Orf virus DNA prime-protein boost strategy is superior to adenovirus-based vaccination in mice and sheep.

Contagious ecthyma (Orf), an acute and highly contagious zoonosis, is prevalent worldwide. Orf is caused by Orf virus (ORFV), which mainly infects sheep/goats and humans. Therefore, effective and safe vaccination strategies for Orf prevention are needed. Although immunization with single-type Orf vaccines has been tested, heterologous prime-boost strategies still need to be studied. In the present study, ORFV B2L and F1L were selected as immunogens, based on which DNA, subunit and adenovirus vaccine candidates were generated. Of note, heterologous immunization strategies using DNA prime-protein boost and DNA prime-adenovirus boost in mice were performed, with single-type vaccines as controls. We have found that the DNA prime-protein boost strategy induces stronger humoral and cellular immune responses than DNA prime-adenovirus boost strategy in mice, which was confirmed by the changes in specific antibodies, lymphocyte proliferation and cytokine expression. Importantly, this observation was also confirmed when these heterologous immunization strategies were performed in sheep. In summary, by comparing the two immune strategies, we found that DNA prime-protein boost strategy can induce a better immune response, which provides a new attempt for exploring Orf immunization strategy.

Parapoxvirus-based therapy eliminates SARS-CoV-2-loaded fine aerosol and blocks viral transmission in hamster models.

Currently, it is believed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an airborne virus, and virus-containing aerosol particles have been found concurrent with the onset of COVID-19, which may contribute to the noncontact transmission of SARS-CoV-2. Exploring agents to block SARS-CoV-2 transmission is of great importance to prevent the COVID-19 pandemic. In this study, we found that inactivated Parapoxvirus ovis (iORFV), a kind of immunomodulator, could compress the proportion of small particle aerosols exhaled by Syrian golden hamsters. Notably, the concentration of SARS-CoV-2 RNA-containing aerosol particles was significantly reduced by iORFV in the early stages after viral inoculation. Importantly, smaller aerosol particles (<4.7 µm) that carry infectious viruses were completely cleared by iORFV. Consistently, iORFV treatment completely blocked viral noncontact (aerosol) transmission. In summary, iORFV may become a repurposed agent for the prevention and control of COVID-19 by affecting viral aerosol exhalation and subsequent viral transmission.

A Dual Role of Complement Activation in the Development of Fulminant Hepatic Failure Induced by Murine-Beta-Coronavirus Infection.

With the epidemic of betacoronavirus increasing frequently, it poses a great threat to human public health. Therefore, the research on the pathogenic mechanism of betacoronavirus is becoming greatly important. Murine hepatitis virus strain-3 (MHV-3) is a strain of betacoronavirus which cause tissue damage especially fulminant hepatic failure (FHF) in mice, and is commonly used to establish models of acute liver injury. Recently, MHV-3-infected mice have also been introduced to a mouse model of COVID-19 that does not require a Biosafety Level 3 (BSL-3) facility. FHF induced by MHV-3 is a type of severe liver damage imbalanced by regenerative hepatocellular activity, which is related to numerous factors. The complement system plays an important role in host defense and inflammation and is involved in first-line immunity and/or pathogenesis of severe organ disorders. In this study, we investigated the role of aberrant complement activation in MHV-3 infection-induced FHF by strategies that use C3-deficient mice and intervene in the complement system. Our results showed that mice deficient in C3 had more severe liver damage, a higher viral load in the liver and higher serum concentrations of inflammatory cytokines than wild-type controls. Treatment of C57BL/6 mice with C3aR antagonist or anti-C5aR antibody reduced liver damage, viral load, and serum IFN-γ concentration compared with the control group. These findings indicated that complement system acts as a double-edged sword during acute MHV-3 infection. However, its dysregulated activation leads to sustained inflammatory responses and induces extensive liver damage. Collectively, by investigating the role of complement activation in MHV-3 infection, we can further understand the pathogenic mechanism of betacoronavirus, and appropriate regulation of immune responses by fine-tuning complement activation may be an intervention for the treatment of diseases induced by betacoronavirus infection.

A glucose-like metabolite deficient in diabetes inhibits cellular entry of SARS-CoV-2.

