Targeted next-generation sequencing of pathogens reveals the profile of secondary infections in COVID-19 patients (preprint)

PURPOSE: To use targeted next-generation sequencing (tNGS) of pathogens for analysing the etiological distribution of secondary infections in patients with severe and critical novel coronavirus pneumonia (COVID-19), to obtain microbial epidemiological data on secondary infections in patients with COVID-19, and to provide a reference for early empirical antibiotic treatment of such patients. METHODS: Patients with infections secondary to severe and critical COVID-19 and hospitalised at the First Affiliated Hospital of Shandong First Medical University between 1 December 2022 and 30 June 2023 were included in the study. The characteristics and etiological distribution of secondary infections in these patients were analysed using tNGS. RESULTS: A total of 95 patients with COVID-19 secondary infections were included in the study, of whom 87.37% had one or more underlying diseases. Forty-eight pathogens were detected, the most common being HSV-4, Candida albicans, Klebsiella pneumoniae, Enterococcus faecium, HSV-1, Staphylococcus aureus, Aspergillus fumigatus, Acinetobacter baumannii, HSV-5, and Stenotrophomonas maltophilia, with Pneumocystis jirovecii being detected in 14.29% of cases. The majority (76.84%) of COVID-19 secondary infections were mixed infections, with mixed viral-bacterial-fungal infections being the most common (28.42%). CONCLUSION: Most secondary infections in severe and critical COVID-19 patients are mixed, with high rates of viral and fungal infections. In clinical settings, monitoring for reactivation or secondary infections by Herpesviridae viruses is crucial; additionally, these patients have a significantly higher rate of P. jirovecii infection. tNGS testing on bronchoalveolar lavage fluid can help determine the aetiology of secondary infections early in COVID-19 patients and assist in choosing appropriate antibiotics.

Targeting ARF4-mediated intracellular transport as broad-spectrum antivirals (preprint)

Host factors that regulate cellular vesicular trafficking also contribute to progeny virions’ destination, thus representing as potential antiviral drug targets. Here we demonstrate that genetic deletion of ARF4, a regulator in vesicle transport, repressed multiple pathogenic RNA viral infections including Zika virus (ZIKV), influenza A virus (IAV), SARS-CoV-2 and Vesicular Stomatitis virus (VSV). ARF4 activation was stimulated upon viral infection, and viral production was rescued when reconstituted with the activated ARF4, but not the inactivated mutants. Mechanically, ARF4 deletion obstructed viral normal translocation into Golgi complex, but led to mis-sorting for lysosomal degradation, consequently caused the blockage of final release. More importantly, ARF4 targeting peptides achieved significant therapeutic efficacy against ZIKV and IAV challenge in mice by blocking ARF4 activation. Hence, we clarify the critical role of ARF4 during viral infection, providing a broad-spectrum antiviral target and the basis for further pharmaceutical development.

Analysis of risk factors of Omicron infection and antibody response in people with HIV: a multi-center cross-sectional study (preprint)

[Purpose]Analyse the factors that influence the rate of Omicron infection, symptom severity, symptom duration and antibody levels in PLWH, in particular the role of vaccination. [Methods]We randomly investigated the Omicron infection status in 338 PLWH at multiple centers and examined their WT and Omicron specific antibodies, separately. [Results]Age was the only factor that affected the rate of Omicron infection, severity of symptoms, and duration of symptoms in PLWH. Infection, number of vaccination, ART regimen and time all affect antibody levels.With the increase of number of vaccination, both WT and BA.5 specific antibodies were gradually increased, and the overall antibody levels of PLWH with hybrid immunity were higher than those of PLWH with only vaccine immune. [Conclusions]The prevalence of Omicron is higher and symptoms is more severe in PLWH younger than 40 years, but the duration of symptoms is shorter in PLWH younger than 40 years. Although vaccination does not reduce the infection rate of SARS-CoV-2, it can significantly not only enhance the antibody level against the original strain, but also expand the antibody response against the newly emerged virus variant strain. What’s more, although breakthrough infections still occur, antibody levels can be significantly increased after hybrid immunization. For PLWH at high risk of infection, booster vaccination may be beneficial for them to cope with SARS-CoV-2 normalized infection.

