Non-classical HLA class I molecules and their potential role in viral infections.

Human Leukocyte Antigens (HLA) are classified in three different classes I, II and III, and represent the key mediators of immune responses, self-tolerance development and pathogen recognition. Among them, non-classical subtypes (HLA-Ib), e.g. HLA-E and HLA-G, are characterize by tolerogenic functions that are often exploited by viruses to evade the host immune responses. In this perspective, we will review the main current data referred to HLA-G and HLA-E and viral infections, as well as the impact on immune response. Data were selected following eligibility criteria accordingly to the reviewed topic. We used a set of electronic databases (Medline/PubMed, Scopus, Web of Sciences (WOS), Cochrane library) for a systematic search until November 2022 using MeSH keywords/terms (i.e. HLA, HLA-G, HLA-E, viral infection, SARS-CoV-2, etc.…). Recent studies support the involvement of non-classical molecules, such as HLA-E and HLA-G, in the control of viral infection. On one side, viruses exploit HLA-G and HLA-E molecule to control host immune activation. On the other side, the expression of these molecules might control the inflammatory condition generated by viral infections. Hence, this review has the aim to summarize the state of art of literature about the modulation of these non-classical HLA-I molecules, to provide a general overview of the new strategies of viral immune system regulation to counteract immune defenses.

SARS-CoV-2 viral entry and replication is impaired in Cystic Fibrosis airways due to ACE2 downregulation.

As an inherited disorder characterized by severe pulmonary disease, cystic fibrosis could be considered a comorbidity for coronavirus disease 2019. Instead, current clinical evidence seems to be heading in the opposite direction. To clarify whether host factors expressed by the Cystic Fibrosis epithelia may influence coronavirus disease 2019 progression, here we describe the expression of SARS-CoV-2 receptors in primary airway epithelial cells. We show that angiotensin converting enzyme 2 (ACE2) expression and localization are regulated by Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Consistently, our results indicate that dysfunctional CFTR channels alter susceptibility to SARS-CoV-2 infection, resulting in reduced viral entry and replication in Cystic Fibrosis cells. Depending on the pattern of ACE2 expression, the SARS-CoV-2 spike (S) protein induced high levels of Interleukin 6 in healthy donor-derived primary airway epithelial cells, but a very weak response in primary Cystic Fibrosis cells. Collectively, these data support that Cystic Fibrosis condition may be at least partially protecting from SARS-CoV-2 infection.

Cell cycle block by p53 activation reduces SARS-CoV-2 release in infected alveolar basal epithelial A549-hACE2 cells.

SARS-CoV viruses have been shown to downregulate cellular events that control antiviral defenses. They adopt several strategies to silence p53, key molecule for cell homeostasis and immune control, indicating that p53 has a central role in controlling their proliferation in the host. Specific actions are the stabilization of its inhibitor, MDM2, and the interference with its transcriptional activity. The aim of our work was to evaluate a new approach against SARS-CoV-2 by using MDM2 inhibitors to raise p53 levels and activate p53-dependent pathways, therefore leading to cell cycle inhibition. Experimental setting was performed in the alveolar basal epithelial cell line A549-hACE2, expressing high level of ACE2 receptor, to allow virus entry, as well as p53 wild-type. Cells were treated with several concentrations of Nutlin-3 or RG-7112, two known MDM2 inhibitors, for the instauration of a cell cycle block steady-state condition before and during SARS-CoV-2 infection, and for the evaluation of p53 activation and impact on virus release and related innate immune events. The results indicated an efficient cell cycle block with inhibition of the virion release and a significant inhibition of IL-6, NF-kB and IFN-λ expression. These data suggest that p53 is an efficient target for new therapies against the virus and that MDM2 inhibitors deserve to be further investigated in this field.