The severity and mortality of COVID-19 are associated with pre-existing medical comorbidities such as diabetes mellitus. However, the underlying causes for increased susceptibility to viral infection in patients with diabetes is not fully understood. Here we identify several small-molecule metabolites from human blood with effective antiviral activity against SARS-CoV-2, one of which, 1,5-anhydro-D-glucitol (1,5-AG), is associated with diabetes mellitus. The serum 1,5-AG level is significantly lower in patients with diabetes. In vitro, the level of SARS-CoV-2 replication is higher in the presence of serum from patients with diabetes than from healthy individuals and this is counteracted by supplementation of 1,5-AG to the serum from patients. Diabetic (db/db) mice undergo SARS-CoV-2 infection accompanied by much higher viral loads and more severe respiratory tissue damage when compared to wild-type mice. Sustained supplementation of 1,5-AG in diabetic mice reduces SARS-CoV-2 loads and disease severity to similar levels in nondiabetic mice. Mechanistically, 1,5-AG directly binds the S2 subunit of the SARS-CoV-2 spike protein, thereby interrupting spike-mediated virus-host membrane fusion. Our results reveal a mechanism that contributes to COVID-19 pathogenesis in the diabetic population and suggest that 1,5-AG supplementation may be beneficial to diabetic patients against severe COVID-19.

Long-term stability and protection efficacy of the RBD-targeting COVID-19 mRNA vaccine in nonhuman primates.

Messenger RNA (mRNA) vaccine technology has shown its power in preventing the ongoing COVID-19 pandemic. Two mRNA vaccines targeting the full-length S protein of SARS-CoV-2 have been authorized for emergency use. Recently, we have developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor-binding domain (RBD) of SARS-CoV-2 (termed ARCoV), which confers complete protection in mouse model. Herein, we further characterized the protection efficacy of ARCoV in nonhuman primates and the long-term stability under normal refrigerator temperature. Intramuscular immunization of two doses of ARCoV elicited robust neutralizing antibodies as well as cellular response against SARS-CoV-2 in cynomolgus macaques. More importantly, ARCoV vaccination in macaques significantly protected animals from acute lung lesions caused by SARS-CoV-2, and viral replication in lungs and secretion in nasal swabs were completely cleared in all animals immunized with low or high doses of ARCoV. No evidence of antibody-dependent enhancement of infection was observed throughout the study. Finally, extensive stability assays showed that ARCoV can be stored at 2-8 °C for at least 6 months without decrease of immunogenicity. All these promising results strongly support the ongoing clinical trial.

MERS-CoV infection causes brain damage in human DPP4-transgenic mice through complement-mediated inflammation.

The highly pathogenic Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a severe respiratory virus. Recent reports indicate additional central nervous system (CNS) involvement. In this study, human DPP4 transgenic mice were infected with MERS-CoV, and viral antigens were first detected in the midbrain-hindbrain 4 days post-infection, suggesting the virus may enter the brainstem via peripheral nerves. Neurons and astrocytes throughout the brain were infected, followed by damage of the blood brain barrier (BBB), as well as microglial activation and inflammatory cell infiltration, which may be caused by complement activation based on the observation of deposition of complement activation product C3 and high expression of C3a receptor (C3aR) and C5a receptor (C5aR1) in neurons and glial cells. It may be concluded that these effects were mediated by complement activation in the brain, because of their reduction resulted from the treatment with mouse C5aR1-specific mAb. Such mAb significantly reduced nucleoprotein expression, suppressed microglial activation and decreased activation of caspase-3 in neurons and p38 phosphorylation in the brain. Collectively, these results suggest that MERS-CoV infection of CNS triggers complement activation, leading to inflammation-mediated damage of brain tissue, and regulating of complement activation could be a promising intervention and adjunctive treatment for CNS injury by MERS-CoV and other coronaviruses.

Screening HLA-A-restricted T cell epitopes of SARS-CoV-2 and the induction of CD8+ T cell responses in HLA-A transgenic mice.

Since severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific T cells have been found to play essential roles in host immune protection and pathology in patients with coronavirus disease 2019 (COVID-19), this study focused on the functional validation of T cell epitopes and the development of vaccines that induce specific T cell responses. A total of 120 CD8+ T cell epitopes from the E, M, N, S, and RdRp proteins were functionally validated. Among these, 110, 15, 6, 14, and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; in addition, four epitopes from the S protein displayed one amino acid that was distinct from the current SARS-CoV-2 variants. Then, 31 epitopes restricted by the HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or poly (lactic-co-glycolic acid) nanoparticles, and these vaccines elicited robust and specific CD8+ T cell responses in HLA-A2/DR1 transgenic mice as well as wild-type mice. In contrast to previous research, this study established a modified DC-peptide-PBL cell coculture system using healthy donor PBMCs to validate the in silico predicted epitopes, provided an epitope library restricted by nine of the most prevalent HLA-A allotypes covering broad Asian populations, and identified the HLA-A restrictions of these validated epitopes using competitive peptide binding experiments with HMy2.CIR cell lines expressing the indicated HLA-A allotype, which initially confirmed the in vivo feasibility of 9- or 10-mer peptide cocktail vaccines against SARS-CoV-2. These data will facilitate the design and development of vaccines that induce antiviral CD8+ T cell responses in COVID-19 patients.

