Conversion of monoclonal IgG to dimeric and secretory IgA restores neutralizing ability and prevents infection of Omicron lineages (preprint)

The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal IgA response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further show that the intramuscular mRNA and/or inactivated vaccines cannot sufficiently boost the mucosal sIgA response in uninfected individuals, particularly against the Omicron variant. We thus engineered and characterized recombinant monomeric, dimeric and secretory IgA1 antibodies derived from four neutralizing IgG monoclonal antibodies targeting the receptor-binding domain of the spike protein (01A05, rmAb23, DXP-604 and XG014). Compared to their parental IgG antibodies, dimeric and secretory IgA1 antibodies showed a higher neutralizing activity against different variants of concern (VOCs), in part due to an increased avidity. Importantly, the dimeric or secretory IgA1 form of the DXP-604 antibody significantly outperformed its parental IgG antibody, and neutralized the Omicron lineages BA.1, BA.2 and BA.4/5 with a 50-150-fold increase in potency, reaching the level of the most potent monoclonal antibodies described till date. In hACE2 transgenic mice, a single intranasal dose of the dimeric IgA DXP-604 conferred prophylactic and therapeutic protection against Omicron BA.5. Conversion of IgA and dimerization further enhanced or restored the neutralizing ability against the emerging Omicron sub-variants (DXP-604 for BQ.1, BQ.1.1 and BA2.75; 01A05 for BA2.75, BA.2.75.2 and XBB.1). Thus, dimeric or secretory IgA delivered by nasal administration may potentially be exploited for the treatment and prevention of Omicron infection, thereby providing an alternative tool for combating immune evasion by subvariants and, potentially, future VOCs.

Rational identification of potent and broad sarbecovirus-neutralizing antibody cocktails from SARS convalescents (preprint)

SARS-CoV-2 Omicron sublineages have escaped most RBD-targeting therapeutic neutralizing antibodies (NAbs), which proves the previous NAb drug screening strategies deficient against the fast-evolving SARS-CoV-2. Better broad NAb drug candidate selection methods are needed. Here, we describe a rational approach for identifying RBD-targeting broad SARS-CoV-2 NAb cocktails. Based on high-throughput epitope determination, we propose that broad NAb drugs should target non-immunodominant RBD epitopes to avoid herd immunity-directed escape mutations. Also, their interacting antigen residues should focus on sarbecovirus conserved sites and associate with critical viral functions, making the antibody-escaping mutations less likely to appear. Following the criteria, a featured non-competing antibody cocktail, SA55+SA58, is identified from a large collection of broad sarbecovirus NAbs isolated from SARS convalescents. SA55+SA58 potently neutralizes ACE2-utilizing sarbecoviruses, including circulating Omicron variants, and could serve as broad SARS-CoV-2 prophylactics to offer long-term protection. Our screening strategy can also be further applied to identify broad-spectrum NAb drugs against other fast-evolving viruses.

Spatiotemporal landscape of SARS-CoV-2 pulmonary infection reveals Slamf9+Spp1+ macrophages promoting viral clearance and inflammation resolution (preprint)

