Receptor-binding Region Research Articles

Seropositive Reaction Rates of 9 B-Cell Epitopes of the SARS-CoV-2 Spike Protein and the Relationship between the Epitopes and Neutralizing Antibody

Objective: The aim of the study was to analyze the relationship between serum antibody and neutralizing antibody titers in convalescent coronavirus disease 2019 (COVID-19) patients with different disease severities, and the seropositive reaction rates of 9 reported B-cell epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: Serum IgG and total antibody titers of 165 convalescent COVID-19 patients were determined by chemiluminescence, the serum neutralization antibody titers were determined by microneutralization assay, and the S/CO values of 9 peptides were detected by indirect enzyme-linked immunosorbent assay. Correlations between the aforementioned indexes were statistically analyzed, and differences in patients with different diseases severities were evaluated. Results: IgG, total antibody, and neutralizing antibody titers increased with disease severity. The positive rate of the receptor-binding region (RBD) was 100%, and the average positive rate for all the 9 peptides was above 50% in 165 patients. IDf showed the highest rate of positivity (86.06%), with a rate of 95% for the (IDf + IDa) pattern. Moreover, S/CO values of RBD and mix (IDh) were significantly correlated with IgG, total antibody titers, and neutralizing antibody titers (p < 0.001), whereas the S/CO values for other 8 peptides showed no obvious correlation. Conclusion: In this study, a large sample was used to confirm that the peptide IDf had a high positive reaction rate for all patients (86.06%) and also had the highest detection rate in asymptomatic patients (86.67%). Only long peptide and mixed peptide showed correlation with neutralizing antibody titers, suggesting that the ability of SARS-CoV-2 antibody to neutralize virus infectivity may require the interaction of multiple sites.

B cell receptor (BCR) guided mechanotransduction: Critical hypothesis to instruct SARS-CoV-2 specific B cells to trigger proximal signalling and antibody reshaping

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, has spread around the globe with remarkable consequences for the health of millions of people. Despite the approval of mRNA vaccines to prevent the spread of infection, long-term immunity must still be monitored. Targeting and modifying virus receptor binding regions to activate B cell receptors (BCRs) is a promising way to develop long-term immunity against SARS-CoV-2. After the interaction of antigens, BCRs undergo series of signal transduction events through phosphorylation of immune receptor tyrosine activation motifs (ITAMs) to produce neutralizing antibodies against pathogens. BCRs intricate entity displays remarkable capability to translate the external mechanosensing cues to reshape the immune mechanism. However, potential investigations suggesting how SARS-CoV-2 specific B cells respond to mechanosensing cues remain obscure. This study proposes a sophisticated hypothesis explaining how B cells isolated from the CP of SARS-CoV-2 infected patients may undergo a triggered series of B cell activation, BCR dynamics, proximal signalling, and antibody production on PDMS-embedded in-vitro antigen-presenting structures (APCs). These studies could provide detailed insights in the future for the development of structural and therapeutic entanglements to fight against pathogens.

Abstract 1298: Designing novel small molecule inhibitors as possible targeted therapeutics to prevent breast cancer metastasis