Competition for dominance within replicating quasispecies during prolonged SARS-CoV-2 infection in an immunocompromised host.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) emerge for their capability to better adapt to the human host aimed and enhance human-to-human transmission. Mutations in spike largely contributed to adaptation. Viral persistence is a prerequisite for intra-host virus evolution, and this likely occurred in immunocompromised patients who allow intra-host long-term viral replication. The underlying mechanism leading to the emergence of variants during viral persistence in the immunocompromised host is still unknown. Here, we show the existence of an ensemble of minor mutants in the early biological samples obtained from an immunocompromised patient and their dynamic interplay with the master mutant during a persistent and productive long-term infection. In particular, after 222 days of active viral replication, the original master mutant, named MB610, was replaced by a minor quasispecies (MB61222) expressing two critical mutations in spike, namely Q493K and N501T. Isolation of the two viruses allowed us to show that MB61222 entry into target cells occurred mainly by the fusion at the plasma membrane (PM), whereas endocytosis characterized the entry mechanism used by MB610. Interestingly, coinfection of two human cell lines of different origin with the SARS-CoV-2 isolates highlighted the early and dramatic predominance of MB61222 over MB610 replication. This finding may be explained by a faster replicative activity of MB61222 as compared to MB610 as well as by the capability of MB61222 to induce peculiar viral RNA-sensing mechanisms leading to an increased production of interferons (IFNs) and, in particular, of IFN-induced transmembrane protein 1 (IFITM1) and IFITM2. Indeed, it has been recently shown that IFITM2 is able to restrict SARS-CoV-2 entry occurring by endocytosis. In this regard, MB61222 may escape the antiviral activity of IFITMs by using the PM fusion pathway for entry into the target cell, whereas MB610 cannot escape this host antiviral response during MB61222 coinfection, since it has endocytosis as the main pathway of entry. Altogether, our data support the evidence of quasispecies fighting for host dominance by taking benefit from the cell machinery to restrict the productive infection of competitors in the viral ensemble. This finding may explain, at least in part, the extraordinary rapid worldwide turnover of VOCs that use the PM fusion pathway to enter into target cells over the original pandemic strain.

Aerosols modification with H2O2 reduces airborne contamination by dental handpieces.

Objective: We designed an in vitro study to evaluate the efficiency of an 0.5 vol% hydrogen peroxide-based spray in reducing Coronavirus 229E spread during a conventional dental procedure. Methods: A class III cabinet-like chamber was custom-built, using phantoms for both patient and operator. A suspension of HCoV-229E in artificial saliva having a similar viral load to SARS-CoV-2 asymptomatic patients was inoculated inside the patient's phantom mouth. A 10 s-lasting dental procedure was performed using an aerosol-generating air-turbine, with or without high-volume evacuation (HVE). The effect of 0.5 vol% H2O2 cooling spray in reducing viral loads was tested. Viral presence on the operator phantom was assessed by Real-Time quantitative PCR on the mask's outer surface, on the phantom's forehead, and inside its mouth. Results: When the H2O2 cooling spray was used, as compared to the conventional spray, viral loads were significantly lower on all tested sites, falling below the detection limit. Viral loads did not significantly change in any tested site when HVE was used. Conclusion: The use of 0.5 vol% H2O2 cooling spray by dental handpieces drastically reduced the possibility of coronaviruses spread during aerosol-generating dental procedures. This strategy deserves further consideration among the preventive measures to be adopted during the SARS-CoV-2 pandemic.

Viral Load Difference between Symptomatic and Asymptomatic COVID-19 Patients: Systematic Review and Meta-Analysis.

