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.

Single-nucleus transcriptomic profiling of multiple organs in a rhesus macaque model of SARS-CoV-2 infection.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes diverse clinical manifestations and tissue injuries in multiple organs. However, cellular and molecular understanding of SARS-CoV-2 infection-associated pathology and immune defense features in different organs remains incomplete. Here, we profiled approximately 77 000 single-nucleus transcriptomes of the lung, liver, kidney, and cerebral cortex in rhesus macaques ( Macaca mulatta) infected with SARS-CoV-2 and healthy controls. Integrated analysis of the multi-organ dataset suggested that the liver harbored the strongest global transcriptional alterations. We observed prominent impairment in lung epithelial cells, especially in AT2 and ciliated cells, and evident signs of fibrosis in fibroblasts. These lung injury characteristics are similar to those reported in patients with coronavirus disease 2019 (COVID-19). Furthermore, we found suppressed MHC class I/II molecular activity in the lung, inflammatory response in the liver, and activation of the kynurenine pathway, which induced the development of an immunosuppressive microenvironment. Analysis of the kidney dataset highlighted tropism of tubule cells to SARS-CoV-2, and we found membranous nephropathy (an autoimmune disease) caused by podocyte dysregulation. In addition, we identified the pathological states of astrocytes and oligodendrocytes in the cerebral cortex, providing molecular insights into COVID-19-related neurological implications. Overall, our multi-organ single-nucleus transcriptomic survey of SARS-CoV-2-infected rhesus macaques broadens our understanding of disease features and antiviral immune defects caused by SARS-CoV-2 infection, which may facilitate the development of therapeutic interventions for COVID-19.

A combination vaccine against SARS-CoV-2 and H1N1 influenza based on receptor binding domain trimerized by six-helix bundle fusion core.

BACKGROUND: Increasing severe morbidity and mortality by simultaneous or sequential infections with SARS-CoV-2 and influenza A viruses (IAV), especially in the elderly and obese patients, highlight the urgency of developing a combination vaccine against COVID-19 and influenza. METHODS: Self-assembling SARS-CoV-2 RBD-trimer and Influenza H1N1 HA1-trimer antigens were constructed, upon the stable fusion core in post-fusion conformation. Immunogenicity of SARS-CoV-2 RBD-trimer vaccine and H1N1 HA1-trimer antigens candidates were evaluated in mice. Protection efficacy of a combination vaccine candidate against SARS-CoV-2 and IAV challenge was identified using the K18-hACE2 mouse model. FINDINGS: Both the resultant RBD-trimer for SARS-CoV-2 and HA1-trimer for H1N1 influenza fully exposed receptor-binding motifs (RBM) or receptor-binding site (RBS). Two-dose RBD-trimer induced significantly higher binding and neutralizing antibody titers, and also a strong Th1/Th2 balanced cellular immune response in mice. Similarly, the HA1-trimer vaccine was confirmed to exhibit potent immunogenicity in mice. A combination vaccine candidate, composed of RBD-trimer and HA1-trimer, afforded high protection efficacy in mouse models against stringent lethal SARS-CoV-2 and homogenous H1N1 influenza co-infection, characterized by 100% survival rate. INTERPRETATION: Our results represent a proof of concept for a combined vaccine candidate based on trimerized receptor binding domain against co-epidemics of COVID-19 and influenza. FUNDING: This project was funded by the Strategic Priority Research Program of CAS (XDB29040201), the National Natural Science Foundation of China (81830050, 81901680, and 32070569) and China Postdoctoral Science Foundation (2021M703450).

Optimization of Milk Substitutes for the Artificial Rearing of Chinese Tree Shrews (Tupaia belangeri chinensis).

The Chinese tree shrew (Tupaia belangeri chinensis) has the potential to replace the use of non-human primates in biomedical research. To increase the availability of this species, we have undertaken the ambitious task of establishing inbred lines of the Chinese tree shrew; however, we have been hindered by a low survival rate of inbred pups. Here, we report our artificial rearing (AR) of Chinese tree shrew pups using four different milk substitutes: the formula described by Tsang and Collins (milk TC) and three commercially available milk substitutes intended for possums (milk A and milk C) and for guinea pigs (milk B). We compared the effects of these milk substitutes and maternal milk on the daily milk consumption, growth performance, and survival of the pups. We also assessed the life span and reproductive performance of the F1 individuals given the best milk substitute as compared to the maternally reared (MR) pups. Milk B was found to be appropriate for AR. Pups fed with milk B had a high survival rate at the weaning age compared to those fed with the other milk substitutes. The AR pups fed with milk B had a life span similar to that of MR pups. AR females fed with milk B had an earlier age of the first reproduction, a larger number of litters, and a higher rate of survival of the offspring at the weaning age compared with the MR females. The successful optimization of a milk substitute for AR of Chinese tree shrew pups will undoubtedly facilitate the wide usage of this experimental animal.

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.).

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.

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.

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.