Abstract Breast cancer is currently the second leading cause of cancer-related deaths and the most commonly diagnosed cancer among women in the Unites States. Localized breast cancer has a five-year survival rate of 99%, however the survival rate for patients with distant metastasis is 27%, despite improved screening and detection methods. Currently, there are few targeted therapies that specifically inhibit metastasis. Research performed by our lab and others has shown that inflammatory cytokines play a crucial role in promoting cancer growth and metastatic potential in cancer cells. While efforts have been made to develop FDA approved therapeutics for inflammatory cytokines, very few have successfully made it through clinical trials and several cytokines still lack effective clinical inhibitors. Our research has developed a stepwise approach towards designing and testing novel small molecule inhibitors (SMIs) against inflammatory cytokines. In silico computational screening of compound libraries towards specific inflammatory cytokines revealed potential molecules that interact with receptor binding regions and inhibit cytokine biological function. Top candidate inhibitors were analyzed for direct interaction with cytokines using differential scanning fluorimetry, fluorescent quenching, and Chemical Shift Pertubation NMR (CSP-NMR). Additionally, to quantitatively measure cytokine-receptor interference, a novel ligand-receptor binding assay was developed highlighting small molecule inhibitors that effectively prevent biological cytokine function. In vitro efficacy of the SMIs were analyzed by measuring inhibition of various signaling cascades activated by inflammatory cytokines using enzyme-linked immunosorbent assay (ELISA) and immunoblot assays. Inhibition of tumor cell proliferation, epithelial to mesenchymal transition (EMT), and cytokine-induced secretion of pro-tumorigenic molecules was also assessed. From these in silico and in vitro methods, SMI's are structurally modified with effective functional groups to increase efficacy of binding interaction and reduce toxicity for living systems. Finally, small molecule inhibitors that have been sufficiently tested for efficacy are then examined in vivo to evaluate toxicity and determine the maximum tolerated dose. SMI's that prove to be non-toxic are evaluated further in vivo by mouse mammary breast cancer models to analyze reduction of distant metastases. Our interdisciplinary team has designed, synthesized, and tested small molecule inhibitors (SMIs) to target inflammatory cytokines shown to promote metastasis in cancer with the long-term goal of understanding lethal breast cancer metastasis, as well as providing a novel FDA approved therapeutic for breast cancer patients. Citation Format: Cody L. Wolf, Clyde Pruett, Darren Lighter, Cooper McGrath, Amberlyn Nemeth, Joey Tuccinardi, Grace Coughlin, Samuel Randall, Joey Day, Matthew King, Lisa Warner, Don Warner, Cheryl L. Jorcyk. Designing novel small molecule inhibitors as possible targeted therapeutics to prevent breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1298.

First report of field cases of Y280-like LPAI H9N2 strains in South Korean poultry farms: pathological findings and genetic characterization

ABSTRACT H9N2 low-pathogenic avian influenza (LPAI) viruses have long been circulating in the world poultry industry, resulting in substantial economic losses. In addition to bird health consequences, viruses from specific lineages such as G1 and Y280 are also known to have the potential to cause a pandemic within the human population. In South Korea, after introducing inactivated H9N2 vaccines in 2007, there were no field outbreaks of H9N2 LPAI since 2009. However, in June 2020, an H9N2 virus was isolated from an outbreak in a Korean chicken farm. This strain was distinct from the predominant Korean/Y439 lineage and was believed to be part of the Y280-like lineage. Since the first case of this new H9N2 LPAI, nine more cases of field infections in poultry farms were documented through July and December of 2020. Phylogenetic analysis of the haemagglutinin (HA) and neuraminidase genes of these case isolates revealed that all strains were grouped with exotic Y280-like strains that did not previously exist in South Korea and were emerging into a new cluster. Serological assays also confirmed the existence of antibodies to Y280-like viruses in field sera collected from infected birds, and that they had seroconverted. Further analysis of the receptor-binding region in the HA protein also revealed that these isolates harboured a human-like motif that could potentially affect mammals and humans, demonstrating a possible public health risk. This is the first report of field cases caused by Y280-like H9N2 LPAI in the Korean poultry industry. RESEARCH HIGHLIGHTS Field outbreaks caused by Y280-like H9N2 avian influenza viruses were confirmed. A human-like motif was found at the HA receptor-binding region of all isolates.

Branching out the aerolysin, ETX/MTX-2 and Toxin_10 family of pore forming proteins

Organisms have evolved mechanisms in which cellular membranes can both be targeted and punctured thereby killing the targeted cell. One such mechanism involves the deployment of pore forming proteins (PFPs) which function by oligomerizing on cell membranes and inserting a physical pore spanning the membrane. This pore can lead to cell death by either causing osmotic flux or allowing the delivery of a secondary toxin. Pore forming proteins can be broadly classified into different families depending on the structure of the final pore; either α-PFPs using channels made from α -helices or β-PFPs using channels made from β-barrels. There are many different β-PFPs and an emerging superfamily is the aerolysin-ETX/MTX-2 superfamily. A comparison between the members of this superfamily reveals the pore forming domain is a common module yet the receptor binding region is highly variable. These structural and architectural variations lead to differences in the target recognition and determine the site of activity. Closer investigation of the topology of the family also suggests that the Toxin_10 family of PFPs could be considered as part of the aerolysin-ETX/MTX-2 superfamily. Comparatively, far less is known about how Toxin_10 proteins assemble into the final pore structure than aerolysin-ETX/MTX-2 proteins. This review aims to collate the pore forming protein members and bridge the structural similarities between the aerolysin-ETX/MTX-2 superfamily and the insecticidal Toxin_10 subfamily.