We conducted a systematic review and meta-analysis to investigate the possible difference in the SARS-CoV-2 viral load between asymptomatic and symptomatic COVID-19 patients. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed in abstracting data and assessing validity. We searched MEDLINE, Scopus, Web of Science and Google Scholar for all investigations in the English language, reporting data on the threshold cycle (Ct) from real-time RT-PCR assays for the RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) SARS-CoV-2 genes in asymptomatic and symptomatic COVID-19 patients. Results: Overall, 703 COVID-19 patients (553 symptomatic and 150 asymptomatic) were analyzed. Five investigations reported the mean age of patients, evidencing that asymptomatic patients were younger than symptomatic patients (34.0 vs. 40.3 years, respectively). Pooled data regarding the levels of expression of the RdRp gene revealed no significant difference between symptomatic and asymptomatic subjects. Similarly, no differences were observed comparing the mean Ct values for the E and N genes. Based on real-time RT-PCR data, no differences exist in the viral load between symptomatic and asymptomatic COVID-19 subjects considering Ct values for RdRp, E and N genes' expression. Asymptomatic subjects may represent a reservoir of the infection and significantly contribute to the maintenance of the pandemic.

Relevance of VEGF and CD147 in different SARS-CoV-2 positive digestive tracts characterized by thrombotic damage.

Several evidence suggests that, in addition to the respiratory tract, also the gastrointestinal tract is a main site of severe acute respiratory syndrome CoronaVirus 2 (SARS-CoV-2) infection, as an example of a multi-organ vascular damage, likely associated with poor prognosis. To assess mechanisms SARS-CoV-2 responsible of tissue infection and vascular injury, correlating with thrombotic damage, specimens of the digestive tract positive for SARS-CoV-2 nucleocapsid protein were analyzed deriving from three patients, negative to naso-oro-pharyngeal swab for SARS-CoV-2. These COVID-19-negative patients came to clinical observation due to urgent abdominal surgery that removed different sections of the digestive tract after thrombotic events. Immunohistochemical for the expression of SARS-CoV-2 combined with a panel of SARS-CoV-2 related proteins angiotensin-converting enzyme 2 receptor, cluster of differentiation 147 (CD147), human leukocyte antigen-G (HLA-G), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 was performed. Tissue samples were also evaluated by electron microscopy for ultrastructural virus localization and cell characterization. The damage of the tissue was assessed by ultrastructural analysis. It has been observed that CD147 expression levels correlate with SARS-CoV-2 infection extent, vascular damage and an increased expression of VEGF and thrombosis. The confirmation of CD147 co-localization with SARS-CoV-2 Spike protein binding on gastrointestinal tissues and the reduction of the infection level in intestinal epithelial cells after CD147 neutralization, suggest CD147 as a possible key factor for viral susceptibility of gastrointestinal tissue. The presence of SARS-CoV-2 infection of gastrointestinal tissue might be consequently implicated in abdominal thrombosis, where VEGF might mediate the vascular damage.

Transparent Polymeric Formulations Effective against SARS-CoV-2 Infection.

The main route of the transmission of the SARS-CoV-2 virus is through airborne small aerosol particles containing viable virus as well as through droplets transmitted between people within close proximity. Transmission via contaminated surfaces has also been recognized as an important route for the spread of SARS-CoV-2 coronavirus. Among a variety of antimicrobial agents currently in use, polymers represent a class of biocides that have become increasingly important as an alternative to existing biocidal approaches. Two transparent polymeric compounds, containing silver and benzalkonium ions electrostatically bound to a polystyrene sulfonate backbone, were synthesized, through simple procedures, and evaluated for their antimicrobial properties against Gram-positive and Gram-negative bacteria and Candida albicans (ISO EN 1276) and for their antiviral activity toward 229E and SARS-CoV-2 coronaviruses (ISO UNI EN 14476:2019). The results showed that the two tested formulations are able to inhibit the growth of (1.5-5.5) × 1011 CFU of Gram-positive bacteria, Gram-negative bacteria, and of the fungal species Candida albicans. Both compounds were able to control the 229E and SARS-CoV-2 infection of a target cell in a time contact of 5 min, with a virucidal effect from 24 to 72 h postinfection, according to the European Medicines Agency (EMA) guidelines, where a product is considered virucidal upon achieving a reduction of 4 logarithms. This study observed a decrease of more than 5 logarithms, which implies that these formulations are likely ideal candidates for the realization of transparent surface coatings that are capable of maintaining remarkable antibacterial activity and SARS-CoV-2 antiviral properties over time.