While SARS-CoV-2 pathogenesis has been intensively investigated, the host mechanisms of viral clearance and inflammation resolution are still elusive because of the ethical limitation of human studies based on COVID-19 convalescents. Here we infected Syrian hamsters by authentic SARS-CoV-2 and built an ideal model to simulate the natural recovery process of SARS-CoV-2 infection from severe pneumonia. We developed and applied a spatial transcriptomic sequencing technique with subcellular resolution and tissue-scale extensibility, i.e., Stereo-seq, together with single-cell RNA sequencing (scRNA-seq), to the entire lung lobes of 45 hamsters and obtained an elaborate map of the pulmonary spatiotemporal changes from acute infection, severe pneumonia to the late viral clearance and inflammation resolution. While SARS-CoV-2 infection caused massive damages to the hamster lungs, including naive T cell infection and deaths related to lymphopenia, we identified a group of monocyte-derived proliferating Slamf9+Spp1+ macrophages, which were SARS-CoV-2 infection-inducible and cell death-resistant, recruiting neutrophils to clear viruses together. After viral clearance, the Slamf9+Spp1+ macrophages differentiated into Trem2+ and Fbp1+ macrophages, both responsible for inflammation resolution and replenishment of alveolar macrophages. The existence of this specific macrophage subpopulation and its descendants were validated by RNAscope in hamsters, immunofluorescence in hACE2 mice, and public human autopsy scRNA-seq data of COVID-19 patients. The spatiotemporal landscape of SARS-CoV-2 infection in hamster lungs and the identification of Slamf9+Spp1+ macrophages that is pivotal to viral clearance and inflammation resolution are important to better understand the critical molecular and cellular players of COVID-19 host defense and also develop potential interventions of COVID-19 immunopathology.

Binding affinity improvement analysis of multiple-mutant Omicron on 2019-nCov to human ACE2 by In-silico predictions (preprint)

Since the outbreak of COVID-19 in 2019, the 2019-nCov coronavirus has appeared diverse mutational characteristics due to its own flexible conformation. One multiple-mutant strain (Omicron) with surprisingly infective activity outburst, and affected the biological activities of current drugs and vaccines, making the epidemic significantly difficult to prevent and control, and seriously threaten health around the world. Importunately exploration of mutant characteristics for novel coronavirus Omicron can supply strong theoretical guidance for learning binding mechanism of mutant viruses. What’s more, full acknowledgement of key mutated-residues on Omicron strain can provide new methodology of the novel pathogenic mechanism to human ACE2 receptor, as well as the subsequent vaccine development. In this research, 3D structures of 32 single-point mutations of 2019-nCov were firstly constructed, and 32-sites multiple-mutant Omicron were finally obtained based one the wild-type virus by homology modeling method. One total number of 33 2019-nCov/ACE2 complex systems were acquired by protein-protein docking, and optimized by using preliminary molecular dynamics simulations. Binding free energies between each 2019-nCov mutation system and human ACE2 receptor were calculated, and corresponding binding patterns especially the regions adjacent to mutation site were analyzed. The results indicated that one total number of 6 mutated sites on the Omicron strain played crucial role in improving binding capacities from 2019-nCov to ACE2 protein. Subsequently, we performed long-term molecular dynamic simulations and protein-protein binding energy analysis for the selected 6 mutations. 3 infected individuals, the mutants T478K, Q493R and G496S with lower binding energies − 66.36, -67.98 and − 67.09 kcal/mol also presents the high infectivity. These findings indicated that the 3 mutations T478K, Q493R and G496S play the crucial roles in enhancing binding affinity of Omicron to human ACE2 protein. All these results illuminate important theoretical guidance for future virus detection of the Omicron epidemic, drug research and vaccine development.

A subset of Memory B-derived antibody repertoire from 3-dose vaccinees is ultrapotent against diverse and highly transmissible SARS-CoV-2 variants, including Omicron (preprint)

Omicron, the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. We examined whether sera from individuals who received two or three doses of inactivated vaccine, could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2/60) and 95% (57/60) for 2- and 3-dose vaccinees, respectively. For three-dose recipients, the geometric mean neutralization antibody titer (GMT) of Omicron was 15, 16.5-fold lower than that of the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in 3-dose vaccinees, half of which recognize the receptor binding domain (RBD) and show that a subset of them (24/163) neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron, potently. Therapeutic treatments with representative broadly neutralizing mAbs individually or antibody cocktails were highly protective against SARS-CoV-2 Beta infection in mice. Atomic structures of the Omicron S in complex with three types of all five VOC-reactive antibodies defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to one major class of antibodies bound at the right shoulder of RBD through altering local conformation at the binding interface. Our results rationalize the use of 3-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are a rational target for a universal sarbecovirus vaccine. One sentence summary A sub-set of antibodies derived from memory B cells of volunteers vaccinated with 3 doses of an inactivated SARS-CoV-2 vaccine work individually as well as synergistically to keep variants, including Omicron, at bay.

