Extracellular vesicles mediate antibody-resistant transmission of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Antibody resistance dampens neutralizing antibody therapy and threatens current global Coronavirus (COVID-19) vaccine campaigns. In addition to the emergence of resistant SARS-CoV-2 variants, little is known about how SARS-CoV-2 evades antibodies. Here, we report a novel mechanism of extracellular vesicle (EV)-mediated cell-to-cell transmission of SARS-CoV-2, which facilitates SARS-CoV-2 to escape from neutralizing antibodies. These EVs, initially observed in SARS-CoV-2 envelope protein-expressing cells, are secreted by various SARS-CoV-2-infected cells, including Vero E6, Calu-3, and HPAEpiC cells, undergoing infection-induced pyroptosis. Various SARS-CoV-2-infected cells produce similar EVs characterized by extra-large sizes (1.6-9.5 µm in diameter, average diameter > 4.2 µm) much larger than previously reported virus-generated vesicles. Transmission electron microscopy analysis and plaque assay reveal that these SARS-CoV-2-induced EVs contain large amounts of live virus particles. In particular, the vesicle-cloaked SARS-CoV-2 virus is resistant to neutralizing antibodies and able to reinfect naïve cells independent of the reported receptors and cofactors. Consistently, the constructed 3D images show that intact EVs could be taken up by recipient cells directly, supporting vesicle-mediated cell-to-cell transmission of SARS-CoV-2. Our findings reveal a novel mechanism of receptor-independent SARS-CoV-2 infection via cell-to-cell transmission, provide new insights into antibody resistance of SARS-CoV-2 and suggest potential targets for future antiviral therapeutics.

A variant-proof SARS-CoV-2 vaccine targeting HR1 domain in S2 subunit of spike protein.

The emerging SARS-CoV-2 variants, commonly with many mutations in S1 subunit of spike (S) protein are weakening the efficacy of the current vaccines and antibody therapeutics. This calls for the variant-proof SARS-CoV-2 vaccines targeting the more conserved regions in S protein. Here, we designed a recombinant subunit vaccine, HR121, targeting the conserved HR1 domain in S2 subunit of S protein. HR121 consisting of HR1-linker1-HR2-linker2-HR1, is conformationally and functionally analogous to the HR1 domain present in the fusion intermediate conformation of S2 subunit. Immunization with HR121 in rabbits and rhesus macaques elicited highly potent cross-neutralizing antibodies against SARS-CoV-2 and its variants, particularly Omicron sublineages. Vaccination with HR121 achieved near-full protections against prototype SARS-CoV-2 infection in hACE2 transgenic mice, Syrian golden hamsters and rhesus macaques, and effective protection against Omicron BA.2 infection in Syrian golden hamsters. This study demonstrates that HR121 is a promising candidate of variant-proof SARS-CoV-2 vaccine with a novel conserved target in the S2 subunit for application against current and future SARS-CoV-2 variants.

A nanomaterial targeting the spike protein captures SARS-CoV-2 variants and promotes viral elimination.

The global emergency caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic can only be solved with effective and widespread preventive and therapeutic strategies, and both are still insufficient. Here, we describe an ultrathin two-dimensional CuInP2S6 (CIPS) nanosheet as a new agent against SARS-CoV-2 infection. CIPS exhibits an extremely high and selective binding capacity (dissociation constant (KD) < 1 pM) for the receptor binding domain of the spike protein of wild-type SARS-CoV-2 and its variants of concern, including Delta and Omicron, inhibiting virus entry and infection in angiotensin converting enzyme 2 (ACE2)-bearing cells, human airway epithelial organoids and human ACE2-transgenic mice. On association with CIPS, the virus is quickly phagocytosed and eliminated by macrophages, suggesting that CIPS could be successfully used to capture and facilitate virus elimination by the host. Thus, we propose CIPS as a promising nanodrug for future safe and effective anti-SARS-CoV-2 therapy, and as a decontamination agent and surface-coating material to reduce SARS-CoV-2 infectivity.

Microplastics interact with SARS-CoV-2 and facilitate host cell infection

Microplastics (MP) pollution is a global issue that raises concerns about potential toxicity for environmental and human health. The notion that SARS-CoV-2 is more stable when adsorbed on plastic surfaces urged us to examine whether the virus can attach to MP, which may facilitate infection upon inhalation or ingestion. Here, we describe that MP can bind SARS-CoV-2 pseudovirus on their surface and enhance infection of human cells in vitro. This enhanced in vitro infectivity was confirmed with authentic SARS-CoV-2, in parallel with increased expression of inflammation-related caspase-3, IL-8 and TNF-alpha genes. These results suggest that the presence of MP in the environment or in our respiratory or gastrointestinal tracts has the potential to interact with SARS-CoV-2, and potentially increase viral infectivity and spreading.

