De novo design of site-specific protein interactions with learned surface fingerprints (preprint)

Physical interactions between proteins are essential for most biological processes governing life. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic, and structural data grows. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein-protein interaction (PPI) networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications. We exploit a geometric deep learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features critical to drive PPIs. We hypothesized these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof-of-principle, we computationally designed four de novo protein binders to engage three protein targets: SARS-CoV-2 spike, PD-1, and PD-L1. The designs bound the target sites with nanomolar affinity upon experimental optimization, structural and mutational characterization showed highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling a novel approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.

An Imported Case of COVID-19 Delta Variant B.1.617.2---Guangxi Zhuang Autonomous Region, China, June 8, 2021 (preprint)

A male passenger arriving at Nanning Wuxu Airport in Guangxi on an international flight from Jakarta, Indonesia, was found to be positive for SARS-CoV-2 nucleic acid on a routine test at the airport on June 8 2021. The passenger was sent to Fourth People’s Hospital of Nanning immediately for further isolation and observation. On the day of admission, the test for SARS-CoV-2 nucleic acid of nasopharyngeal swabs, pharyngeal swabs and sputum specimens were positive (CT values of N gene and ORF1ab gene were between 20 and 30). After 8 weeks of hospitalization, the patient’s test for SARS-CoV-2 nucleic acid of all specimens turned to negative. We isolated a SARS-CoV-2 variant strain from the nasal swab of the patient, and then we found that the genome sequence of the variant strain had 13 base deletions and 38 nucleotide mutations compared with that of the Novel Coronavirus Wuhan strain after sequencing, comparison and analysis. The deletions and mutations of the variant strain resulted in four amino acid deletions and 30 amino acid mutations. Furthermore, we found that the variant strain was similar to those from Indonesia, South Korea and The United Kingdom after conducting BLAST analysis on GISAID platform, among them, hCOV-19 /Indonesia/ Ji-ITD-43591N /2021 was the most similar, with 99.98% similarity and only 8 base differences. The maximum likelihood phylogenetic tree was constructed taking the Wuhan strain as the root and including most the reference sequence contained most of the epidemic strains. The result showed that the strains isolated in our laboratory belonged to Delta strain.

Surveillance of SARS-CoV-2 in the environment and animal samples of the Huanan Seafood Market (preprint)

Emerging in December 2019, coronavirus disease 2019 (COVID-19) eventually became a pandemic and has posed a tremendous threat to global public health. However, the origins of SARS-CoV-2, the causative agent of COVID-19, remain to be determined. It has reported that a certain number of the early case clusters had a contact history with Huanan Seafood Market. Therefore, surveillance of SARS-CoV-2 within the market is of vital importance. Herein, we presented the SARS-CoV-2 detection results of 1380 samples collected from the environment and the animals within the market in early 2020. By SARS-CoV-2-specific RT-qPCR, 73 environmental samples tested positive for SARS-CoV-2 and three live viruses were successfully isolated. The viruses from the market shared nucleotide identity of 99.980% to 99.993% with the human isolate HCoV/Wuhan/IVDC-HB-01. In contrast, no virus was detected in the animal swabs covering 18 species of animals in the market. The SARS-COV-2 nucleic acids in the positive environmental samples showed significant correlation of abundance of Homo sapiens with SARS-CoV-2. In summary, this study provided convincing evidence of the prevalence of SARS-CoV-2 in the Huanan Seafood Market during the early stage of COVID-19 outbreak.