Single-shot rAAV5-based Vaccine Provides Long-term Protective Immunity against SARS-CoV-2 and Its Variants (preprint)

The COVID-19 pandemic and the SARS-CoV-2 with its variants have posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against the SARS-CoV-2 variants. Therefore, novel vaccines to match current mutated viral lineages with long-term protective immunity are urgently in demand. In the current study, we for the first time designed a recombinant Adeno-Associated Virus 5 (rAAV5)-based vaccine named as rAAV-COVID-19 vaccine (Covacinplus) by using RBD-plus of spike protein with both the single-stranded and the self-complementary AAV5 delivering vectors (ssAAV5 and scAAAV5), which provides excellent protection from SARS-CoV-2 infection. A single dose vaccination induced the strong immune response against SARS-CoV-2. The induced neutralizing antibodies (NAs) titers were maintained at a high peak level of over 1:1024 even after more than one year of injection and accompanied with functional T-cells responses in mice. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines exhibited high levels of serum NAs against current circulating variants including variants Alpha, Beta, Gamma and Delta. SARS-CoV-2 virus challenge test showed that ssAAV5-RBD-plus vaccine protected both young and old age mice from SARS-CoV-2 infection in the upper and the lower respiratory tracts. Moreover, whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genome of the vaccinated mice after one year vaccination, demonstrating excellent safety of the vaccine. Taken together, this study suggests that rAAV5-based vaccine is powerful against SARS-CoV-2 and its variants with long-term protective immunity and excellent safety, which has great potential for development into prophylactic vaccination in human to end this global pandemic.

Comparison of Wild Type DNA Sequence of Spike Protein from SARS-CoV-2 with Optimized Sequence on The Induction of Protective Responses Against SARS-Cov-2 Challenge in Mouse Model (preprint)

COVID-19 caused by SARS-CoV-2 has been spreading worldwide. To date, several vaccine candidates moved into EUA or CA applications. Although DNA vaccine is on phase III clinical trial, it is a promised technology platform with many advantages. Here, we showed that the pGX9501 DNA vaccine encoded the spike full-length protein-induced strong humoral and cellular immune responses in mice with higher neutralizing antibodies, blocking the hACE2-RBD binding against live virus infection in vitro. Importantly, higher levels of IFN-{gamma} expression in CD8+ and CD4+ T cell and specific cytotoxic lymphocyte (CTL) killings effect were also observed in the pGX9501-immunized group. It provided subsequent protection against virus challenges in the hACE2 transgenic mouse model. Overall, pGX9501 was a promising DNA vaccine candidate against COVID-19, inducing strong humoral immunity and cellular immunity that contributed to the vaccines protective effects.

The Genetic Diversity Mice Models in SARS-CoV-2 Infection (preprint)

The SARS-CoV-2 has led to a worldwide health crisis. The ACE2 has been identified as the entry receptor in a species-specific manner. Classic laboratory mice were insusceptible since the virus cannot use murine ACE2 orthologue. Animal models rely on gene modification on the virus or the host. However, these mice were restricted in limited genetic backgrounds and did not support natural infection. Here we showed two wild-type inbred lines (CAST and FEW) from Genetic Diversity mice supported authentic SARS-CoV-2 infection, and developed mild to moderate interstitial pneumonia, along with infiltrating inflammatory cells. Particularly, FEW featured age-dependent damages, while CAST charactered by pulmonary fibrosis. Genome and transcriptome comparative analysis suggested the mutated ACE2 was not responsible for SARS-CoV-2 infection in CAST and FEW, and the differential gene expressions in immune response and immune cell may be risk factors for the infection. In summary, the GD mice, derived from the multi-parental panel, provided promising murine models for exploring sophisticated pathogenesis in SARS-CoV-2.

