SARS-CoV-2 Variant Delta Potently Suppresses Innate Immune Response and Evades Interferon-Activated Antiviral Responses

Delta variant of SARS-CoV-2 has caused more severe infections than its previous variants. We studied the host innate immune response to Delta, Alpha and two earlier variants to map the evolution of the recent ones. Our biochemical and transcriptomic studies reveal that Alpha and Delta have progressively evolved over the ancestral variants by silencing innate immune response, thereby limiting cytokine and chemokine production. Though Alpha silenced RLR pathway just as Delta, it failed to persistently silence the innate immune response unlike Delta. Both Alpha and Delta have evolved to resist IFN treatment while they are still susceptible to RLR activation, further highlighting the importance of RLR-mediated, IFN-independent mechanisms in restricting SARS-CoV-2. Our studies reveal that SARS-CoV-2 Delta has integrated multiple mechanisms to silence host innate immune response and evade IFN response. Deltas silent replication and sustained suppression of host innate immune response, possibly resulting in delayed or reduced intervention by the adaptive immune response, could potentially contribute to the severe symptoms and poor recovery index associated with it.

Pharmacological Activation of Autophagy Restores Cellular Homeostasis in Ultraviolet-(B)-Induced Skin Photodamage.

Ultraviolet (UV) exposure to the skin causes photo-damage and acts as the primary etiological agent in photo-carcinogenesis. UV-B exposure induces cellular damage and is the major factor challenging skin homeostasis. Autophagy allows the fundamental adaptation of cells to metabolic and oxidative stress. Cellular dysfunction has been observed in aged tissues and in toxic insults to cells undergoing stress. Conversely, promising anti-aging strategies aimed at inhibiting the mTOR pathway have been found to significantly improve the aging-related disorders. Recently, autophagy has been found to positively regulate skin homeostasis by enhancing DNA damage recognition. Here, we investigated the geno-protective roles of autophagy in UV-B-exposed primary human dermal fibroblasts (HDFs). We found that UV-B irradiation to HDFs impairs the autophagy response in a time- and intensity-independent manner. However, improving autophagy levels in HDFs with pharmacological activators regulates the UV-B-induced cellular stress by decreasing the induction of DNA photo-adducts, promoting the DNA repair process, alleviating oxidative and ER stress responses, and regulating the expression levels of key cell cycle regulatory proteins. Autophagy also prevents HDFs from UV-B-induced nuclear damage as is evident in TUNEL assay and Acridine Orange/Ethidium Bromide co-staining. Salubrinal (an eIF2α phosphatase inhibitor) relieves ER stress response in cells and also significantly alleviates DNA damage and promotes the repair process in UV-B-exposed HDFs. P62-silenced HDFs show enhanced DNA damage response and also disturb the tumor suppressor PTEN/pAKT signaling axis in UV-B-exposed HDFs whereas Atg7-silenced HDFs reveal an unexpected consequence by decreasing the UV-B-induced DNA damage. Taken together, these results suggest that interventional autophagy offers significant protection against UV-B radiation-induced photo-damage and holds great promise in devising it as a suitable therapeutic strategy against skin pathological disorders.

Suppression of Global Protein Translation in SARS-CoV-2 Infection

The relationship of SARS-CoV-2 with the host translation remains largely unexplored. Using polysome profiling of SARS-CoV-2 infected Caco2 cells, we here demonstrate that the virus induces a strong suppression of global translation by 48 hours of infection. Heavy polysome fractions displayed substantial depletion in the infected cells, indicating the loss of major translational activities in them. Further assessment of the major pathways regulating translation in multiple permissive cell lines revealed strong eIF4E dephosphorylation accompanied by Mnk1 depletion and ERK1/2 dephosphorylations. p38MAPK showed consistent activation and its inhibition lowered viral titers, indicating its importance in viral survival. mTORC1 pathway showed the most profound inhibition, indicating its potential contribution to the suppression of global translation associated with the infection. Pharmacological activation of mTORC1 caused a drop in viral titers while inhibition resulted in higher viral RNA levels, confirming a critical role of mTORC1 in regulating viral replication. Surprisingly, the infection did not cause a general suppression of 5-TOP translation, as evident from the continued expression of ribosomal proteins. Our results collectively indicate that the differential suppression of mTORC1 might allow SARS-CoV-2 to hijack translational machinery in its favor and specifically target a set of host mRNAs.