Rescue of Infectious Rotavirus Reassortants by a Reverse Genetics System Is Restricted by the Receptor-Binding Region of VP4.

The rotavirus species A (RVA) capsid contains the spike protein VP4, which interacts with VP6 and VP7 and is involved in cellular receptor binding. The capsid encloses the genome consisting of eleven dsRNA segments. Reassortment events can result in novel strains with changed properties. Using a plasmid-based reverse genetics system based on simian RVA strain SA11, we previously showed that the rescue of viable reassortants containing a heterologous VP4-encoding genome segment was strain-dependent. In order to unravel the reasons for the reassortment restrictions, we designed here a series of plasmids encoding chimeric VP4s. Exchange of the VP4 domains interacting with VP6 and VP7 was not sufficient for rescue of viable viruses. In contrast, the exchange of fragments encoding the receptor-binding region of VP4 resulted in virus rescue. All parent strains and the rescued reassortants replicated efficiently in MA-104 cells used for virus propagation. In contrast, replication in BSR T7/5 cells used for plasmid transfection was only efficient for the SA11 strain, whereas the rescued reassortants replicated slowly, and the parent strains failing to produce reassortants did not replicate. While future research in this area is necessary, replication in BSR T7/5 cells may be one factor that affects the rescue of RVAs.

Emerging new avian reovirus variants from cases of enteric disorders and arthritis/tenosynovitis in Brazilian poultry flocks

ABSTRACT 1. The objective of this study was to characterise circulating Brazilian avian reovirus (ARV) strains by genetic analysis of the σC protein encoded by segment 1 of the viral genome and compare these with those of viral strains used for immunising commercial poultry. 2. The analysis detected the presence of ARV genomes by quantitative reverse transcriptase PCR (RT-qPCR) in the enteric samples and the joint tissues (JT) of birds with signs of viral arthritis/tenosynovitis. Nucleotide sequencing used 16 strains (three commercial vaccines, 10 from enteric tissues and three from JT). The results indicated high variability in the amino acid sequences of 13 wild strains, showing between 40% and 75% similarity compared with the vaccine strains (S1133 and 2177). 3. The sequences were grouped into three well-defined clusters in a phylogenetic tree, two of these clusters together with previous Brazilian σC ARV sequences, and one cluster (VII) that was novel for Brazilian strains. Antigenic analysis showed that there were amino acids within putative epitopes located on the surface of the receptor-binding region of the σC protein with a high degree of variability. 4. The study confirmed the presence of ARV genetic variants circulating in commercial birds in Brazil, and according to the antigenic prediction, the possibility of antigenic variants appears to be high.

Anti-CfaE nanobodies provide broad cross-protection against major pathogenic enterotoxigenic Escherichia coli strains, with implications for vaccine design

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naïve yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope.

No Higher Infectivity But Immune Escape of SARS-CoV-2 501Y.V2 Variants

The 501Y.V2 variants contain multiple mutations in the spike region. We found that they had no significant increased effect on infectivity to human cell lines and cells overexpressing human ACE2, but increased infectivity in cells overexpressing mouse ACE2 based on the pseudotyped viruses. The susceptibility of 501Y.V2 variants to many neutralizing monoclonal antibodies decreased significantly. These variants also had a significant reduced effect on the neutralization ability of convalescent and RBD protein immunized sera. The immune resistances were mainly caused by E484K and N501Y mutations located in receptor binding region. These data suggest that the enhancement of infectivity in mouse may lead to the spillover of 501Y.V2 to mouse. The immune resistance of 501Y.V2 may compromise the efficacy of monoclonal antibodies, convalescent sera and vaccines. Funding: This work was supported by General Program ofNational Natural Science Foundation of China [grant number 82073621], BILL & MELINDA GATES FOUNDATION [Investment ID INV-006379], National Science and Technology Major Projects of Drug Discovery [grant number 2018ZX09101001] and National Science and Technology Major Projects of Infectious Disease [grant number 2017ZX10304402]. Conflict of Interest: All the authors declare no competing interests. Ethical Approval: The protocol of the animal study was approved by Ethical review Committee for Animal Welfare of The National Institutes for Food and Drug Control.

