Receptor-binding Region Research Articles

Structural analysis of the stable form of fibroblast growth factor 2 – FGF2-STAB

Fibroblast growth factor 2 (FGF2) is a signaling protein that plays a significant role in tissue development and repair. FGF2 binds to fibroblast growth factor receptors (FGFRs) alongside its co-factor heparin, which protects FGF2 from degradation. The binding between FGF2 and FGFRs induces intracellular signaling pathways such as RAS-MAPK, PI3K-AKT, and STAT. FGF2 has strong potential for application in cell culturing, wound healing, and cosmetics but the potential is severely limited by its low protein stability. The thermostable variant FGF2-STAB was constructed by computer-assisted protein engineering to overcome the natural limitation of FGF2. Previously reported characterization of FGF2-STAB revealed an enhanced ability to induce MAP/ERK signaling while having a lower dependence on heparin when compared with FGF2-wt. Here we report the crystal structure of FGF2-STAB solved at 1.3 Å resolution. Protein stabilization is achieved by newly formed hydrophobic interactions, polar contacts, and one additional hydrogen bond. The overall structure of FGF2-STAB is similar to FGF2-wt and does not reveal information on the experimentally observed lower dependence on heparin. A noticeable difference in flexibility in the receptor binding region can explain the differences in signaling between FGF2-STAB and its wild-type counterpart. Our structural analysis provided molecular insights into the stabilization and unique biological properties of FGF2-STAB.

Design, synthesis, and biological evaluation of novel imidazole derivatives as analgesic and anti-inflammatory agents: experimental and molecular docking insights

Imidazole moieties exhibit a broad range of biological activities, including analgesic, anti-depressant, anticancer, anti-fungal, anti-tubercular, anti-inflammatory, antimicrobial, antiviral, and antifungal properties. In this study, we explored the use of Schiff base for the synthesis of new imidazole derivatives as anti-inflammatory and pain-relieving agents. A series of eight novel imidazole analogues (2a–h) were prepared in three steps with excellent yields. All compounds were characterized using IR, NMR, and mass spectral data. Their analgesic and anti-inflammatory activities were evaluated using hot plate and paw oedema methods. Compound 2 g (1-(2,3-dichlorophenyl)-2-(3-nitrophenyl)-4,5-diphenyl-1H-imidazole) showed significant analgesic activity (89% at 100 mg/kg b.w.), while compounds 2a (2-(2,6-dichlorophenyl)-1-(4-ethoxyphenyl)-4,5-diphenyl-1H-imidazole) and 2b (2-(2,3-dichlorophenyl)-1-(2-chlorophenyl)-4,5-diphenyl-1H-imidazole) exhibited good anti-inflammatory activity (100% at 100 mg/kg b.w.), comparable to diclofenac salt (100% at 50 mg/kg b.w.). Molecular docking studies were conducted using Schrödinger software version 2021-2, employing the OPLS4 force field for both receptor and ligand preparation. The results were visualized using molecular visualization software such as PyMOL. These studies revealed that compound 2g exhibited the highest binding affinity with the COX-2 receptor (−5.516 kcal/mol). Compound 2g formed three conventional hydrogen bonds with residues GLN-242 (bond length: 2.3 Å) and ARG-343 (bond lengths: 2.2 Å & 2.4 Å). This binding affinity was comparable to that of Diclofenac salt, which showed the highest binding affinity of −5.627 kcal/mol with the COX-2 receptor. Diclofenac salt formed two conventional hydrogen bonds with the residues ARG-344 (bond length: 2.0 Å) and TRP-140 (bond length: 1.7 Å). Later, molecular dynamic simulations confirmed the stable binding affinity of compound 2g with the protein. Furthermore, other compounds also demonstrated potential binding to the receptor-binding pocket region. The anti-inflammatory potential of the synthesized compounds was evaluated using the carrageenan-induced rat hind paw oedema model, while the analgesic potential was assessed using the hot plate method. These evaluations were conducted in comparison with Diclofenac sodium, serving as the standard compound. However, compound 2g stood out for its superior analgesic activity, as confirmed by in-vivo examination. These findings suggest that these novel imidazole derivatives have potential as anti-inflammatory and analgesic agents.