Competitive fitness and homologous recombination of SARS-CoV-2 variants of concern (preprint)

SARS-CoV-2 variants continue to emerge and cocirculate in humans and wild animals. The factors driving the emergence and replacement of novel variants and recombinants remain incompletely understood. Herein, we comprehensively characterized the competitive fitness of SARS-CoV-2 wild type (WT) and three variants of concern (VOCs), Alpha, Beta and Delta, by coinfection and serial passaging assays in different susceptible cells. Deep sequencing analyses revealed cell-specific competitive fitness: the Beta variant showed enhanced replication fitness during serial passage in Caco-2 cells, whereas the WT and Alpha variant showed elevated fitness in Vero E6 cells. Interestingly, a high level of neutralizing antibody sped up competition and completely reshaped the fitness advantages of different variants. More importantly, single clone purification identified a significant proportion of homologous recombinants that emerged during the passage history, and immune pressure reduced the frequency of recombination. Interestingly, a recombination hot region located between nucleotide sites 22995 and 28866 of the viral genomes could be identified in most of the detected recombinants. Our study not only profiled the variable competitive fitness of SARS-CoV-2 under different conditions, but also provided direct experimental evidence of homologous recombination between SARS-CoV-2 viruses, as well as a model for investigating SARS-CoV-2 recombination.

Characterization and Evaluation of the Pathogenicity of a Natural Gene-Deleted Transmissible Gastroenteritis Virus in China

Porcine transmissible gastroenteritis virus is the major pathogen that causes fatal diarrhea in newborn piglets. In this study, a TGEV strain was isolated from the small intestine of diarrhea piglets in Sichuan Province, China, and designated SC2021. The complete genomic sequence of TGEV SC2021 was 28561 bp, revealing a new natural deletion TGEV strain. Based on phylogenetic analyses, TGEV SC2021 belonged to the Miller cluster and was closely related to CN strains. The newborn piglets orally challenged with TGEV SC2021 showed typical watery diarrhea. In addition, macro and micropathological changes in the lungs and intestines were observed. In conclusion, we isolated a new natural deletion virus strain and confirmed that the virus strain has high pathogenicity in newborn piglets. Moreover, macroscopic and microscopic lesions were observed in the lungs and intestines of all TGEV SC2021-infected piglets. In summary, we isolated a new natural deletion TGEV strain and demonstrated that the natural deletion strain showed high pathogenicity in newborn piglets. These data enrich the diversity of TGEV strains and help us to understand the genetic evolution and molecular pathogenesis of TGEV.

Safety and superior immunogenicity of heterologous boosting with an RBD-based SARS-CoV-2 mRNA vaccine in Chinese adults (preprint)

Homologous and heterologous booster with COVID-19 mRNA vaccines represent the most effective strategy to prevent the ongoing Omicron pandemic. The additional protection from these prototype SARS-CoV-2 S-targeting vaccine was attributed to the increased RBD-specific memory B cells with expanded potency and breadth. Herein, we show the safety and immunogenicity of heterologous boosting with the RBD-targeting mRNA vaccine AWcorna (also term ARCoV) in Chinese adults who have received two doses inactivated vaccine. The superiority over inactivated vaccine in neutralization antibodies, as well as the safety profile, support the use of AWcorna as heterologous booster in China.

Dynamical Behavior of SEIR-SVS Epidemic Models with Nonlinear Incidence and Vaccination

For some infectious diseases such as mumps, HBV, there is evidence showing that vaccinated individuals always lose their immunity at different rates depending on the inoculation time. In this paper, we propose an age-structured epidemic model using a step function to describe the rate at which vaccinated individuals lose immunity and reduce the age-structured epidemic model to the delay differential model. For the age-structured model, we consider the positivity, boundedness, and compactness of the semiflow and study global stability of equilibria by constructing appropriate Lyapunov functionals. Moreover, for the reduced delay differential equation model, we study the existence of the endemic equilibrium and prove the global stability of equilibria. Finally, some numerical simulations are provided to support our theoretical results and a brief discussion is given.

Rational development of a combined mRNA vaccine against COVID-19 and influenza (preprint)

As the world continues to experience the COVID-19 pandemic, seasonal influenza remain a cause of severe morbidity and mortality globally. Worse yet, coinfection with SARS-CoV-2 and influenza A virus (IAV) leads to more severe clinical outcomes. The development of a combined vaccine against both COVID-19 and influenza is thus of high priority. Based on our established lipid nanoparticle (LNP)-encapsulated mRNA vaccine platform, we developed and characterized a novel mRNA vaccine encoding the HA antigen of influenza A (H1N1) virus, termed ARIAV. Then, ARIAV was combined with our COVID-19 mRNA vaccine ARCoV, which encodes the receptor binding domain (RBD) of the SARS-CoV-2 S protein, to formulate the final combined vaccine, AR-CoV/IAV. Further characterization demonstrated that immunization with two doses of AR-CoV/IAV elicited robust protective antibodies as well as antigen-specific cellular immune responses against SARS-CoV-2 and IAV. More importantly, AR-CoV/IAV immunization protected mice from coinfection with IAV and the SARS-CoV-2 Alpha and Delta variants. Our results highlight the potential of the LNP-mRNA vaccine platform in preventing COVID-19 and influenza, as well as other respiratory diseases.