Increased sHLA-G Is Associated with Improved COVID-19 Outcome and Reduced Neutrophil Adhesion.

Human leukocyte antigen (HLA) is a group of molecules involved in inflammatory and infective responses. We evaluated blood sHLA-E and sHLA-G levels in hospitalized COVID-19 patients with respiratory failure and their relationship with clinical evolution, changes in endothelial activation biomarker profile, and neutrophil adhesion. sHLA-E, sHLA-G, and endothelial activation biomarkers were quantified by ELISA assay in plasma samples. Neutrophil adhesion to endothelium was assessed in the presence/absence of patients' plasma samples. At admission, plasma levels of sHLA-G and sHLA-E were significantly higher in COVID-19 patients with respiratory failure compared to controls. COVID-19 clinical improvement was associated with increased sHLA-G plasma levels. In COVID-19, but not in control patients, an inverse correlation was found between serum sICAM-1 and E-selectin levels and plasma sHLA-G values. The in vitro analysis of activated endothelial cells confirmed the ability of HLA-G molecules to control sICAM-1 and sE-selectin expression via CD160 interaction and FGF2 induction and consequently neutrophil adhesion. We suggest a potential role for sHLA-G in improving COVID-19 patients' clinical condition related to the control of neutrophil adhesion to activated endothelium.

TLR3 and TLR7 RNA Sensor Activation during SARS-CoV-2 Infection.

(1) Background: Acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent for the coronavirus disease (COVID-19) that has led to a pandemic that began in March 2020. The role of the SARS-CoV-2 components on innate and adaptive immunity is still unknown. We investigated the possible implication of pathogen-associated molecular patterns (PAMPs)-pattern recognition receptors (PRRs) interaction. (2) Methods: We infected Calu-3/MRC-5 multicellular spheroids (MTCSs) with a SARS-CoV-2 clinical strain and evaluated the activation of RNA sensors, transcription factors, and cytokines/interferons (IFN) secretion, by quantitative real-time PCR, immunofluorescence, and ELISA. (3) Results: Our results showed that the SARS-CoV-2 infection of Calu-3/MRC-5 multicellular spheroids induced the activation of the TLR3 and TLR7 RNA sensor pathways. In particular, TLR3 might act via IRF3, producing interleukin (IL)-1α, IL-1ß, IL-4, IL-6, and IFN-α and IFN-ß, during the first 24 h post-infection. Then, TLR3 activates the NFκB transduction pathway, leading to pro-inflammatory cytokine secretion. Conversely, TLR7 seems to mainly act via NFκB, inducing type 1 IFN, IFN-γ, and IFN-λ3, starting from the 48 h post-infection. (4) Conclusion: We showed that both TLR3 and TLR7 are involved in the control of innate immunity during lung SARS-CoV-2 infection. The activation of TLRs induced pro-inflammatory cytokines, such as IL-1α, IL-1ß, IL-4, and IL-6, as well as interferons. TLRs could be a potential target in controlling the infection in the early stages of the disease.

COVID-19 Ocular Prophylaxis: The Potential Role of Ozonated-Oils in Liposome Eyedrop Gel.

Purpose: To assess whether ozonated-oil in liposome eyedrop gel (OED) could be used to prevent the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection in an in vitro infection model. Methods: First, we tested the efficacy of OED on in vitro cell regeneration and dry eye resolution in human corneal epithelial cells (hCE-2). Second, we assessed the in vitro anti-SARS-CoV-2 infection efficacy of OED using Vero E6 cells. Tissues were examined to assess different parameters: morphology, histology, and mRNA expression at 24 hours after treatment. Results: OED could restore 50% of the scratch in the monolayer of hCE-2 cells in vitro compared with the 25% obtained with phosphate-buffered saline solution (PBS). At 24 hours after treatment with OED, the number of microvilli and the mucin network were restored, as observed using scanning electron microscopy. In Vero E6 cells infected with a primary SARS-CoV-2 strain and treated with OED two times/day, viral replication was found to be inhibited, with a 70-fold reduction observed at 72 hours after infection compared with that under the untreated and PBS-treated conditions. Conclusions: SARS-CoV-2 transmission through the ocular surface should not be ignored. Although the prevalence of coronavirus disease 2019 conjunctivitis infection is low, the need for a barrier to prevent possible viral infection is warranted. OED treatment may prevent the risk of SARS-CoV-2 infection after 72 hours of twice-daily applications. Translational Relevance: Dry eye condition might be a risk factor for SARS-CoV-2 infection and OED treatment may have a preventive role.