N440K variant of SARS-CoV-2 has Higher Infectious Fitness

Several variants of SARS-CoV-2 have been emerging across the globe, continuing to threaten the efforts to end COVID-19 pandemic. Recent data indicate the prevalence of variants with N440K Spike substitution in several parts of India, which is under the second wave of the pandemic. Here, we first analyze the prevalence of N440K variants within the sequences submitted from India and identify a rising trend of its spread across various clusters. We then compare the replicative fitness and infectivity of a prototype of this variant with two other previously prevalent strains. The N440K variant produced ten times higher infectious viral titers than a prevalent A2a strain, and over 1000 folds higher titers than a much less prevalent A3i strain prototype in Caco2 cells. Similar results were detected in Calu-3 cells as well, confirming the increased potency of the N440K variant. Interestingly, A3i strain showed the highest viral RNA levels, but the lowest infectious titers in the culture supernatants, indicating the absence of correlation between the RNA content and the infectivity of the sample. N440K mutation has been reported in several viral sequences across India and based on our results, we predict that the higher infectious titers achieved by N440K variant could possibly lead to its higher rate of transmission. Availability of more sequencing data in the immediate future would help understand the potential spread of this variant in more detail.

Inactivation of SARS-CoV-2 by β-propiolactone Causes Aggregation of Viral Particles and Loss of Antigenic Potential

Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated whole-virus vaccines are also considered effective. {beta}-propiolactone (BPL) is a widely used chemical inactivator of several viruses. Here, we analyze various concentrations of BPL to effectively inactivate SARS-CoV-2 and their effects on the biochemical properties of the virion particles. BPL at 1:2000 (v/v) concentrations effectively inactivated SARS-CoV-2. However, higher BPL concentrations resulted in the loss of both protein content as well as the antigenic integrity of the structural proteins. Higher concentrations also caused substantial aggregation of the virion particles possibly causing undesirable outcomes including a potential immune escape by infectious virions, and a loss in antigenic potential. We also identify that the viral RNA content in the culture supernatants can be a direct indicator of their antigenic content. Our findings may have important implications in the vaccine and antisera industry during COVID-19 pandemic.

Development of Equine Immunoglobulin Fragment F(ab')2 with High Neutralizing Capability against SARS-CoV-2

The ongoing pandemic, COVID-19, caused by SARS-CoV-2 has taken the world, and especially the scientific community by storm. While vaccines are being introduced into the market, there is also a pressing need to find potential drugs and therapeutic modules. Remdesivir is one of the antivirals currently being used with a limited window of action. As more drugs are being vetted, passive immunotherapy in the form of neutralizing antibodies can provide immediate action to combat the increasing numbers of COVID-positive cases. Herein, we demonstrate that equines hyper-immunized with chemically inactivated SARS-CoV-2 generate high titers of antibody with a strong virus neutralizing potential. ELISA performed with pooled antisera displayed highest immunoglobulin titer on 42 days post-immunization, at 1:51,200 dilutions. F(ab)2 immunoglobulin fragments generated from the pools also showed very high, antigen-specific affinity at 1:102,400 dilutions. Finally, in vitro virus neutralization assays confirmed that different pools of F(ab)2 fragments could successfully neutralize SARS-CoV-2 with titers well above 25,000, indicating the potential of this strategy in treating severe COVID-19 cases with high titers. The F(ab)2 was able to cross neutralize another SARS-CoV-2 strain, demonstrating its efficacy against the emerging viral variants and the importance of this approach in our efforts of eradication of COVID-19. In conclusion, this study demonstrates that virus-neutralizing antibodies raised in equines can potentially be used as a treatment regimen in the form of effective passive immunotherapy to combat COVID-19.

Inhibition of Ultraviolet-B Radiation Induced Photodamage by Trigonelline Through Modulation of Mitogen Activating Protein Kinases and Nuclear Factor-κB Signaling Axis in Skin.

Cutaneous photodamage is incited via exposure of ultraviolet-B (UV-B) radiation to skin, characterized by the manifestation of oxidative stress, inflammation, collagen degradation and apoptosis which translates to external aging signs such as wrinkle formation and leathery skin appearance. Meanwhile, it increases cellular susceptibility to photocarcinogenesis. Several studies have accumulated evidence regarding the usage of natural agents in reversing the clinical signs of photoaging as well as preventing photo-toxicity at molecular level. In this study, we have explored the therapeutic potential of natural agent Trigonelline (TG) against UV-B radiation mediated skin photodamage. Various parameters modulated by the exposure of UV-B radiation were investigated in human skin cells and chronic photodamage mice model (Balb/c). We found that TG alleviates UV-B radiation induced photodamage in human skin cells and Balb/c skin mice. TG treatment in UV-B irradiated skin cells abates UV-B radiation mediated phototoxicity, oxidative stress, inflammation and apoptosis. At molecular level, we observed TG treatment significantly prevents the reactive oxygen species (ROS) generation and lipid peroxidation, restores collagen synthesis and matrix metalloproteinase (MMPs) levels. The in vitro findings were replicated in the in vivo model. We found that the TG acts potentially via modulation of ROS-MAPKs-NF-κB axis. Collectively, we propose that TG acts antagonistically against UV-B mediated skin damage and has strong potential to be developed as a therapeutic and cosmetical agent against photodamage disorders.

