The ongoing COVID-19 pandemic has prioritized the development of small animal models for SARS-CoV-2. Herein, 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 (termed MASCp6) showed increased infectivity in mouse lung, and led to interstitial pneumonia and inflammatory responses in both young and aged mice following 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 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.

Human intestinal tract as an alternative route to acquire MERS-CoV infection.

The mortality of human infection by influenza A/H5N1 virus can exceed 80%. The high mortality and its poor response to the neuraminidase inhibitor oseltamivir have been attributed to uncontrolled virus-induced cytokine storm. We challenged BALB/c mice with 1,000 LD 50 of influenza A/Vietnam/1194/04. Survival, body weight, histopathology, inflammatory markers, viral loads, T lymphocyte counts, and neutralizing antibody response were documented in infected mice treated individually or in combination with zanamvir, celecoxib, gemfibrozil, and mesalazine. To imitate the real-life scenario, treatment was initiated at 48 h after viral challenge. There were significant improvements in survival rate (P ‫؍‬ 0.02), survival time (P < 0.02), and inflammatory markers (P < 0.01) in the group treated with a triple combination of zanamivir, celecoxib, and mesalazine when compared with zanamivir alone. Zanamivir with or without immunomodulators reduced viral load to a similar extent. Insignificant prolongation of survival was observed when individual agents were used alone. Significantly higher levels of CD4 ؉ and CD8 ؉ T lymphocytes and less pulmonary inflammation were also found in the group receiving triple therapy. Zanamivir alone reduced viral load but not inflammation and mortality. The survival benefits of adding celecoxib and mesalazine to zanamivir could be caused by their synergistic effects in reducing cytokine dysfunction and preventing apoptosis. Combinations of a neuraminidase inhibitor with these immunomodulators should be considered in randomized controlled treatment trials of patients suffering from H5N1 infection.zanamivir ͉ celecoxib ͉ mesalazine

The pathogenesis of highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) remains poorly understood. In a previous study, we established an hDPP4-transgenic (hDPP4-Tg) mouse model in which MERS-CoV infection causes severe acute respiratory failure and high mortality accompanied by an elevated secretion of cytokines and chemokines. Since excessive complement activation is an important factor that contributes to acute lung injury after viral infection, in this study, we investigated the role of complement in MERS-CoV-induced lung damage. Our study showed that complement was excessively activated in MERS-CoV-infected hDPP4-Tg mice through observations of increased concentrations of the C5a and C5b-9 complement activation products in sera and lung tissues, respectively. Interestingly, blocking C5a production by targeting its receptor, C5aR, alleviated lung and spleen tissue damage and reduced inflammatory responses. More importantly, anti-C5aR antibody treatment led to decreased viral replication in lung tissues. Furthermore, compared with the sham treatment control, apoptosis of splenic cells was less pronounced in the splenic white pulp of treated mice, and greater number of proliferating splenic cells, particularly in the red pulp, was observed. These data indicate that (1) dysregulated host immune responses contribute to the severe outcome of MERS; (2) excessive complement activation, triggered by MERS-CoV infection, promote such dysregulation; and (3) blockade of the C5a–C5aR axis lead to the decreased tissue damage induced by MERS-CoV infection, as manifested by reduced apoptosis and T cell regeneration in the spleen. Therefore, the results of this study suggest a new strategy for clinical intervention and adjunctive treatment in MERS-CoV cases.

Development of effective vaccines against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is still a priority in prevention of re-emergence of SARS. Our previous studies have shown that the receptor-binding domain (RBD) of SARS-CoV spike (S) protein elicits highly potent neutralizing antibody responses in the immunized animals. But it is unknown whether RBD can also induce protective immunity in an animal model, a key aspect for vaccine development. In this study, BALB/c mice were vaccinated intramuscularly (i.m.) with 10microg of RBD-Fc (RBD fused with human IgG1 Fc) and boosted twice at 3-week intervals and one more time at 12th month. Humoral immune responses of vaccinated mice were investigated for up to 12 months at a 1-month interval and the neutralizing titers of produced antibodies were reported at months 0, 3, 6 and 12 post-vaccination. Mice were challenged with the homologous strain of SARS-CoV 5 days after the last boost, and sacrificed 5 days after the challenge. Mouse lung tissues were collected for detection of viral load, virus replication and histopathological effects. Our results showed that RBD-Fc vaccination induced high titer of S-specific antibodies with long-term and potent SARS-CoV neutralizing activity. Four of five vaccinated mice were protected from subsequent SARS-CoV challenge because no significant virus replication, and no obvious histopathological changes were found in the lung tissues of the vaccinated mice challenged with SARS-CoV. Only one vaccinated mouse had mild alveolar damage in the lung tissues. In contrast, high copies of SARS-CoV RNA and virus replication were detected, and pathological changes were observed in the lung tissues of the control mice. In conclusion, our findings suggest that RBD, which can induce protective antibodies to SARS-CoV, may be further developed as a safe and effective SARS subunit vaccine.