Isolation and molecular characterization of the hemagglutinin gene of H9N2 avian influenza viruses from poultry in Java, Indonesia.

Objective:The avian influenza virus (AIV) subtype H9N2 circulating in Indonesia has raised increasing concern about its impact on poultry and its public health risks. In this study, the H9N2 virus from chicken poultry farms in Java was isolated and characterized molecularly.Materials and Methods:Thirty-three pooled samples of chicken brain, cloacal swab, trachea, and oviduct were taken from multiple chickens infected with AIV in five regions of Java, Indonesia. The samples were isolated from specific pathogenic-free embryonated eggs that were 9 days old. Reverse transcription polymerase chain reaction and sequencing were used to identify H9N2 viruses.Results:This study was successful in detecting and characterizing 13 H9N2 isolates. The sequencing analysis of hemagglutinin genes revealed a 96.9%–98.8% similarity to the H9N2 AIV isolated from Vietnam in 2014 (A/muscovy duck/Vietnam/LBM719/2014). According to the phylogenetic analysis, all recent H9N2 viruses were members of the lineage Y280 and clade h9.4.2.5. Nine of the H9N2 isolates studied showed PSKSSR↓GLF motifs at the cleavage site, while four had PSKSSR↓GLF. Notably, all contemporary viruses have leucine (L) at position 216 in the receptor-binding region, indicating that the virus can interact with a human-like receptor.Conclusion:This study described the features of recent H9N2 viruses spreading in Java’s poultry industry. Additionally, H9N2 infection prevention and management must be implemented to avoid the occurrence of virus mutations in the Indonesian poultry industry.

Grafting Methionine on 1F1 Ab Increases the Broad-Activity on HA Structural-Conserved Residues of H1, H2, and H3 Influenza a Viruses.

A high level of mutation enables the influenza A virus to resist antibiotics previously effective against the influenza A virus. A portion of the structure of hemagglutinin HA is assumed to be well-conserved to maintain its role in cellular fusion, and the structure tends to be more conserved than sequence. We designed peptide inhibitors to target the conserved residues on the HA surface, which were identified based on structural alignment. Most of the conserved and strongly similar residues are located in the receptor-binding and esterase regions on the HA1 domain In a later step, fragments of anti-HA antibodies were gathered and screened for the binding ability to the found conserved residues. As a result, Methionine amino acid got the best docking score within the −2.8 Å radius of Van der Waals when it is interacting with Tyrosine, Arginine, and Glutamic acid. Then, the binding affinity and spectrum of the fragments were enhanced by grafting hotspot amino acid into the fragments to form peptide inhibitors. Our peptide inhibitor was able to form in silico contact with a structurally conserved region across H1, H2, and H3 HA, with the binding site at the boundary between HA1 and HA2 domains, spreading across different monomers, suggesting a new target for designing broad-spectrum antibody and vaccine. This research presents an affordable method to design broad-spectrum peptide inhibitors using fragments of an antibody as a scaffold.

Molecular evolution and characterization of hemagglutinin and neuraminidase of influenza A(H1N1)pdm09 viruses isolated in Beijing, China, during the 2017-2018 and 2018-2019 influenza seasons.