Harnessing Natural Products by a Pharmacophore-Oriented Semisynthesis Approach for the Discovery of Potential Anti-SARS-CoV-2 Agents.

Natural products possessing unique scaffolds may have antiviral activity but their complex structures hinder facile synthesis. A pharmacophore-oriented semisynthesis approach was applied to (-)-maoelactone A (1) and oridonin (2) for the discovery of anti-SARS-CoV-2 agents. The Wolff rearrangement/lactonization cascade (WRLC) reaction was developed to construct the unprecedented maoelactone-type scaffold during semisynthesis of 1. Further mechanistic study suggested a concerted mechanism for Wolff rearrangement and a water-assisted stepwise process for lactonization. The WRLC reaction then enabled the creation of a novel family by assembly of the maoelactone-type scaffold and the pharmacophore of 2, whereby one derivative inhibited SARS-CoV-2 replication in HPA EpiC cells with a low EC50 value (19±1 nM) and a high TI value (>1000), both values better than those of remdesivir.

Novel nucleocapsid protein-targeting phenanthridine inhibitors of SARS-CoV-2.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented in human history. As a major structural protein, nucleocapsid protein (NPro) is critical to the replication of SARS-CoV-2. In this work, 17 NPro-targeting phenanthridine derivatives were rationally designed and synthesized, based on the crystal structure of NPro. Most of these compounds can interact with SARS-CoV-2 NPro tightly and inhibit the replication of SARS-CoV-2 in vitro. Compounds 12 and 16 exhibited the most potent anti-viral activities with 50% effective concentration values of 3.69 and 2.18 µM, respectively. Furthermore, site-directed mutagenesis of NPro and Surface Plasmon Resonance (SPR) assays revealed that 12 and 16 target N-terminal domain (NTD) of NPro by binding to Tyr109. This work found two potent anti-SARS-CoV-2 bioactive compounds and also indicated that SARS-CoV-2 NPro-NTD can be a target for new anti-virus agents.

Pro-inflammatory microenvironment and systemic accumulation of CXCR3+ cell exacerbate lung pathology of old rhesus macaques infected with SARS-CoV-2.

Understanding the pathological features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in an animal model is crucial for the treatment of coronavirus disease 2019 (COVID-19). Here, we compared immunopathological changes in young and old rhesus macaques (RMs) before and after SARS-CoV-2 infection at the tissue level. Quantitative analysis of multiplex immunofluorescence staining images of formalin-fixed paraffin-embedded (FFPE) sections showed that SARS-CoV-2 infection specifically induced elevated levels of apoptosis, autophagy, and nuclear factor kappa-B (NF-κB) activation of angiotensin-converting enzyme 2 (ACE2)+ cells, and increased interferon α (IFN-α)- and interleukin 6 (IL-6)-secreting cells and C-X-C motif chemokine receptor 3 (CXCR3)+ cells in lung tissue of old RMs. This pathological pattern, which may be related to the age-related pro-inflammatory microenvironment in both lungs and spleens, was significantly correlated with the systemic accumulation of CXCR3+ cells in lungs, spleens, and peripheral blood. Furthermore, the ratio of CXCR3+ to T-box protein expression in T cell (T-bet)+ (CXCR3+/T-bet+ ratio) in CD8+ cells may be used as a predictor of severe COVID-19. These findings uncovered the impact of aging on the immunopathology of early SARS-CoV-2 infection and demonstrated the potential application of CXCR3+ cells in predicting severe COVID-19.

SARS-CoV-2 Mpro inhibitors with antiviral activity in a transgenic mouse model.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continually poses serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing Mpro inhibitors derived from either boceprevir or telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 Mpro activity in vitro, with 50% inhibitory concentration values ranging from 7.6 to 748.5 nM. The cocrystal structure of Mpro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a transgenic mouse model of SARS-CoV-2 infection, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.

