Entangling of Peptide Nanofibers Reduces the Invasiveness of SARS-CoV-2.

The viral spike (S) protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to angiotensin-converting enzyme 2 (ACE2) receptors on the host cells, facilitating its entry and infection. Here, functionalized nanofibers targeting the S protein with peptide sequences of IRQFFKK, WVHFYHK and NSGGSVH, which are screened from a high-throughput one-bead one-compound screening strategy, are designed and prepared. The flexible nanofibers support multiple binding sites and efficiently entangle SARS-CoV-2, forming a nanofibrous network that blocks the interaction between the S protein of SARS-CoV-2 and the ACE2 on host cells, and efficiently reduce the invasiveness of SARS-CoV-2. In summary, nanofibers entangling represents a smart nanomedicine for the prevention of SARS-CoV-2.

Preventive and therapeutic benefits of nelfinavir in rhesus macaques and human beings infected with SARS-CoV-2.

Effective drugs with broad spectrum safety profile to all people are highly expected to combat COVID-19 caused by SARS-CoV-2. Here we report that nelfinavir, an FDA approved drug for the treatment of HIV infection, is effective against SARS-CoV-2 and COVID-19. Preincubation of nelfinavir could inhibit the activity of the main protease of the SARS-CoV-2 (IC50 = 8.26 µM), while its antiviral activity in Vero E6 cells against a clinical isolate of SARS-CoV-2 was determined to be 2.93 µM (EC50). In comparison with vehicle-treated animals, rhesus macaque prophylactically treated with nelfinavir had significantly lower temperature and significantly reduced virus loads in the nasal and anal swabs of the animals. At necropsy, nelfinavir-treated animals had a significant reduction of the viral replication in the lungs by nearly three orders of magnitude. A prospective clinic study with 37 enrolled treatment-naive patients at Shanghai Public Health Clinical Center, which were randomized (1:1) to nelfinavir and control groups, showed that the nelfinavir treatment could shorten the duration of viral shedding by 5.5 days (9.0 vs. 14.5 days, P = 0.055) and the duration of fever time by 3.8 days (2.8 vs. 6.6 days, P = 0.014) in mild/moderate COVID-19 patients. The antiviral efficiency and clinical benefits in rhesus macaque model and in COVID-19 patients, together with its well-established good safety profile in almost all ages and during pregnancy, indicated that nelfinavir is a highly promising medication with the potential of preventative effect for the treatment of COVID-19.

Characteristics of replication and pathogenicity of SARS-CoV-2 Alpha and Delta isolates.

The continuously arising of SARS-CoV-2 variants has been posting a great threat to public health safety globally, from B.1.17 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta) to B.1.1.529 (Omicron). The emerging or re-emerging of the SARS-CoV-2 variants of concern is calling for the constant monitoring of their epidemics, pathogenicity and immune escape. In this study, we aimed to characterize replication and pathogenicity of the Alpha and Delta variant strains isolated from patients infected in Laos. The amino acid mutations within the spike fragment of the isolates were determined via sequencing. The more efficient replication of the Alpha and Delta isolates was documented than the prototyped SARS-CoV-2 in Calu-3 and Caco-2 â€‹cells, while such features were not observed in Huh-7, Vero E6 and HPA-3 â€‹cells. We utilized both animal models of human ACE2 (hACE2) transgenic mice and hamsters to evaluate the pathogenesis of the isolates. The Alpha and Delta can replicate well in multiple organs and cause moderate to severe lung pathology in these animals. In conclusion, the spike protein of the isolated Alpha and Delta variant strains was characterized, and the replication and pathogenicity of the strains in the cells and animal models were also evaluated.

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.

Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice.