An emerging respiratory infectious disease with high mortality, Middle East respiratory syndrome (MERS), is caused by a novel coronavirus (MERS-CoV). It was first reported in 2012 in Saudi Arabia and has now spread to eight countries. Development of effective therapeutics and vaccines is crucial to save lives and halt the spread of MERS-CoV. Here, we show that a recombinant protein containing a 212-amino acid fragment (residues 377-588) in the truncated receptor-binding domain (RBD: residues 367–606) of MERS-CoV spike (S) protein fused with human IgG Fc fragment (S377-588-Fc) is highly expressed in the culture supernatant of transfected 293T cells. The purified S377-588-Fc protein efficiently binds to dipeptidyl peptidase 4 (DPP4), the receptor of MERS-CoV, and potently inhibited MERS-CoV infection, suggesting its potential to be further developed as a therapeutic modality for treating MERS-CoV infection and saving the patients’ lives. The recombinant S377-588-Fc is able to induce in the vaccinated mice strong MERS-CoV S-specific antibodies, which blocks the binding of RBD to DPP4 receptor and effectively neutralizes MERS-CoV infection. These findings indicate that this truncated RBD protein shows promise for further development as an effective and safe vaccine for the prevention of MERS-CoV infection.

A novel human Middle East respiratory syndrome coronavirus (MERS-CoV) caused outbreaks of severe acute respiratory syndrome (SARS)-like illness with a high mortality rate, raising concerns of its pandemic potential. Dipeptidyl peptidase-4 (DPP4) was recently identified as its receptor. Here we showed that residues 377 to 662 in the S protein of MERS-CoV specifically bound to DPP4-expressing cells and soluble DPP4 protein and induced significant neutralizing antibody responses, suggesting that this region contains the receptor-binding domain (RBD), which has a potential to be developed as a MERS-CoV vaccine. In 2003, Farzan and colleagues successfully identified the receptor of SARS-CoV, angiotensin-converting enzyme 2 (ACE2) (7), and a 193-amino-acid fragment in the spike (S) protein (residues 318 to 510) as the receptor-binding domain (RBD) (8). We found that SARS-CoV S-RBD contains a critical neutralizing site (9) which induces potent neutralizing antibodies and protection against SARS-CoV infection in an animal model (10).Since MERS-CoV is genetically related to SARS-CoV (1), we compared their S protein sequences and predicted that the RBD of MERS-CoV might be located in the region spanning residues 377 to 662 of the S1 subunit (Fig. 1). Using the Swiss-Model Workplace homology modeling server (11) and basing our work on the X-ray crystal structure of the SARS-CoV S-RBD (Protein Data Bank [PDB] identification no. 2DD8) (12), we predicted the conformational structure of the region consisting of residues 377 to 662 in the S1 subunit of the MERS-CoV S protein (13). We noticed that the SARS-CoV S-RBD and the predicted MERS-CoV S-RBD possessed similar core structures but had an extended secondary structure consisting predominantly of the receptor-binding motifs (RBM) (12,14). The extended region in MERS-CoV S-RBD is much longer than that in SARS-CoV S-RBD, suggesting that MERS-CoV and SARS-CoV use different receptors. Indeed, it has been proven that dipeptidyl peptidase-4 (DPP4; also known as CD26) is the functional receptor of MERS-CoV (15).We then constructed MERS-CoV S-RBD based on the synthesized codon-optimized MERS-CoV S sequences (GenBank accession no. AFS88936.1) and fused it to Fc of human IgG using pFUSE-hIgG1-Fc2 expression vector (here named Fc) (InvivoGen, San Diego, CA). The SARS-CoV S-RBD-Fc was constructed by fusing RBD of codon-optimized SARS-CoV S sequence into the Fc vector referred to above as a control (Fig. 1) (16). The S-RBD-Fc proteins were expressed in 293T cell culture supernatant and purified by protein A affinity chromatography (GE Healthcare, Piscataway, NJ) (17). We found that both MERS-CoV and SARS-CoV S-RBD-Fc proteins were highly purified from transfected culture supernatants ( Fig. 2A, panel a). MERS-CoV S-RBD-Fc could be recognized by an MERS-CoV S-specific polyclonal antibody (1:1,000), while SARS-CoV S-RBD-Fc could not react with this antibody, as detected by Western blotting (Fig. 2A, panel b).Using analysis performed by Western blotting, we found that DPP4 was highly express...

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) was originally identified in Saudi Arabia in 2012. It has caused MERS outbreaks with high mortality in the Middle East and Europe, raising a serious concern about its pandemic potential. Therefore, development of effective vaccines is crucial for preventing its further spread and future pandemic. Our previous study has shown that subcutaneous (s.c.) vaccination of a recombinant protein containing receptor-binding domain (RBD) of MERS-CoV S fused with Fc of human IgG (RBD-Fc) induced strong systemic neutralizing antibody responses in vaccinated mice. Here, we compared local and systemic immune responses induced by RBD-Fc via intranasal (i.n.) and s.c. immunization pathways. We found that i.n. vaccination of MERS-CoV RBD-Fc induced systemic humoral immune responses comparable to those induced by s.c. vaccination, including neutralizing antibodies, but more robust systemic cellular immune responses and significantly higher local mucosal immune responses in mouse lungs. This study suggests the potential of developing MERS-CoV RBD protein into an effective and safe mucosal candidate vaccine for prevention of respiratory tract infections caused by MERS-CoV.