Northern pig-tailed macaques ( Macaca leonina) infected with SARS-CoV-2 show rapid viral clearance and persistent immune response.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), has become an unprecedented global health emergency. At present, SARS-CoV-2-infected nonhuman primates are considered the gold standard animal model for COVID-19 research. Here, we showed that northern pig-tailed macaques ( Macaca leonina, NPMs) supported SARS-CoV-2 replication. Furthermore, compared with rhesus macaques, NPMs showed rapid viral clearance in lung tissues, nose swabs, throat swabs, and rectal swabs, which may be due to higher expression of interferon (IFN)-α in lung tissue. However, the rapid viral clearance was not associated with good outcome. In the second week post infection, NPMs developed persistent or even more severe inflammation and body injury compared with rhesus macaques. These results suggest that viral clearance may have no relationship with COVID-19 progression and SARS-CoV-2-infected NPMs could be considered as a critically ill animal model in COVID-19 research.

Lower respiratory tract samples are reliable for severe acute respiratory syndrome coronavirus 2 nucleic acid diagnosis and animal model study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19) continue to impact countries worldwide. At present, inadequate diagnosis and unreliable evaluation systems hinder the implementation and development of effective prevention and treatment strategies. Here, we conducted a horizontal and longitudinal study comparing the detection rates of SARS-CoV-2 nucleic acid in different types of samples collected from COVID-19 patients and SARS-CoV-2-infected monkeys. We also detected anti-SARS-CoV-2 antibodies in the above clinical and animal model samples to identify a reliable approach for the accurate diagnosis of SARS-CoV-2 infection. Results showed that, regardless of clinical symptoms, the highest detection levels of viral nucleic acid were found in sputum and tracheal brush samples, resulting in a high and stable diagnosis rate. Anti-SARS-CoV-2 immunoglobulin M (IgM) and G (IgG) antibodies were not detected in 6.90% of COVID-19 patients. Furthermore, integration of nucleic acid detection results from the various sample types did not improve the diagnosis rate. Moreover, dynamic changes in SARS-CoV-2 viral load were more obvious in sputum and tracheal brushes than in nasal and throat swabs. Thus, SARS-CoV-2 nucleic acid detection in sputum and tracheal brushes was the least affected by infection route, disease progression, and individual differences. Therefore, SARS-CoV-2 nucleic acid detection using lower respiratory tract samples alone is reliable for COVID-19 diagnosis and study.

Delayed severe cytokine storm and immune cell infiltration in SARS-CoV-2-infected aged Chinese rhesus macaques.

As of June 2020, Coronavirus Disease 2019 (COVID-19) has killed an estimated 440 000 people worldwide, 74% of whom were aged ≥65 years, making age the most significant risk factor for death caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To examine the effect of age on death, we established a SARS-CoV-2 infection model in Chinese rhesus macaques ( Macaca mulatta) of varied ages. Results indicated that infected young macaques manifested impaired respiratory function, active viral replication, severe lung damage, and infiltration of CD11b + and CD8 + cells in lungs at one-week post infection (wpi), but also recovered rapidly at 2 wpi. In contrast, aged macaques demonstrated delayed immune responses with a more severe cytokine storm, increased infiltration of CD11b + cells, and persistent infiltration of CD8 + cells in the lungs at 2 wpi. In addition, peripheral blood T cells from aged macaques showed greater inflammation and chemotaxis, but weaker antiviral functions than that in cells from young macaques. Thus, the delayed but more severe cytokine storm and higher immune cell infiltration may explain the poorer prognosis of older aged patients suffering SARS-CoV-2 infection.

COVID-19-like symptoms observed in Chinese tree shrews infected with SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues to pose a global threat to the human population. Identifying animal species susceptible to infection with the SARS-CoV-2/ HCoV-19 pathogen is essential for controlling the outbreak and for testing valid prophylactics or therapeutics based on animal model studies. Here, different aged Chinese tree shrews (adult group, 1 year old; old group, 5-6 years old), which are close relatives to primates, were infected with SARS-CoV-2. X-ray, viral shedding, laboratory, and histological analyses were performed on different days post-inoculation (dpi). Results showed that Chinese tree shrews could be infected by SARS-CoV-2. Lung infiltrates were visible in X-ray radiographs in most infected animals. Viral RNA was consistently detected in lung tissues from infected animals at 3, 5, and 7 dpi, along with alterations in related parameters from routine blood tests and serum biochemistry, including increased levels of aspartate aminotransferase (AST) and blood urea nitrogen (BUN). Histological analysis of lung tissues from animals at 3 dpi (adult group) and 7 dpi (old group) showed thickened alveolar septa and interstitial hemorrhage. Several differences were found between the two different aged groups in regard to viral shedding peak. Our results indicate that Chinese tree shrews have the potential to be used as animal models for SARS-CoV-2 infection.