative analysis showed that the expression of SARS-CoV-2 entry factors on corneal and limbal cells is likely to be modulated in an age-dependent manner, in agreement with the increased susceptibility to COVID-19 in the elderly. Moreover, we found a relationship between the expression of TMPRSS proteases with the activation state of limbal cells in 80-year-old donors. Conclusion: This study provides information on the expression of SARS-CoV-2 entry factors on the ocular surface of 10 adult human donors and is a first observation of a possible age-dependent modulation on corneal and limbal tissues. Our data pave the way to further investigate the susceptibility to the infection through the ocular surface in the elderly.

Expression and characterization of SARS-CoV-2 spike proteins.

The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.

Serial co-expression analysis of host factors from SARS-CoV viruses highly converges with former high-throughput screenings and proposes key regulators.

The current genomics era is bringing an unprecedented growth in the amount of gene expression data, only comparable to the exponential growth of sequences in databases during the last decades. This data allow the design of secondary analyses that take advantage of this information to create new knowledge. One of these feasible analyses is the evaluation of the expression level for a gene through a series of different conditions or cell types. Based on this idea, we have developed Automatic and Serial Analysis of CO-expression, which performs expression profiles for a given gene along hundreds of heterogeneous and normalized transcriptomics experiments and discover other genes that show either a similar or an inverse behavior. It might help to discover co-regulated genes, and common transcriptional regulators in any biological model. The present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is an opportunity to test this novel approach due to the wealth of data that are being generated, which could be used for validating results. Thus, we have identified 35 host factors in the literature putatively involved in the infectious cycle of SARS-CoV viruses and searched for genes tightly co-expressed with them. We have found 1899 co-expressed genes whose assigned functions are strongly related to viral cycles. Moreover, this set of genes heavily overlaps with those identified by former laboratory high-throughput screenings (with P-value near 0). Our results reveal a series of common regulators, involved in immune and inflammatory responses that might be key virus targets to induce the coordinated expression of SARS-CoV-2 host factors.

Developing effective siRNAs to reduce the expression of key viral genes of COVID-19.

The COVID-19 pandemic has been raging worldwide for more than a year. Many efforts have been made to create vaccines and develop new antiviral drugs to cope with the disease. Here, we propose the application of short interfering RNAs (siRNAs) to degrade the viral genome, thus reducing viral infection. By introducing the concept of the probability of binding efficiency (PBE) and combining the secondary structures of RNA molecules, we designed 11 siRNAs that target the consensus regions of three key viral genes: the spike (S), nucleocapsid (N) and membrane (M) genes of SARS-CoV-2. The silencing efficiencies of the siRNAs were determined in human lung and endothelial cells overexpressing these viral genes. The results suggested that most of the siRNAs could significantly reduce the expression of the viral genes with inhibition rates above 50% in 24 hours. This work not only provides a strategy for designing potentially effective siRNAs against target genes but also validates several potent siRNAs that can be used in the clinical development of preventative medication for COVID-19 in the future.

The rationale for a multi-step therapeutic approach based on antivirals, drugs and nutrients with immunomodulatory activity in patients with coronavirus-SARS2-induced disease of different severities.

In December 2019, a novel human-infecting coronavirus, named Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2), was recognised to cause a pneumonia epidemic outbreak with different degrees of severity in Wuhan, Hubei Province in China. Since then, this epidemic has spread worldwide; in Europe, Italy has been involved. Effective preventive and therapeutic strategies are absolutely required to block this serious public health concern. Unfortunately, few studies about SARS-CoV-2 concerning its immunopathogenesis and treatment are available. On the basis of the assumption that the SARS-CoV-2 is genetically related to SARS-CoV (about 82 % of genome homology) and that its characteristics, like the modality of transmission or the type of the immune response it may stimulate, are still poorly known, a literature search was performed to identify the reports assessing these elements in patients with SARS-CoV-induced infection. Therefore, we have analysed: (1) the structure of SARS-CoV-2 and SARS-CoV; (2) the clinical signs and symptoms and pathogenic mechanisms observed during the development of acute respiratory syndrome and the cytokine release syndrome; (3) the modification of the cell microRNome and of the immune response in patients with SARS infection; and (4) the possible role of some fat-soluble compounds (such as vitamins A, D and E) in modulating directly or indirectly the replication ability of SARS-CoV-2 and host immune response.

Coronavirus disease-19 and fertility: viral host entry protein expression in male and female reproductive tissues.

OBJECTIVE: To identify cell types in the male and female reproductive systems at risk for SARS-CoV-2 infection because of the expression of host genes and proteins used by the virus for cell entry. DESIGN: Descriptive analysis of transcriptomic and proteomic data. SETTING: Academic research department and clinical diagnostic laboratory. PATIENT(S): Not applicable (focus was on previously generated gene and protein expression data). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Identification of cell types coexpressing the key angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) genes and proteins as well as other candidates potentially involved in SARS-CoV-2 cell entry. RESULT(S): On the basis of single-cell RNA sequencing data, coexpression of ACE2 and TMPRSS2 was not detected in testicular cells, including sperm. A subpopulation of oocytes in nonhuman primate ovarian tissue was found to express ACE2 and TMPRSS2, but coexpression was not observed in ovarian somatic cells. RNA expression of TMPRSS2 in 18 samples of human cumulus cells was shown to be low or absent. There was general agreement between publicly available bulk RNA and protein datasets in terms of ACE2 and TMPRSS2 expression patterns in testis, ovary, endometrial, and placental cells. CONCLUSION(S): These analyses suggest that SARS-CoV-2 infection is unlikely to have long-term effects on male and female reproductive function. Although the results cannot be considered definitive, they imply that procedures in which oocytes are collected and fertilized in vitro are associated with very little risk of viral transmission from gametes to embryos and may indeed have the potential to minimize exposure of susceptible reproductive cell types to infection in comparison with natural conception.