Differential transcriptomic landscapes of multiple organs from SARS-CoV-2 early infected rhesus macaques (preprint)

SARS-CoV-2 infection causes complicated clinic manifestations with variable multi-organ injuries, however, the underlying mechanism, in particular immune responses in different organs, remains elusive. In this study, comprehensive transcriptomic alterations of 14 tissues from rhesus macaque infected with SARS-CoV-2 were analyzed. Compared to normal controls, SARS-CoV-2 infection resulted in dysregulation of genes involving diverse functions in various tissues/organs examined, with drastic transcriptomic changes in cerebral cortex and right ventricle. Intriguingly, cerebral cortex exhibited a hyperinflammatory state evidenced by significant upregulation of inflammation response-related genes. Meanwhile, expressions of coagulation, angiogenesis and fibrosis factors were also up-regulated in cerebral cortex. Neuronal receptor NRP1 expression showed a significant induction by SARS-CoV-2 in cerebral cortex, which might be responsible for a higher infectivity and consequent inflammatory response. Overall, our study depicts a multi-tissue/organ transcriptomic landscapes of rhesus macaque with early infection of SARS-CoV-2, and provides important insights into the mechanistic basis for COVID-19-associated clinical complications.

Alarmin S100A8/A9 Mediates Activation of Aberrant Neutrophils in the Pathogenesis of COVID-19 (preprint)

The SARS-CoV-2 pandemic poses an unprecedented public health crisis. Accumulating evidences suggest that SARS-CoV-2 infection causes dysregulation of immune system. However, the unique signature of early immune responses remains elusive. We characterized the transcriptome of rhesus macaques and mice infected with SARS-CoV-2. Alarmin S100A8 was robustly induced by SARS-CoV-2 in animal models as well as in COVID-19 patients. Paquinimod, a specific inhibitor of S100A8/A9, could reduce inflammatory response and rescue the pneumonia with substantial reduction of viral titers in SASR-CoV-2 infected animals. Remarkably, Paquinimod treatment resulted in 100% survival of mice in a lethal model of mouse coronavirus (MHV) infection. A novel group of neutrophils that contributed to the uncontrolled inflammation and onset of COVID-19 were dramatically induced by coronavirus infections. Paquinimod treatment could reduce these neutrophils and regain antiviral responses, unveiling key roles of S100A8/A9 and noncanonical neutrophils in the pathogenesis of COVID-19, highlighting new opportunities for therapeutic intervention.Funding: This work was supported by the National Natural Science Foundation of China (31570891; 31872736), the National Key Research and Development Program of China (2016YFA0500302; 2020YFA0707800), the National Key Research and Development Program (2020YFA0707500) and the Strategic Priority Research Program (XDB29010000). Xiangxi Wang was supported by Ten Thousand Talent Program and the NSFS Innovative Research Group (81921005). We thank National Mega projects of China for Major Infectious Diseases (2017ZX10304402), CAMS initiative for Innovative Medicine of China (2016-12M-2-006) and The National Natural Science Foundation of China (82041008) for the support on the animal model study. Conflict of Interest: The authors have no conflicts of interest to declare.Ethical Approval: All experiments with live SARS-CoV-2 viruses were carried out in the enhanced biosafety level 3 (P3+) facilities in the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) approved by the National Health Commission of the People’s Republic of China. All animals care and use were in accordance with the Guide for the Care and Use of Laboratory Animals of the Chinese Association for Laboratory Animal Science. All procedures of animal handling were approved by the Animal Care Committee of Peking University Health Science Center.