Early Detection of COVID-19 Epidemic Using Search Engine Query Data (preprint)

Background: Early warnings of emerging infectious disease are crucial to prevent epidemics. However, in the early stage of the COVID-19 pandemic, traditional infectious disease surveillance failed to deliver a warning alert. The aim of this work is to develop search-engine-based surveillance methods for the early warning and prediction of COVID-19 outbreaks. Methods: By using more than 444 million Baidu search queries from China as training set, we collected 32 keywords from the Baidu Search Index that may related to COVID-19 outbreak from 18 December 2019 to 11 February 2020. The Beijing Xinfadi outbreak from 30 May 2020 to 30 July 2020 was used as independent test set. A multiple linear regression was applied to model the relationship between the daily query frequencies of keywords and the daily new cases. Findings: Our results show that 11 keywords in search queries were highly correlated to the daily numbers of confirmed cases (r =0.96, P <0.01). An abnormal initial peak (1.46 times the normal volume) in queries appeared on 31 December 2019, which could have served as an early warning signal for an outbreak. Of particular concern, on this day, the volume of the query “Wuhan Seafood Market” increased by over 240 times (from 10 to 2410), the volume of the query “Wuhan outbreak” increased by over 622 times (from 7 to 4359), and 17.5% of China’s query volume originated from Hubei Province, 51.15% of which was from Wuhan city. The quantitative model using four keywords (“Epidemic”, “Masks”, “Coronavirus” and “Clustered pneumonia”) successfully predicted the daily numbers of cases for the next two days, and detected an early signal during the Beijing Xinfadi outbreak (R2 =0.80). Interpretation: Our study demonstrates the ability of search engine query data to detect COVID-19 outbreaks, and suggests that abnormalities in query volume can serve as early warning signals.

Neutralization of recombinant RBD-subunit vaccine ZF2001-elicited antisera to SARS-CoV-2 variants including Delta (preprint)

SARS-CoV-2 variants brought new waves of infection worldwide. In particular, Delta variant (B.1.617.2 lineage) has become predominant in many countries. These variants raised the concern for their potential immune escape to the currently approved vaccines. ZF2001 is a subunit vaccine received emergency use authorization (EUA) in both China and Uzbekistan, with more than 100-million doses administrated with a three-dose regimen. The tandem-repeat dimer of SARS-CoV-2 spike protein receptor binding domain (RBD) was used as the antigen. In this work, we evaluated the neutralization of ZF2001-elicited antisera to SARS-CoV-2 variants including all four variants of concern (Alpha, Beta, Gamma and Delta) and other three variants of interest (Epsilon, Eta and Kappa) by pseudovirus-based assay. We found antisera preserved majority of the neutralizing activity against these variants. E484K/Q substitution is the key mutation to reduce the RBD-elicited sera neutralization. Moreover, ZF2001-elicited sera with a prolonged intervals between the second and third dose enhanced the neutralizing titers and resilience to SARS-CoV-2 variants.

Changes in Notifiable Infectious Diseases in China During the COVID-19 Pandemic: A National Surveillance Study (preprint)

Background: Nationwide nonpharmaceutical interventions (NPI) were used to combat the novel coronavirus disease (COVID-19) during 2020 in the mainland of China. These NPIs have proven effective on mitigating the spread of COVID-19, but their broad impact on other diseases remains under-investigated. In this study, we aim to assess whether such broad impact exists on notifiable diseases in China.Methods: Weekly incidence and mortality data for 31 major notifiable infectious diseases at the province level were extracted from the China Information System for Disease Control and Prevention from 2014 to 2020. We assessed the impact of NPIs by contrasting the incidences of each infectious disease in predefined COVID-19 phases during 2020 to the average incidences in the corresponding time intervals during 2014-2019.Findings: We observed decreased incidences of most diseases during the phases after the lockdown of Wuhan. In general, respiratory diseases and gastrointestinal or enteroviral diseases were more affected than sexually transmitted or bloodborne diseases and vector-borne or zoonotic diseases. Seasonal flu and rubella were the most sensitive to the NPIs, with reductions of 67-99% in incidence rates throughout the NPI-implemented phases in China (Jan 27-Dec 31, 2020). Among gastrointestinal or enteroviral diseases, the hand, foot and mouth disease (HFMD) was subject to the largest declines during Jan 27-Aug 31, 2020, with >90% reduction in incidence rate. Phases with more stringent NPIs were associated with more reductions. Non-respiratory diseases, particularly HFMD, gonorrhea and brucellosis, rebounded towards the end of the year as the NPIs were relaxed.Interpretation: NPIs are broadly effective in containing infectious diseases. Less disruptive NPIs such as wearing face masks are still effective in mitigating respiratory diseases but are not adequate for containing non-respiratory diseases.Funding Statement: This work was supported by grants from the National Natural Science Funds [91846302, 81825019], the China Mega-Project on Infectious Disease Prevention [2018ZX10713001, 2018ZX10713002, 2018ZX10201001 and 2017ZX10103004], and the US National Institutes of Health [R56 AI148284].Declaration of Interests: All authors declare no competing interests.Ethics Approval Statement: Missing.