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.

GP73 is a glucogenic hormone that contributes to SARS-CoV-2-induced hyperglycemia (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces new-onset diabetes and severe metabolic complications of pre-existing diabetes. The pathogenic mechanism underlying this is incompletely understood. Here, we provided evidence linking circulating GP73 with the exaggerated gluconeogenesis triggered by SARS-CoV-2 infection. We found that SARS-CoV-2 infection or glucotoxic conditions increased GP73 production and secretion. Secreted GP73 then trafficked to the liver and kidney to stimulate gluconeogenesis through the cAMP/PKA pathway. By using global phosphoproteomics, we found a drastic remodeling of the PKA kinase hub exerted by GP73. Notably, plasma GP73 levels were elevated and positively correlated with blood glucose in patients with COVID19 and diabetes. Neutralization of circulating GP73 in serum of individuals infected with SARS-CoV-2 or with diabetes reduced excessive gluconeogenesis in cultured hepatocytes, and lowered blood glucose levels in animal models of diabetes. Ablation of GP73 from whole animals has a profound glucose-lowering effect secondary to reduced gluconeogenesis. Thus, GP73 is a key glucogenic hormone contributing to SARS-CoV-2-induced glucose abnormality.

Nanometer resolution in situ structure of SARS-CoV-2 post-fusion spike (preprint)

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of viral genome into host cell. To understand its detailed entry mechanism and develop specific entry inhibitor, the in situ structural information of SARS-CoV-2 spikes in different states are urgently important. Here, by using the cryo-electron microscopic tomograms, we observed spikes of inactivated SARS-CoV-2 virions in both pre-fusion and post-fusion states and solved the nanometer resolution structure of in situ post-fusion spike. With a more complete model compared to previous reports, the relative spatial position between fusion peptide and transmembrane domain was discovered. Novel oligomerizations of spikes on viral membrane were observed, likely suggesting a new mechanism of fusion pore formation.

Structure and function analysis of a potent human neutralizing antibody CA521LALA against SARS-CoV-2 (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which has resulted in ~1,119,431 deaths. There is currently no approved vaccines or therapeutics for treating COVID-19. The SARS-CoV-2 Spike protein promotes entry into host cells and is considered a key therapeutic target by many researchers. Here we describe the identification of several monoclonal antibodies that target the SARS-CoV-2 Spike protein. One human antibody, CA521LALA, demonstrated neutralization potential by immunizing human antibody transgenic mice. CA521LALA showed potent SARS-CoV-2-specific neutralization activity against SARS-CoV-2 pseudovirus and authentic SARS-CoV-2 infection in vitro. The LALA mutation introduced to CA521 abrogates the binding with Fc receptors or complement receptors reducing antibody-dependent enhancement seen with anti-SARS-CoV antibodies. CA521LALA also demonstrated having a long half-life of 9.5 days in mice and 9.3 days in rhesus monkeys. CA521LALA inhibited SARS-CoV-2 infection in SARS-CoV-2 susceptible mice at a therapeutic setting with the virus titer of the lung reduced by 4.5 logs. Structural analysis by cryo-EM revealed that CA521LALA recognizes an epitope overlapping with angiotensin converting enzyme 2 (ACE2)-binding sites in SARS-CoV-2 receptor binding domain (RBD) in the Spike protein. CA521LALA blocks the interaction by binding all three RBDs of one SARS CoV-2 spike trimer simultaneously. These results demonstrate the importance for antibody-based therapeutic interventions in the treatment of COVID-19 and identifies CA521LALA a promising antibody that reacts with SARS-CoV-2 Spike protein to strongly neutralize its activity. 

SARS-CoV-2 infection causes transient olfactory dysfunction in mice (preprint)

Olfactory dysfunction caused by SARS-CoV-2 infection represents as one of the most predictive and common symptoms in COVID-19 patients. However, the causal link between SARS-CoV-2 infection and olfactory disorders remains lacking. Herein we demonstrate intranasal inoculation of SARS-CoV-2 induces robust viral replication in the olfactory epithelium (OE), resulting in transient olfactory dysfunction in humanized ACE2 mice. The sustentacular cells and Bowman's gland cells in OE were identified as the major targets of SARS-CoV-2 before the invasion into olfactory sensory neurons. Remarkably, SARS-CoV-2 infection triggers cell death and immune cell infiltration, and impairs the uniformity of OE structure. Combined transcriptomic and proteomic analyses reveal the induction of antiviral and inflammatory responses, as well as the downregulation of olfactory receptors in OE from the infected animals. Overall, our mouse model recapitulates the olfactory dysfunction in COVID-19 patients, and provides critical clues to understand the physiological basis for extrapulmonary manifestations of COVID-19.