We investigated and analysed the molecular evolution of hemagglutinin (HA) and neuraminidase (NA) of influenza A(H1N1)pdm09 virus during the 2017-2018 and 2018-2019 influenza seasons in Beijing, China. We collected and extracted RNA from influenza A(H1N1)pdm09 strains from Peking University People's Hospital and analyzed their HA and NA genes by RT-PCR and sequencing. Phylogenetic analysis of HA and NA sequences was used to compare the amino acid sequences of 51 strains with those of reference strains. All strains belonged to subclade 6B.1, with S162N and I216T substitutions (H1 numbering). Our strains differed from strain A/Michigan/45/2015, with the substitutions S91R, S181T and I312V in the HA antigenic epitope. An E189G mutation was detected in the 190 helix of the receptor binding region of HA. A new potential glycosylation site, 179 (NQT), which was not detected before the 2015 influenza season, was identified. Two strains were mutated at I223, the NA inhibitor resistance site. During 2012-2019, amino acids of HA and NA mutated over time. Co-occurrence mutations N146D, S200P, S202I and A273T in HA appeared along with Q51K, F74S and D416N in NA in six strains during two influenza seasons. Our work reveals the molecular changes and phylogenetic characteristics of influenza A(H1N1)pdm09 virus and suggests that a vaccine probably provides suboptimal protection. The biological characteristics of the new glycosylation and drug-resistance sites detected in this work need to be studied further. The co-occurrence of mutations in HA and NA might affect the characteristics of the virus and need to be given more attention.

Identification of Clinically and Pathophysiologically Relevant Rheumatoid Factor Epitopes by Engineered IgG Targets.

Rheumatoid factors (RFs), which are anti-IgG autoantibodies strongly associated with rheumatoid arthritis (RA), are also found in other diseases and in healthy individuals. RFs bind to various epitopes in the constant (Fc-) domain of IgG. Therefore, disease-specific reactivity patterns may exist. This study was undertaken in order to develop a new approach to dissecting RF epitope binding patterns across different diseases. We analyzed RF reactivity patterns in serum from patients with seropositive arthralgia, patients with RA, and patients with primary Sjögren's syndrome (SS) using bioengineered, natively folded IgG-Fc targets that demonstrated selective RF binding toward several distinct regions of the IgG-Fc domain. Rheumatoid factor responses primarily bound the Fc Elbow region, with a smaller number of RFs binding the Fc Tail region, while the Fc receptor binding region was hardly targeted. A restricted reactivity against the IgG-Fc Tail region was associated with less positivity for anti-citrullinated protein antibodies (ACPAs) and less arthritis development in arthralgia, whereas combined reactivity toward IgG-Fc Tail and Elbow regions was associated with more arthritis development. Reactivity toward the IgG-Fc Tail region was observed far more frequently in RA than in primary SS. Bioengineered IgG targets enable serologic characterization of RF reactivity patterns, and use of this approach appears to reveal patterns associated with ACPA detection and arthritis development in patients with arthralgia. These patterns are able to distinguish RA patients from primary SS patients. This new methodology improves the clinical value of RFs and our understanding of their pathophysiologic processes.

Potential protease inhibitors and their combinations to block SARS-CoV-2

COVID-19, which has emerged recently as a pandemic viral infection caused by SARS-coronavirus 2 has spread rapidly around the world, creating a public health emergency. The current situation demands an effective therapeutic strategy to control the disease using drugs that are approved, or by inventing new ones. The present study examines the possible repurposing of existing anti-viral protease inhibitor drugs. For this, the structural features of the viral spike protein, the substrate for host cell protease and main protease of the available SARS CoV-2 isolates were established by comparing with related viruses for which antiviral drugs are effective. The results showed 97% sequence similarity among SARS and SARS-CoV-2 main protease and has same cleavage site positions and ACE2 receptor binding region as in the SARS–CoV spike protein. Though both are N-glycosylated, unlike SARS-CoV, human SARS-CoV-2 S-protein was O-glycosylated as well. Molecular docking studies were done to explore the role of FDA approved protease inhibitors to control SARS-CoV-2 replication. The results indicated that, Ritonavir has the highest potency to block SARS-CoV-2 main protease and human TMPRSS2, a host cell factor that aids viral infection. Other drugs such as Indinavir and Atazanavir also showed favourable binding with Cathepsin B/L that helped viral fusion with the host cell membrane. Further molecular dynamics simulation and MM-PBSA binding free energy calculations confirmed the stability of protein-drug complexes. These results suggest that protease inhibitors particularly Ritonavir, either alone or in combination with other drugs such as Atazanavir, have the potential to treat COVID 19. Communicated by Ramaswamy H. Sarma

Global distribution of single amino acid polymorphisms in Plasmodium vivax Duffy-binding-like domain and implications for vaccine development efforts.