Potent Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by photosensitizers (preprint)

The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has exploded since December 2019, and causes more than 2 million death with more than 95 million people infected as of Jan. 21th, 2021 globally1,2. Angiotensin-converting enzyme 2 (ACE2), expressed in the lungs, arteries, heart, kidney, intestines, and nasal epithelium3, has been shown to be the primary entry point targeted by the surface spike protein of SARS-CoV-2. Currently, no proven antiviral treatment for SARS-CoV-2 infection is available. In this study, we screened a number of photosensitizers for photodynamic viral inactivation, and found compounds pentalysine β-carbonylphthalocyanine zinc (ZnPc5K) and chlorin e6 (ce6) potently inhibited the viral infection and replication in vitro with half-maximal effective concentrations (EC50) values at nanomolar level. Such viral inactivation strategy is implementable, and has unique advantages, including resistance to virus mutations, affordability compared to the monoclonal antibodies, and lack of long-term toxicity.

Landscapes and dynamic diversifications of B-cell receptor repertoires in COVID-19 patients (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of coronavirus disease 2019 (COVID-19). Great international efforts have been put into the development of prophylactic vaccines and neutralizing antibodies. However,the knowledge about the B cell immune response induced by the SARS-CoV-2 virus is still limited. Here,we report a comprehensive characterization of the dynamics of immunoglobin heavy chain (IGH) repertoire in COVID-19 patients. By using next-generation sequencing technology, we examined the temporal changes in the landscape of the patient's immunological status, and found dramatic changes in the IGH within the patient's immune system after the onset of COVID-19 symptoms. Although different patients have distinct immune responses to SARS-CoV-2 infection, by employing clonotype overlap, lineage expansion and clonotype network analyses, we observed a higher clonotype overlap and substantial lineage expansion of B cell clones during 2-3 weeks of illness, which is of great importance to B-cell immune responses. Meanwhile, for preferences of V gene usage during SARS-CoV-2 infection, IGHV3-74 and IGHV4-34 and IGHV4-39 in COVID-19 patients were more abundant than that of healthy controls. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic development as well as mechanistic research.

Structural dynamics of the SARS-CoV-2 frameshift-stimulatory pseudoknot reveal topologically distinct conformers (preprint)

The RNA pseudoknot that stimulates -1 programmed ribosomal frameshifting in SARS coronavirus-2 (SARS-CoV-2) is a possible drug target. To understand how this 3-stemmed pseudoknot responds to the mechanical tension applied by ribosomes during translation, which is thought to play a key role during frameshifting, we probed its structural dynamics under tension using optical tweezers. Unfolding curves revealed that the frameshift signal formed multiple different structures: at least two distinct pseudoknotted conformers with different unfolding forces and energy barriers, as well as alternative stem-loop structures. Refolding curves showed that stem 1 formed first in the pseudoknotted conformers, followed by stem 3 and then stem 2. By extending the handle holding the RNA to occlude the 5' end of stem 1, the proportion of the different pseudoknot conformers could be altered systematically, consistent with structures observed in cryo-EM images and computational simulations that had distinct topologies: the 5' end of the RNA threaded through the 3-helix junction to form a ring-knot, or unthreaded as in more standard H-type pseudoknots. These results resolve the folding mechanism of the frameshift signal in SARS-CoV-2 and highlight the dynamic conformational heterogeneity of this RNA, with important implications for structure-based drug-discovery efforts.