Small molecules inhibit SARS-COV-2 induced aberrant inflammation and viral replication in mice by targeting S100A8/A9-TLR4 axis (preprint)

The SARS-CoV-2 pandemic poses an unprecedented public health crisis. Accumulating evidences suggest that SARS-CoV-2 infection causes dysregulation of immune system. However, the unique signature of early immune responses remains elusive. We characterized the transcriptome of rhesus macaques and mice infected with SARS-CoV-2. Alarmin S100A8 was robustly induced by SARS-CoV-2 in animal models as well as in COVID-19 patients. Paquinimod, a specific inhibitor of S100A8/A9, could reduce inflammatory response and rescue the pneumonia with substantial reduction of viral titers in SASR-CoV-2 infected animals. Remarkably, Paquinimod treatment resulted in 100% survival of mice in a lethal model of mouse coronavirus (MHV) infection. A novel group of neutrophils that contributed to the uncontrolled inflammation and onset of COVID-19 were dramatically induced by coronavirus infections. Paquinimod treatment could reduce these neutrophils and regain antiviral responses, unveiling key roles of S100A8/A9 and noncanonical neutrophils in the pathogenesis of COVID-19, highlighting new opportunities for therapeutic intervention.

Immunogenicity and Safety of a SARS-CoV-2 Inactivated Vaccine in Healthy Adults Aged 18-59 years: Report of the Randomized, Double-blind, and Placebo-controlled Phase 2 Clinical Trial (preprint)

BACKGROUND The top priority for the control of COVID-19 pandemic currently is the development of a vaccine. A phase 2 trial conducted to further evaluate the immunogenicity and safety of a SARS-CoV-2 inactivated vaccine (CoronaVac). METHODS We conducted a randomized, double-blind, placebo-controlled trial to evaluate the optimal dose, immunogenicity and safety of the CoronaVac. A total of 600 healthy adults aged 18-59 years were randomly assigned to receive 2 injections of the trial vaccine at a dose of 3 g/0.5 mL or 6 g /0.5mL, or placebo on Day 0,14 schedule or Day 0,28 schedule. For safety evaluation, solicited and unsolicited adverse events were collected after each vaccination within 7 days and 28 days, respectively. Blood samples were taken for antibody assay. RESULTS CoronaVac was well tolerated, and no dose-related safety concerns were observed. Most of the adverse reactions fell in the solicited category and were mild in severity. Pain at injection site was the most frequently reported symptoms. No Grade 3 adverse reaction or vaccine related SAEs were reported. CoronaVac showed good immunogenicity with the lower 3 g dose eliciting 92.4% seroconversion under Day 0,14 schedule and 97.4% under Day 0,28 schedule. 28 days after two-dose vaccination, the Nab levels of individual schedules range from 23.8 to 65.4 among different dosage and vaccination schedules. CONCLUSIONS Favorable safety and immunogenicity of CoronaVac was demonstrated on both schedules and both dosages, which support the conduction of phase 3 trial with optimum schedule/dosage per different scenarios.

Development and External Evaluation of Predictions Models for Mortality of COVID-19 Patients using Machine Learning Method (preprint)

Background To develop and evaluate the prognostic machine-learning model for mortality in patients with coronavirus disease 2019 (COVID-19).Methods Clinical data of confirmed COVID-19 were retrospectively collected from Wuhan between 18th January and 29th March 2020. Gradient Boosting Decision Tree (GBDT), logistic regression (LR) model, and simplified LR with selected 5 features (LR-5) model were built to predict the mortality of COVID-19. 5-fold area under curve (AUC), accuracy, positive predictive value (PPV), and negative predictive value (NPV) were calculated and compared between models.Results A total of 2,924 patients were included in the final analysis, 257(8.8%) of whom died during hospitalization and 2,667 (91.2%) survived. There were 21(0.7%) mild cases, 2,051(70.1%) moderate case, 779(26.6%) severe cases, and 73(2.5%) critically severe cases of COVID-19 on admission. The overall 5-fold AUC was observed highest in GBDT model (0.941), followed by LR (0.928) and LR-5 (0.913). The diagnostic accuracy were 0.889 in GBDT, 0.868 in LR and 0.887 in LR-5. GBDT model also showed the highest sensitivity (0.899) and speciality (0.889). The NPV of all three models exceeded 97%, while the PPV were relatively low in all models, 0.381 for LR, 0.402 for LR-5 and 0.432 for GBDT. In subgroups analysis with severe cases only, GBDT model also performed the best with a accuracy of 0.799 and 5-fold AUC (0.918).Conclusion The finding revealed that mortality prediction performance of the GBDT was superior to the LR models in confirmed cases of COVID-19, regardless of disease severity.