Characterization and structural basis of a lethal mouse-adapted SARS-CoV-2.

There is an urgent need for animal models to study SARS-CoV-2 pathogenicity. Here, we generate and characterize a novel mouse-adapted SARS-CoV-2 strain, MASCp36, that causes severe respiratory symptoms, and mortality. Our model exhibits age- and gender-related mortality akin to severe COVID-19. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three RBD mutations significantly enhance binding affinity to its endogenous receptor, ACE2. Cryo-electron microscopy analysis of human ACE2 (hACE2), or mouse ACE2 (mACE2), in complex with the RBD of MASCp36, at 3.1 to 3.7 Å resolution, reveals the molecular basis for the receptor-binding switch. N501Y and Q493H enhance the binding affinity to hACE2, whereas triple mutations at N501Y/Q493H/K417N decrease affinity and reduce infectivity of MASCp36. Our study provides a platform for studying SARS-CoV-2 pathogenesis, and unveils the molecular mechanism for its rapid adaptation and evolution.

Rational development of a human antibody cocktail that deploys multiple functions to confer Pan-SARS-CoVs protection.

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.

Large-Sized Graphene Oxide Nanosheets Increase DC-T-Cell Synaptic Contact and the Efficacy of DC Vaccines against SARS-CoV-2.

Dendritic cell (DC) vaccines are used for cancer and infectious diseases, albeit with limited efficacy. Modulating the formation of DC-T-cell synapses may greatly increase their efficacy. The effects of graphene oxide (GO) nanosheets on DCs and DC-T-cell synapse formation are evaluated. In particular, size-dependent interactions are observed between GO nanosheets and DCs. GOs with diameters of >1 µm (L-GOs) demonstrate strong adherence to the DC surface, inducing cytoskeletal reorganization via the RhoA-ROCK-MLC pathway, while relatively small GOs (≈500 nm) are predominantly internalized by DCs. Furthermore, L-GO treatment enhances DC-T-cell synapse formation via cytoskeleton-dependent membrane positioning of integrin ICAM-1. L-GO acts as a "nanozipper," facilitating the aggregation of DC-T-cell clusters to produce a stable microenvironment for T cell activation. Importantly, L-GO-adjuvanted DCs promote robust cytotoxic T cell immune responses against SARS-CoV-2 spike 1, leading to >99.7% viral RNA clearance in mice infected with a clinically isolated SARS-CoV-2 strain. These findings highlight the potential value of nanomaterials as DC vaccine adjuvants for modulating DC-T-cell synapse formation and provide a basis for the development of effective COVID-19 vaccines.

Sensitive and Easy-Read CRISPR Strip for COVID-19 Rapid Point-of-Care Testing.

Rapid and clinically sensitive detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) play an important role in the contact tracing and containment of the COVID-19 pandemic. A recently developed field-deployable clustered regularly interspaced short palindromic repeats (CRISPR) detection assay with lateral flow strips shows promise for point-of-care detection of SARS-CoV-2. However, the limit of detection of paper strip-based assays (10-100 copies/µL) is much lower than that of fluorescence-based detection methods. In this study, we developed an easy-readout and sensitive enhanced (ERASE) strip to visualize the results of CRISPR detection and improve the sensitivity to 1 copy/µL with an unambiguous easy-read result. Using 649 clinical samples from blind specimens collected from patients in China, we validated our ERASE assay for SARS-CoV-2 RNA detection with 90.67% positive predictive agreement and 99.21% negative predictive agreement. In conclusion, our study provided a customized CRISPR strip for use in a simple, rapid, ultrasensitive, and highly specific assay for SARS-CoV-2 detection. (Clinical Trial Registration number: 2020-008-01; [2020]IEC(ZD01); PJ-NBEY-2020-009-01; 2020#34).

CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells.