Efficacy of personal protective equipment against coronavirus transmission via dental handpieces.

BACKGROUND: This laboratory study was done to evaluate the efficacy of personal protective equipment (PPE) and high-volume evacuation (HVE) against the spread of human coronavirus type 229E (HCoV-229E) during a standard dental procedure. METHODS: Patient and operator manikins were used to recreate a dental setting inside a custom-built class III cabinet-like chamber. The mouth of the patient manikin was inoculated with an HCoV-229E suspension, the viral load of which was similar to that of asymptomatic people infected with severe acute respiratory syndrome coronavirus 2. The dental procedure was performed with an air turbine handpiece and HVE for 10 seconds. The efficacy of surgical masks, N95 (filtering facepiece class 2) and filtering facepiece class 3 respirators, and face shields was tested via quantitative real-time polymerase chain reaction. RESULTS: The wide surface on which the inoculum was spread caused low contamination. Over the external surfaces of masks and respirators, when a face shield was not worn, viral loads ranged from 1.2 through 1.4 log10 mean gene copies per cm2. When the shield was worn, viral loads dropped below the detection limit (< 0.317 log10 gene copies/cm2) for all PPE. On the operator's forehead, viral loads were 0.6 through 0.8 log10 gene copies/cm2. Inside the operator manikin's mouth, viral loads were under the detection limit when using any PPE, with or without the shield. HVE did not significantly change viral loads. CONCLUSIONS: All PPE combinations significantly reduced viral loads in the operator manikin's mouth to below the detection limit, but HVE did not decrease viral contamination. PRACTICAL IMPLICATIONS: Although caution is suggested when removing and disposing of PPE to avoid self-contamination, the combination of PPE and face shields drastically decreases the risk of transmitting human coronavirus during aerosol-generating dental procedures.

SARS-CoV-2 Spike 1 Protein Controls Natural Killer Cell Activation via the HLA-E/NKG2A Pathway.

Natural killer cells are important in the control of viral infections. However, the role of NK cells during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has previously not been identified. Peripheral blood NK cells from SARS-CoV and SARS-CoV-2 naïve subjects were evaluated for their activation, degranulation, and interferon-gamma expression in the presence of SARS-CoV and SARS-CoV-2 spike proteins. K562 and lung epithelial cells were transfected with spike proteins and co-cultured with NK cells. The analysis was performed by flow cytometry and immune fluorescence. SARS-CoV and SARS-CoV-2 spike proteins did not alter NK cell activation in a K562 in vitro model. On the contrary, SARS-CoV-2 spike 1 protein (SP1) intracellular expression by lung epithelial cells resulted in NK cell-reduced degranulation. Further experiments revealed a concomitant induction of HLA-E expression on the surface of lung epithelial cells and the recognition of an SP1-derived HLA-E-binding peptide. Simultaneously, there was increased modulation of the inhibitory receptor NKG2A/CD94 on NK cells when SP1 was expressed in lung epithelial cells. We ruled out the GATA3 transcription factor as being responsible for HLA-E increased levels and HLA-E/NKG2A interaction as implicated in NK cell exhaustion. We show for the first time that NK cells are affected by SP1 expression in lung epithelial cells via HLA-E/NKG2A interaction. The resulting NK cells' exhaustion might contribute to immunopathogenesis in SARS-CoV-2 infection.