Rapid development of an inactivated vaccine for SARS-CoV-2 (preprint)

The COVID-19 pandemic caused by SARS-CoV-2 has brought about an unprecedented crisis, taking a heavy toll on human health, lives as well as the global economy. There are no SARS-CoV-2-specific treatments or vaccines available due to the novelty of this virus. Hence, rapid development of effective vaccines against SARS-CoV-2 is urgently needed. Here we developed a pilot-scale production of a purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc), which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats and non-human primates. These antibodies potently neutralized 10 representative SARS-CoV-2 strains, indicative of a possible broader neutralizing ability against SARS-CoV-2 strains circulating worldwide. Immunization with two different doses (3g or 6 g per dose) provided partial or complete protection in macaques against SARS-CoV-2 challenge, respectively, without any antibody-dependent enhancement of infection. Systematic evaluation of PiCoVacc via monitoring clinical signs, hematological and biochemical index, and histophathological analysis in macaques suggests that it is safe. These data support the rapid clinical development of SARS-CoV-2 vaccines for humans. One Sentence SummaryA purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc) confers complete protection in non-human primates against SARS-CoV-2 strains circulating worldwide by eliciting potent humoral responses devoid of immunopathology

Reinfection could not occur in SARS-CoV-2 infected rhesus macaques (preprint)

A global pandemic of Corona Virus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is ongoing spread. It remains unclear whether the convalescing patients have a risk of reinfection. Rhesus macaques were rechallenged with SARS-CoV-2 during an early recovery phase from initial infection characterized by weight loss, interstitial pneumonia and systemic viral dissemination mainly in respiratory and gastrointestinal tracts. The monkeys rechallenged with the identical SARS-CoV-2 strain have failed to produce detectable viral dissemination, clinical manifestations and histopathological changes. A notably enhanced neutralizing antibody response might contribute the protection of rhesus macaques from the reinfection by SARS-CoV-2. Our results indicated that primary SARS-CoV-2 infection protects from subsequent reinfection. One Sentence SummaryNeutralizing antibodies against SARS-CoV-2 might protect rhesus macaques which have undergone an initial infection from reinfection during early recovery days.

Rhesus macaques can be effectively infected with SARS-CoV-2 via ocular conjunctival route (preprint)

The outbreak of Corona Virus Disease 2019 caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) is highly transmitted. The potential extra-respiratory transmission routes remain uncertain. Five rhesus macaques were inoculated with 1x106 TCID50 of SARS-CoV-2 via conjunctival (CJ), intratracheal (IT), and intragastric (IG) routes, respectively. Remarkably, the CJ inoculated-macaques developed mild interstitial pneumonia and viral load was detectable in the conjunctival swabs at 1 days post-inoculation (dpi). Only via IT inoculation, viral load was detected in the anal swab at 1-7 dpi and macaque showed weight loss. However, viral load was undetectable after IG inoculation. Comparatively, viral load was higher in the nasolacrimal system but lesions of lung were relatively mild and local via CJ inoculation compared with that via IT inoculation, demonstrating distinct characteristics of virus dispersion. Both the two routes affected the alimentary tract. Therefore the clinicians need to protect eye while working with patients.

Inhibition of SARS-CoV-2 infection (previously 2019-nCoV) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion (preprint)

The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be confirmed. Therefore, we herein used a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed plasma membrane fusion capacity superior to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. We then generated a series of lipopeptides and found that the EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than that of EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, potently inhibiting replication of 4 live human coronaviruses, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by currently circulating SARS-CoV-2 and emerging SARSr-CoVs.