BACKGROUND: The Coronavirus Disease 2019 (COVID-19) pandemic has been a great threat to global public health since 2020. Although the advance on vaccine development has been largely achieved, a strategy to alleviate immune overactivation in severe COVID-19 patients is still needed. The NLRP3 inflammasome is activated upon SARS-CoV-2 infection and associated with COVID-19 severity. However, the processes by which the NLRP3 inflammasome is involved in COVID-19 disease remain unclear. METHODS: We infected THP-1 derived macrophages, NLRP3 knockout mice, and human ACE2 transgenic mice with live SARS-CoV-2 in Biosafety Level 3 (BSL-3) laboratory. We performed quantitative real-time PCR for targeted viral or host genes from SARS-CoV-2 infected mouse tissues, conducted histological or immunofluorescence analysis in SARS-CoV-2 infected mouse tissues. We also injected intranasally AAV-hACE2 or intraperitoneally NLRP3 inflammasome inhibitor MCC950 before SARS-CoV-2 infection in mice as indicated. FINDINGS: We have provided multiple lines of evidence that the NLRP3 inflammasome plays an important role in the host immune response to SARS-CoV-2 invasion of the lungs. Inhibition of the NLRP3 inflammasome attenuated the release of COVID-19 related pro-inflammatory cytokines in cell cultures and mice. The severe pathology induced by SARS-CoV-2 in lung tissues was reduced in Nlrp3-/- mice compared to wild-type C57BL/6 mice. Finally, specific inhibition of the NLRP3 inflammasome by MCC950 alleviated excessive lung inflammation and thus COVID-19 like pathology in human ACE2 transgenic mice. INTERPRETATION: Inflammatory activation induced by SARS-CoV-2 is an important stimulator of COVID-19 related immunopathology. Targeting the NLRP3 inflammasome is a promising immune intervention against severe COVID-19 disease. FUNDING: This work was supported by grants from the Bureau of Frontier Sciences and Education, CAS (grant no. QYZDJ-SSW-SMC005 to Y.G.Y.), the key project of the CAS "Light of West China" Program (to D.Y.) and Yunnan Province (202001AS070023 to D.Y.).

A pathogen-like antigen-based vaccine confers immune protection against SARS-CoV-2 in non-human primates.

Activation of nucleic acid sensing Toll-like receptors (TLRs) in B cells is involved in antiviral responses by promoting B cell activation and germinal center responses. In order to take advantage of this natural pathway for vaccine development, synthetic pathogen-like antigens (PLAs) constructed of multivalent antigens with encapsulated TLR ligands can be used to activate B cell antigen receptors and TLRs in a synergistic manner. Here we report a PLA-based coronavirus disease 2019 (COVID-19) vaccine candidate designed by combining a phage-derived virus-like particle carrying bacterial RNA as TLR ligands with the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein as the target antigen. This PLA-based vaccine candidate induces robust neutralizing antibodies in both mice and non-human primates (NHPs). Using a NHP infection model, we demonstrate that the viral clearance is accelerated in vaccinated animals. In addition, the PLA-based vaccine induces a T helper 1 (Th1)-oriented response and a durable memory, supporting its potential for further clinical development.

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 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target.

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.

Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global crisis, urgently necessitating the development of safe, efficacious, convenient-to-store, and low-cost vaccine options. A major challenge is that the receptor-binding domain (RBD)-only vaccine fails to trigger long-lasting protective immunity if used alone for vaccination. To enhance antigen processing and cross-presentation in draining lymph nodes (DLNs), we developed an interferon (IFN)-armed RBD dimerized by an immunoglobulin fragment (I-R-F). I-R-F efficiently directs immunity against RBD to DLNs. A low dose of I-R-F induces not only high titers of long-lasting neutralizing antibodies (NAbs) but also more comprehensive T cell responses than RBD. Notably, I-R-F provides comprehensive protection in the form of a one-dose vaccine without an adjuvant. Our study shows that the pan-epitope modified human I-R-F (I-P-R-F) vaccine provides rapid and complete protection throughout the upper and lower respiratory tracts against a high-dose SARS-CoV-2 challenge in rhesus macaques. Based on these promising results, we have initiated a randomized, placebo-controlled, phase I/II trial of the human I-P-R-F vaccine (V-01) in 180 healthy adults, and the vaccine appears safe and elicits strong antiviral immune responses. Due to its potency and safety, this engineered vaccine may become a next-generation vaccine candidate in the global effort to overcome COVID-19.

A recombinant receptor-binding domain in trimeric form generates protective immunity against SARS-CoV-2 infection in nonhuman primates.

A safe and effective vaccine is critical to combat the COVID-19 pandemic. Here, we developed a trimeric SARS-CoV-2 receptor-binding domain (RBD) subunit vaccine candidate that simulates the natural structure of the spike (S) trimer glycoprotein. Immunization with the RBD trimer-induced robust humoral and cellular immune responses, and a high level of neutralizing antibodies was maintained for at least 4.5 months. Moreover, the antibodies that were produced in response to the vaccine effectively cross-neutralized the SARS-CoV-2 501Y.V2 variant (B.1.351). Of note, when the vaccine-induced antibodies dropped to a sufficiently low level, only one boost quickly activated the anamnestic immune response, conferring full protection against a SARS-CoV-2 challenge in rhesus macaques without typical histopathological changes in the lung tissues. These results demonstrated that the SARS-CoV-2 RBD trimer vaccine candidate is highly immunogenic and safe, providing long-lasting, broad, and significant immunity protection in nonhuman primates, thereby offering an optimal vaccination strategy against COVID-19.

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.