Plasmodium vivax (Pv) malaria continues to be geographically widespread with approximately 15 million worldwide cases annually. Along with other proteins, Duffy-binding proteins (DBPs) are used by plasmodium for RBC invasion and the parasite-encoded receptor binding regions lie in their Duffy-binding-like (DBL) domains—thus making it a prime vaccine candidate. This study explores the sequence diversity in PvDBL globally, with an emphasis on India as it remains a major contributor to the global Pv malaria burden. Based on 1358 PvDBL protein sequences available in NCBI, we identified 140 polymorphic sites within 315 residues of PvDBL. Alarmingly, country-wise mapping of SAAPs from field isolates revealed varied and distinct polymorphic profiles for different nations. We report here 31 polymorphic residue positions in the global SAAP profile, most of which map to the PvDBL subdomain 2 (α1–α6). A distinct clustering of SAAPs distal to the DARC-binding sites is indicative of immune evasive strategies by the parasite. Analyses of PvDBL-neutralizing antibody complexes revealed that between 24% and 54% of interface residues are polymorphic. This work provides a framework to recce and expand the polymorphic space coverage in PvDBLs as this has direct implications for vaccine development studies. It also emphasizes the significance of surveying global SAAP distributions before or alongside the identification of vaccine candidates.

Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display.

Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated cross-linking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides using magnetic selection, followed by fluorescence activated cell sorting (FACS) to isolate binders for a model target (lysozyme) with low micromolar binding affinity (KD ∼ 1.2-3.7 μM). The isolated peptides bind lysozyme selectively and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides can be used to efficiently obtain quantitative estimates of binding affinity, circumventing the need for chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that likely binds IL-17 in its receptor binding region with moderate apparent affinity (KD ∼ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.

Analysis of related patents on coronavirus monoclonal antibody therapy

<p indent="0mm">Coronaviruses, such as SARS-CoV, MERS-CoV, and the recently epidemic coronavirus (SARS-CoV-2), seriously threaten human health and cause significant economic losses. Monoclonal antibodies targeting coronaviruses may inhibit viral infection by blocking viral binding to the receptor on cells. In this study, the patent applications and clinical trial results for monoclonal antibody drugs against coronavirus were analyzed primarily to provide ideas and references for the application of monoclonal antibodies in the fight against COVID-19, and to accelerate the drug development process. There are currently no established patents related to the monoclonal antibodies against SARS-CoV-2, both due to COVID-19’s relatively recent emergence, and due to the lengthy patent application and publication process. A thorough global analysis of monoclonal antibody patent applications indicates a total of 194 diagnostic monoclonal antibody techniques, accounting for 63%, and 113 therapeutic monoclonal antibody techniques, accounting for 37%. Among those applications related to therapeutic monoclonal antibody technology, product patent applications dominate, with a total number of 96, accounting for 85%. According to the geographical analysis of applicants for coronavirus therapeutic monoclonal antibodies, there are 35 patent applications from, accounting for 31%, and 78 foreign patent applications, accounting for 69%. The analysis of the target of the coronavirus therapeutic monoclonal antibody-related patent applications indicates that there are 61 patent applications for monoclonal antibodies specific to the virus’s own antigen, accounting for 54%. This type of monoclonal antibody specifically targets viral antigens to suppress viruses, whereas the other 52 patent applications in this category target viral non-specific monoclonal antibodies, accounting for 46%. Amongst current studies on the monoclonal antibodies against SARS-CoV and MERS-CoV, relevant patent applications for therapeutic monoclonal antibodies are mainly based on products, and the target is the S protein or its receptor binding region (RBD), which prevents virus infection of cells. Currently, there are no available therapeutic monoclonal antibody drugs against coronavirus. The monoclonal antibody drug specifically targeting the S protein of MERS-CoV is still in clinical trials. Three monoclonal antibody drugs treating for COVID-19 are also in clinical trials, all of which involve second use of the known monoclonal antibodies in their mechanisms. The RBD of SARS-CoV-2 has common-epitope peptide with SARS-CoV. By verifying the effect of the monoclonal antibody against SARS-CoV in the treatment of COVID-19, patent applications for the use of the monoclonal antibody against COVID-19 can be designed. Methods of blocking monoclonal antibodies against the RBD of SARS-CoV-2 can be used as potential drug mechanisms for the treatment of COVID-19. Related patent applications and drug development will also be hot spots in the future.