Dysregulation of Immune Response in Patients with COVID-19 in Wuhan, China (preprint)

Background: In December 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan and rapidly spread throughout China. The immune response is likely to be highly involved in the pathological process of coronavirus disease 2019 (COVID-19). However, information on specific changes of immune response in COVID-19 are limited. Methods: Demographic and clinical data of all confirmed cases with COVID-19 on admission at Tongji Hospital from January 10 to February 12, 2020, were collected and analyzed. The expression of lymphocytes, lymphocyte subsets, infection related biomarkers and inflammatory cytokines were analyzed and compared between severe cases and non-severe patients. Findings: Of the 452 patients with COVID-19 recruited from January 10 to February 12, 2020, 286 were diagnosed as severe infection. The median age was 58 years and 235 were male. 201 patients had chronic diseases and a higher percentage in the severe cases. The most common symptoms were fever, shortness of breath, expectoration, and fatigue. Severe cases tend to have higher white blood cell and neutrophil lymphopenia ratio (NLR), as well as lower percentages of monocytes, eosinophils, and basophils. Most of severe cases demonstrated elevated levels of infection-related biomarkers, and inflammatory cytokines. The numbers of B cells, T cells and NK cells was significantly decreased in patients with COVID-19, and more severely decreased in the severe cases. T cells were shown to be most affected by SARS-CoV-2, and more hampered in severe cases. Both helper T cells and suppressor T cells in patients with COVID-19 were below normal levels. Helper T cells tend to be more affected in severe cases. The percentage of naïve helper T cells increased and memory helper T cells decreased in severe cases. Patients with COVID-19 have lower level of regulatory T cells, and more obviously damaged in severe cases. Interpretation: SARS-CoV-2 might mainly act on lymphocytes, especially T lymphocytes, and induce a cytokine storm in the body, generate a series of immune responses. Surveillance of NLR and lymphocyte subsets is helpful in the early screening of critical illness, diagnosis and treatment of COVID-19.Funding Statement: None.Declaration of Interests: All authors declare no competing interests.Ethics Approval Statement: The study was performed in accordance with Tongji Hospital Ethics Committee (IRB ID: TJ-C20200121). Written informed consent was waived by the Ethics Commission of the designated hospital for emerging infectious disease.

Protection of Rhesus Macaque from SARS-Coronavirus challenge by recombinant adenovirus vaccine (preprint)

A recombinant adenovirus vaccine against the SARS Coronavirus (SARS-CoV) was constructed, which contains fragments from the S, N, and Orf8 genes. Rhesus Macaques immunized with the recombinant adenovirus generated antigen-specific humoral and cellular response. Furthermore, the vaccine provided significant protection against subsequent live SARS-CoV challenge. In contrast, three out of four monkeys immunized with placebo suffered severe alveolar damage and pulmonary destruction.

The Pathogenicity of 2019 Novel Coronavirus in hACE2 Transgenic Mice (preprint)

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) caused the Corona Virus Disease 2019 (COVID-19) cases in China has become a public health emergency of international concern (PHEIC). Based on angiotensin converting enzyme 2 (ACE2) as cell entry receptor of SARS-CoV, we used the hACE2 transgenic mice infected with SARS-CoV-2 to study the pathogenicity of the virus. Weight loss and virus replication in lung were observed in hACE2 mice infected with SARS-CoV-2. The typical histopathology was interstitial pneumonia with infiltration of significant lymphocytes and monocytes in alveolar interstitium, and accumulation of macrophages in alveolar cavities. Viral antigens were observed in the bronchial epithelial cells, alveolar macrophages and alveolar epithelia. The phenomenon was not found in wild type mice with SARS-CoV-2 infection. The pathogenicity of SARS-CoV-2 in hACE2 mice was clarified and the Kochs postulates were fulfilled as well, and the mouse model may facilitate the development of therapeutics and vaccines against SARS-CoV-2.