A proof of concept for neutralizing antibody-guided vaccine design against SARS-CoV-2 (preprint)

Mutations and transient conformational movements of receptor binding domain (RBD) that make neutralizing epitopes momentarily unavailable, present immune escape routes to SARS-CoV-2. To mitigate viral escape, we developed a cocktail of neutralizing antibodies (NAbs) targeting epitopes located on different domains of spike (S) protein. Screening of a library of monoclonal antibodies generated from peripheral blood mononuclear cells of COVID-19 convalescent patients yielded potent NAbs, targeting N-terminal domain (NTD) and RBD domain of S, effective at nM concentrations. Remarkably, combination of RBD-targeting NAbs and NTD-binding NAb, FC05, dramatically enhanced the neutralization potency in cell-based assays and animal model. Results of competitive SPR assays and cryo-EM structures of Fabs bound to S unveil determinants of immunogenicity. Combinations of immunogens, identified in NTD and RBD of S, when immunized in rabbits elicited potent protective immune responses against SARS-CoV-2. These results provide a proof-of-concept for neutralization-based immunogen design targeting SARS-CoV-2 NTD and RBD.

Development and Validation of a Deep Learning-Based Model Using Computed Tomography Imaging for Predicting Disease Severity of Coronavirus Disease 2019.

OBJECTIVES: Coronavirus disease 2019 (COVID-19) is sweeping the globe and has resulted in infections in millions of people. Patients with COVID-19 face a high fatality risk once symptoms worsen; therefore, early identification of severely ill patients can enable early intervention, prevent disease progression, and help reduce mortality. This study aims to develop an artificial intelligence-assisted tool using computed tomography (CT) imaging to predict disease severity and further estimate the risk of developing severe disease in patients suffering from COVID-19. MATERIALS AND METHODS: Initial CT images of 408 confirmed COVID-19 patients were retrospectively collected between January 1, 2020 and March 18, 2020 from hospitals in Honghu and Nanchang. The data of 303 patients in the People's Hospital of Honghu were assigned as the training data, and those of 105 patients in The First Affiliated Hospital of Nanchang University were assigned as the test dataset. A deep learning based-model using multiple instance learning and residual convolutional neural network (ResNet34) was developed and validated. The discrimination ability and prediction accuracy of the model were evaluated using the receiver operating characteristic curve and confusion matrix, respectively. RESULTS: The deep learning-based model had an area under the curve (AUC) of 0.987 (95% confidence interval [CI]: 0.968-1.00) and an accuracy of 97.4% in the training set, whereas it had an AUC of 0.892 (0.828-0.955) and an accuracy of 81.9% in the test set. In the subgroup analysis of patients who had non-severe COVID-19 on admission, the model achieved AUCs of 0.955 (0.884-1.00) and 0.923 (0.864-0.983) and accuracies of 97.0 and 81.6% in the Honghu and Nanchang subgroups, respectively. CONCLUSION: Our deep learning-based model can accurately predict disease severity as well as disease progression in COVID-19 patients using CT imaging, offering promise for guiding clinical treatment.

Impaired cellular immunity to SARS-CoV-2 in severe COVID-19 patients (preprint)

The World Health Organization has declared SARS-CoV-2 virus outbreak a world-wide pandemic. Individuals infected by the virus exhibited different degrees of symptoms, the basis of which remains largely unclear. Currently, though convalescent individuals have been shown with both cellular and humoral immune responses, there is very limited understanding on the immune responses, especially adaptive immune responses, in patients with severe COVID-19. Here, we examined 10 blood samples from COVID-19 patients with acute respiratory distress syndrome (ARDS). The majority of them (70%) mounted SARS-CoV-2-specific humoral immunity with production of neutralizing antibodies. However, compared to healthy controls, the percentages and absolute numbers of both NK cells and CD8+ T cells were significantly reduced, accompanied with decreased IFN{gamma} expression in CD4+ T cells in peripheral blood from severe patients. Most notably, we failed in detecting SARS-CoV-2-specific IFN{gamma} production by peripheral blood lymphocytes from these patients. Our work thus indicates that COVID-19 patients with severe symptoms are associated with defective cellular immunity, which not only provides insights on understanding the pathogenesis of COVID-19, but also has implications in developing an effective vaccine to SARS-CoV-2.