ACE2 peptide fragment interacts with several sites on the SARS-CoV-2 spike protein S1 (preprint)

The influence of the peptide QAKTFLDKFNHEAEDLFYQ on the kinetics of the SARS-CoV-2 spike protein S1 binding to angiotensin-converting enzyme 2(ACE2) was studied to model the interaction of the virus with its host cell. This peptide corresponds to the sequence 24-42 of the ACE2 1 domain, which is the binding site for the S1 protein. The on-rate and off-rate of S1-ACE2 complex formation were measured in the presence of various peptide concentrations using Bio-Layer Interferometry (BLI). The formation of the S1-ACE2 complex was inhibited when the S1 protein was preincubated with the peptide, however, no significant inhibitory effect was observed in the absence of preincubation. Dissociation kinetics revealed that the peptide remained bound to the S1-ACE2 complex and stabilized this complex. Computational mapping of the S1 protein surface for peptide binding revealed two additional sites, located at some distance from the receptor binding domain (RBD) of S1. These additional binding sites affect the interaction between the peptide, the S1 protein, and ACE2.

Lentiviral vector-based SARS-CoV-2 pseudovirus enables analysis of neutralizing activity in COVID-19 convalescent plasma (preprint)

As the COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to spread around the globe, effective vaccination protocols are under deployment. Alternatively, the use of convalescent plasma (CP) therapy relies on the transfer of the immunoglobulin repertoire of a donor that has recovered from the disease as a means of passive vaccination. While the lack of an effective antiviral treatment inadvertently increases the interest in CP products, initial clinical evaluation on COVID-19 patients revealed that critical factors determining the outcome of CP therapy need to be defined clearly if clinical efficacy is to be expected. Measurement of neutralizing activity against SARS-CoV-2 using live virus presents a reliable functional assay but the availability of suitable BSL3 facilities for live virus culture restricts its applicability. Instead, the use of pseudovirus particles containing elements from the SARS-CoV-2 virus is widely applied to determine the activity of CP or other neutralizing agents such as monoclonal antibodies. In this study, we present our approach to optimize GFP-encoding lentiviral particles pseudotyped with the SARS-CoV-2 Spike and Membrane proteins for use in neutralization assays. Our results show the feasibility of pseudovirus production using a C-terminal truncated Spike protein which is greatly enhanced by the incorporation of the D614G mutation. Moreover, we report that the use of sodium butyrate during lentiviral vector production dramatically increases pseudovirus titers. Analysis of CP neutralizing activity against particles pseudotyped with wildtype or D614G mutant Spike protein in the presence or absence the M protein revealed differential activity in CP samples that did not necessarily correlate with the amount of anti-SARS-CoV-2 antibodies. Our results indicate that the extent of neutralizing activity in CP samples depends on the quality rather than the quantity of the humoral immune responses and varies greatly between donors. Functional screening of neutralizing activity using pseudovirus-based neutralization assays must be accepted as a critical tool for choosing CP donors if clinical efficacy is to be maximized.

Unconventional Monetary Policy and Disaster Risk: Evidence from the Subprime and COVID-19 Crises (preprint)

We contrast the interventions conducted by the Federal Reserve in response to the subprime and COVID-19 crises with respect to their effectiveness in reducing disaster risk. Using model-free measures of disaster risk derived from daily options data, we document that interventions in response to both crises reduced tail risks in domestic equity markets. Spillover effects are notably distinct. While the subprime interventions are generally characterized by negative spillovers to international equity markets, the responses to the COVID--19 crisis are generally associated with positive spillovers. We interpret these results as consistent with different degrees of protagonism played by Central Banks in the two episodes, emphasizing the importance of a broader participation of monetary authorities in expanding their balance sheets to counteract the effects of major crises.