Glycyrrhizic Acid Prevents Oxidative Stress Mediated DNA Damage Response through Modulation of Autophagy in Ultraviolet-B-Irradiated Human Primary Dermal Fibroblasts.

BACKGROUND/AIMS: Excessive exposure to UV radiation negatively affects the human skin, characterized by photo-damage (premature aging & carcinogenesis). UV-B radiation causes about 90% of non-melanoma skin cancers by damaging de-oxy ribonucleic acids (DNA). We have previously reported that UV-B radiation induces skin photodamage through oxidative & Endoplasmic Reticulum (ER) stresses and Glycyrrhizic acid (GA), a natural triterpene, protects skin cells against such stresses. UV-B radiation elicits signalling cascade by activation of proteins involved in sensing, signalling, and repair process of DNA damage. In this study, we explored the effects & mechanisms of Glycyrrhizic acid (GA) against UV-B -induced photodamage using a well established cellular model. METHODS: We used primary human dermal fibroblasts as a cellular model. The cells were cultured in the presence or absence of GA for 3,6, & 24 h. Effect of UV-B was assessed by examining cell viability, cell morphology, oxidative stress, ER stress, DNA damage & cellular autophagy levels through biochemical assays, microscopy & protein expression studies. RESULTS: In this study, we have determined the effect of GA on autophagy mediated DNA damage response system as the main mechanism in preventing photodamage due to UV-B -irradiation to primary human dermal fibroblasts (HDFs). GA treatment to UV-B exposed HDFs, significantly inhibited cell death, oxidative & ER stress responses, prevented Cyclobutane Pyrimidine dimer (CPD) DNA adduct formation, and DNA fragmentation via modulation of UV-B induced autophagic flux. Present results showed that GA treatment quenched reactive oxygen species (ROS), relieved ER stress response, improved autophagy (6 hr's post-UV-B -irradiation) and prevented UV-B induced DNA damage. CONCLUSION: The present study links autophagy induction by GA as the main mechanism in the prevention of DNA damage and provides a mechanistic basis for the photoprotective effect of GA and suggests that GA can be potentially developed as a promising agent against UV-B induced skin photo-damage.

Effects of AR Display Context Switching and Focal Distance Switching on Human Performance.

In augmented reality (AR) environments, information is often distributed between real world and virtual contexts, and often appears at different distances from the user. Therefore, to integrate the information, users must repeatedly switch context and refocus the eyes. To focus at different distances, the user's eyes must accommodate, which when done repeatedly can cause eyestrain and degrade task performance. An experiment was conducted that examined switching context and focal distance between a real and an AR environment, using a text-based visual search task and a monocular optical see-through AR display. Both context switching and focal distance switching resulted in significantly reduced performance. In addition, repeatedly performing the task caused visual fatigue to steadily increase. Performance was particularly poor for virtual text presented at optical infinity, and for target letters that participants tried to read before their eyes had completely accommodated to a new focal distance. The results show that context switching and focal distance switching are important AR user interface design issues.

Interleukin-1B (IL-1B-31 and IL-1B-511) and interleukin-1 receptor antagonist (IL-1Ra) gene polymorphisms in primary immune thrombocytopenia

BACKGROUND: Immune thrombocytopenia (ITP) is an immune-mediated disease caused by autoantibodies against platelets membrane glycoproteins GPIIb/IIIa and GPIb/IX. The etiology of ITP remains unclear. This study evaluated the association of polymorphisms in interleukin (IL)-1B-31, IL-1B-511, and IL-1Ra with ITP. METHODS: Genotyping of IL-1B-31, IL-1B-511, and IL-1Ra was performed in 118 ITP patients and 100 controls by polymerase chain reaction restriction fragment length polymorphism and detection of variable number tandem repeats. RESULTS: Genotype differences in IL-1B-31 and IL-1Ra were significantly associated with ITP. Patients showed a higher frequency of the IL-1B-31 variant allele (T) and a 1.52-fold greater risk of susceptibility to ITP (odds ratio [OR]=1.52, 95% confidence interval [CI]=1.04–2.22, P=0.034). The frequencies of both homozygous and heterozygous variant genotypes of IL-1B-31 were higher (OR=2.33, 95% CI=1.069–5.09, P=0.033 and OR=2.044, 95% CI=1.068–39, P=0.034) among patients and were significantly associated with ITP susceptibility. Both homozygous and heterozygous variant genotypes of IL-1Ra were also more frequent (OR=4.48, 95% CI=1.17–17.05, P=0.0230 and OR=1.80, 95% CI=1.03–3.14, P=0.0494) among patients and were associated with ITP risk. IL-1B-31 and IL-1Ra also showed significant association with severe ITP. However, IL-1B-511 was not associated with ITP. CONCLUSION: IL-1B-31 and IL-1Ra polymorphisms may significantly impact ITP risk, and they could be associated with disease severity, which may contribute to the pathogenesis of ITP.