Phase-adjusted estimation of the COVID-19 outbreak in South Korea under multi-source data and adjustment measures: a modelling study.

Based on the reported data from February 16, 2020 to March 9, 2020 in South Korea including confirmed cases, death cases and recovery cases, the control reproduction number was estimated respectively at different control measure phases using Markov chain Monte Carlo method and presented using the resulting posterior mean and 95% credible interval (CrI). At the early phase from February 16 to February 24, we estimate the basic reproduction number R0 of COVID-19 to be 4.79(95% CrI 4.38 - 5.2). The estimated control reproduction number dropped rapidly to Rc ≈ 0.32(95% CrI 0.19 - 0.47) at the second phase from February 25 to March 2 because of the voluntary lockdown measures. At the third phase from March 3 to March 9, we estimate Rc to be 0.27 (95% CrI 0.14 - 0.42). We predict that the final size of the COVID-19 outbreak in South Korea is 9661 (95% CrI 8660 - 11100) and the whole epidemic will be over by late April. It is found that reducing contact rate and enhancing the testing speed will have the impact on the peak value and the peak time.

Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody (preprint)

The COVID-19 pandemic caused by the SARS-CoV-2 virus has resulted in an unprecedented public health crisis. There are no approved vaccines or therapeutics for treating COVID-19. Here we reported a humanized monoclonal antibody, H014, efficiently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 at nM level by engaging the S receptor binding domain (RBD). Importantly, H014 administration reduced SARS-CoV-2 titers in the infected lungs and prevented pulmonary pathology in hACE2 mouse model. Cryo-EM characterization of the SARS-CoV-2 S trimer in complex with the H014 Fab fragment unveiled a novel conformational epitope, which is only accessible when the RBD is in open conformation. Biochemical, cellular, virological and structural studies demonstrated that H014 prevents attachment of SARS-CoV-2 to its host cell receptors. Epitope analysis of available neutralizing antibodies against SARS-CoV and SARS-CoV-2 uncover broad cross-protective epitopes. Our results highlight a key role for antibody-based therapeutic interventions in the treatment of COVID-19. One sentence summaryA potent neutralizing antibody conferred protection against SARS-CoV-2 in an hACE2 humanized mouse model by sterically blocking the interaction of the virus with its receptor.

Rapid adaptation of SARS-CoV-2 in BALB/c mice: Novel mouse model for vaccine efficacy (preprint)

Coronavirus disease 2019 (COVID-19) threatens global public health and economy. In order to develop safe and effective vaccines, suitable animal models must be established. Here we report the rapid adaption of SARS-CoV-2 in BALB/c mice, based on which a convenient, economical and effective animal model was developed. Specifically, we found that mouse-adapted SARS-CoV-2 at passage 6 (MACSp6) efficiently infected both aged and young wild-type BALB/c mice, resulting in moderate pneumonia as well as inflammatory responses. The elevated infectivity of MACSp6 in mice could be attributed to the substitution of a key residue (N501Y) in the receptorbinding domain (RBD). Using this novel animal model, we further evaluated the in vivo protective efficacy of an RBD-based SARS-CoV-2 subunit vaccine, which elicited highly potent neutralizing antibodies and conferred full protection against SARS-CoV-2 MACSp6 challenge. This novel mouse model is convenient and effective in evaluating the in vivo protective efficacy of SARS-CoV-2 vaccine. SummaryThis study describes a unique mouse model for SARS-CoV-2 infection and confirms protective efficacy of a SARS-CoV-2 RBD subunit vaccine.

Characterization of anti-viral immunity in recovered individuals infected by SARS-CoV-2 (preprint)

The WHO has declared SARS-CoV-2 outbreak a public health emergency of international concern. However, to date, there was hardly any study in characterizing the immune responses, especially adaptive immune responses to SARS-CoV-2 infection. In this study, we collected blood from COVID-19 patients who have recently become virus-free and therefore were discharged, and analyzed their SARS-CoV-2-specific antibody and T cell responses. We observed SARS-CoV-2-specific humoral and cellular immunity in the patients. Both were detected in newly discharged patients, suggesting both participate in immune-mediated protection to viral infection. However, follow-up patients (2 weeks post discharge) exhibited high titers of IgG antibodies, but with low levels of virus-specific T cells, suggesting that they may enter a quiescent state. Our work has thus provided a basis for further analysis of protective immunity to SARS-CoV-2, and understanding the pathogenesis of COVID-19, especially in the severe cases. It has also implications in designing an effective vaccine to protect and treat SARS-CoV-2 infection.