In face of the everlasting battle toward COVID-19 and the rapid evolution of SARS-CoV-2, no specific and effective drugs for treating this disease have been reported until today. Angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, mediates the virus infection by binding to spike protein. Although ACE2 is expressed in the lung, kidney, and intestine, its expressing levels are rather low, especially in the lung. Considering the great infectivity of COVID-19, we speculate that SARS-CoV-2 may depend on other routes to facilitate its infection. Here, we first discover an interaction between host cell receptor CD147 and SARS-CoV-2 spike protein. The loss of CD147 or blocking CD147 in Vero E6 and BEAS-2B cell lines by anti-CD147 antibody, Meplazumab, inhibits SARS-CoV-2 amplification. Expression of human CD147 allows virus entry into non-susceptible BHK-21 cells, which can be neutralized by CD147 extracellular fragment. Viral loads are detectable in the lungs of human CD147 (hCD147) mice infected with SARS-CoV-2, but not in those of virus-infected wild type mice. Interestingly, virions are observed in lymphocytes of lung tissue from a COVID-19 patient. Human T cells with a property of ACE2 natural deficiency can be infected with SARS-CoV-2 pseudovirus in a dose-dependent manner, which is specifically inhibited by Meplazumab. Furthermore, CD147 mediates virus entering host cells by endocytosis. Together, our study reveals a novel virus entry route, CD147-spike protein, which provides an important target for developing specific and effective drug against COVID-19.

Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has prioritized the development of small-animal models for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We adapted a clinical isolate of SARS-CoV-2 by serial passaging in the respiratory tract of aged BALB/c mice. The resulting mouse-adapted strain at passage 6 (called MASCp6) showed increased infectivity in mouse lung and led to interstitial pneumonia and inflammatory responses in both young and aged mice after intranasal inoculation. Deep sequencing revealed a panel of adaptive mutations potentially associated with the increased virulence. In particular, the N501Y mutation is located at the receptor binding domain (RBD) of the spike protein. The protective efficacy of a recombinant RBD vaccine candidate was validated by using this model. Thus, this mouse-adapted strain and associated challenge model should be of value in evaluating vaccines and antivirals against SARS-CoV-2.

A Thermostable mRNA Vaccine against COVID-19.

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.

A Mouse Model of SARS-CoV-2 Infection and Pathogenesis.

Since December 2019, a novel coronavirus SARS-CoV-2 has emerged and rapidly spread throughout the world, resulting in a global public health emergency. The lack of vaccine and antivirals has brought an urgent need for an animal model. Human angiotensin-converting enzyme II (ACE2) has been identified as a functional receptor for SARS-CoV-2. In this study, we generated a mouse model expressing human ACE2 (hACE2) by using CRISPR/Cas9 knockin technology. In comparison with wild-type C57BL/6 mice, both young and aged hACE2 mice sustained high viral loads in lung, trachea, and brain upon intranasal infection. Although fatalities were not observed, interstitial pneumonia and elevated cytokines were seen in SARS-CoV-2 infected-aged hACE2 mice. Interestingly, intragastric inoculation of SARS-CoV-2 was seen to cause productive infection and lead to pulmonary pathological changes in hACE2 mice. Overall, this animal model described here provides a useful tool for studying SARS-CoV-2 transmission and pathogenesis and evaluating COVID-19 vaccines and therapeutics.

Molecular Mechanism for Antibody-Dependent Enhancement of Coronavirus Entry.

Antibody-dependent enhancement (ADE) of viral entry has been a major concern for epidemiology, vaccine development, and antibody-based drug therapy. However, the molecular mechanism behind ADE is still elusive. Coronavirus spike protein mediates viral entry into cells by first binding to a receptor on the host cell surface and then fusing viral and host membranes. In this study, we investigated how a neutralizing monoclonal antibody (MAb), which targets the receptor-binding domain (RBD) of Middle East respiratory syndrome (MERS) coronavirus spike, mediates viral entry using pseudovirus entry and biochemical assays. Our results showed that MAb binds to the virus surface spike, allowing it to undergo conformational changes and become prone to proteolytic activation. Meanwhile, MAb binds to cell surface IgG Fc receptor, guiding viral entry through canonical viral-receptor-dependent pathways. Our data suggest that the antibody/Fc-receptor complex functionally mimics viral receptor in mediating viral entry. Moreover, we characterized MAb dosages in viral-receptor-dependent, Fc-receptor-dependent, and both-receptors-dependent viral entry pathways, delineating guidelines on MAb usages in treating viral infections. Our study reveals a novel molecular mechanism for antibody-enhanced viral entry and can guide future vaccination and antiviral strategies.IMPORTANCE Antibody-dependent enhancement (ADE) of viral entry has been observed for many viruses. It was shown that antibodies target one serotype of viruses but only subneutralize another, leading to ADE of the latter viruses. Here we identify a novel mechanism for ADE: a neutralizing antibody binds to the surface spike protein of coronaviruses like a viral receptor, triggers a conformational change of the spike, and mediates viral entry into IgG Fc receptor-expressing cells through canonical viral-receptor-dependent pathways. We further evaluated how antibody dosages impacted viral entry into cells expressing viral receptor, Fc receptor, or both receptors. This study reveals complex roles of antibodies in viral entry and can guide future vaccine design and antibody-based drug therapy.