What We Need to Consider During and After the SARS-CoV-2 Pandemic.

Even though extreme containment and mitigation strategies were implemented by numerous governments around the world to slow down the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the number of critically ill patients and fatalities keeps rising. This crisis has highlighted the socioeconomic disparities of health care systems within and among countries. As new CoVID policies and responses are implemented to lessen the impact of the virus, it is imperative (1) to consider additional mitigation strategies critical for the development of effective countermeasures, (2) to promote long-term policies and strict regulations of the trade of wildlife and live animal markets, and (3) to advocate for necessary funding and investments in global health, specifically for the prevention of and response to natural and manmade pandemics. This document considers some of these challenges.

Clinical and genetic analysis of lipoprotein glomerulopathy patients caused by APOE mutations.

BackgroundLipoprotein glomerulopathy (LPG) is a rare kidney disease caused by APOE mutations. The aim of this study was to correlate the genetic and clinical features of LPG.MethodsTotally eight LPG patients were recruited in this study and Sanger sequencing of APOE was performed for all available family members. Clinical and histological features were analyzed. A literature review of LPG was also conducted.ResultsGenetic analysis revealed five patients with APOE‐Kyoto, two with APOE‐Osaka/Kurashiki, and one with APOE‐Chicago mutations. LPG patients with urine protein reduced more than 50% had a slower decrease in renal function than those with less urine protein reduction (estimated glomerular filtration rate reduction rate −5.0 ± 0.8 vs. 1.5 ± 0.7 ml/min per 1.73 m2⋅month−1, p = .03). We then enrolled 95 LPG patients from previous studies and this study. LPG patients had higher blood pressure (mean arterial pressure: 109.4 ± 19.4 vs. 94.4 ± 11.1 mmHg, p < .001) than the control group. Interestingly, patients with APOE mutations in the LDL receptor binding region had higher serum apolipoprotein E (apoE) levels [ln(apoE): 2.7 ± 0.4 vs. 2.0 ± 0.5 mg/dl, p < .001] in comparison to other domains.ConclusionHere, we report for the first time APOE‐Osaka/Kurashiki and APOE‐Chicago mutations in the Chinese population. LPG was associated with higher blood pressure and serum apoE levels were higher in patients with mutations in LDL receptor binding region. In addition, the findings further indicated that treatment of proteinuria might slow down renal function progression in these patients.

Noncoding Variants Connect Enhancer Dysregulation with Nuclear Receptor Signaling in Hematopoietic Malignancies.

Mutations in protein-coding genes are well established as the basis for human cancer, yet how alterations within noncoding genome, a substantial fraction of which contain cis-regulatory elements (CRE), contribute to cancer pathophysiology remains elusive. Here, we developed an integrative approach to systematically identify and characterize noncoding regulatory variants with functional consequences in human hematopoietic malignancies. Combining targeted resequencing of hematopoietic lineage-associated CREs and mutation discovery, we uncovered 1,836 recurrently mutated CREs containing leukemia-associated noncoding variants. By enhanced CRISPR/dCas9-based CRE perturbation screening and functional analyses, we identified 218 variant-associated oncogenic or tumor-suppressive CREs in human leukemia. Noncoding variants at KRAS and PER2 enhancers reside in proximity to nuclear receptor (NR) binding regions and modulate transcriptional activities in response to NR signaling in leukemia cells. NR binding sites frequently colocalize with noncoding variants across cancer types. Hence, recurrent noncoding variants connect enhancer dysregulation with nuclear receptor signaling in hematopoietic malignancies. SIGNIFICANCE: We describe an integrative approach to identify noncoding variants in human leukemia, and reveal cohorts of variant-associated oncogenic and tumor-suppressive cis-regulatory elements including KRAS and PER2 enhancers. Our findings support a model in which noncoding regulatory variants connect enhancer dysregulation with nuclear receptor signaling to modulate gene programs in hematopoietic malignancies.See related commentary by van Galen, p. 646.This article is highlighted in the In This Issue feature, p. 627.