A tetravalent peptide efficiently inhibits the intestinal toxicity of heat-labile enterotoxin by targeting the receptor-binding region of the B-subunit pentamer

Infection by enterotoxigenic Escherichia coli (ETEC) causes severe watery diarrhea and dehydration in humans. Heat-labile enterotoxin (LT) is a major virulence factor produced by ETEC. LT is one of AB5-type toxins, such as Shiga toxin (Stx) and cholera toxin (Ctx), and the B-subunit pentamer is responsible for high affinity binding to the LT-receptor, ganglioside GM1, through multivalent interaction. In this report, we found that Glu51 of the B-subunit plays an essential role in receptor binding compared with other amino acids, such as Glu11, Arg13, and Lys91, all of which were previously shown to be involved in the binding. By targeting Glu51, we identified four tetravalent peptides that specifically bind to the B-subunit pentamer with high affinity by screening tetravalent random-peptide libraries, which were tailored to bind to the B-subunit through multivalent interaction. One of these peptides, GGR-tet, efficiently inhibited the cell-elongation phenotype and the elevation of cellular cAMP levels, both induced by LT. Furthermore, GGR-tet markedly inhibited LT-induced fluid accumulation in the mouse ileum. Thus, GGR-tet represents a novel therapeutic agent against ETEC infection.

Aptamer-quantum dots platform for SARS-CoV-2 viral particle detection by fluorescence microscopy

The virus is the smallest known replicative unit, usually in nanometer-range sizes. The most simple and sensitive detection assay involves molecular amplification of nucleic acids. This work shows a novel, straightforward detection based on the interaction of viral particles with fluorescent nanoconstructs without using enzymatic amplification, washing or separation steps.Fluorescent nanoconstructs are prepared with individual quantum dots of different emitting green and red fluorescence as a core. They are decorated with aptamers developed to recognise the receptor-binding region of the SARS-CoV-2 spike protein.Nanoconstructs can recognise SARS-CoV-2 viral particles fixed onto a coverglass generating aggregates. Meanwhile, SARS-CoV-2 viral particles/nanoconstructs complexes in solution yield aggregates and complexes, which a fluorescence microscope can visualise. The multiple molecular recognition allowed the detection of SARS-CoV-2 viral particles from a few microliters of patient swabs. This specific SARS-CoV-2/nanoconstructs interaction generates insoluble and precipitating aggregates. By using a mixture of green and red fluorescent nanoconstructs, upon the viral particle interaction, they yield heterochromatic green, red and yellow spectral fluorescence, easily identifiable by a fluorescence microscope. Washing and separation steps are not required, and aggregates allow one to easily recognise them, offering a sensitive, simple, and cheap alternative for viral detection.

A broadly-neutralizing antibody against Ebolavirus glycoprotein that potentiates the breadth and neutralization potency of other antibodies.

Ebolavirus disease (EVD) is caused by multiple species of Ebolavirus. Monoclonal antibodies (mAbs) against the virus glycoprotein (GP) are the only class of therapeutic approved for treatment of EVD caused by Zaire ebolavirus (EBOV). Therefore, mAbs targeting multiple Ebolavirus species may represent the next generation of EVD therapeutics. Broadly reactive anti-GP mAbs were produced; among these, mAbs 11886 and 11883 were broadly neutralizing in vitro. A 3.0 Å cryo-electron microscopy structure of EBOV GP bound to both mAbs shows that 11886 binds a novel epitope bridging the glycan cap (GC), 310 pocket and GP2 N-terminus, whereas 11883 binds the receptor binding region (RBR) and GC. In vitro, 11886 synergized with a range of mAbs with epitope specificities spanning the RBR/GC, including 11883. Notably, 11886 increased the breadth of neutralization by partner mAbs against different Ebolavirus species. These data provide a strategic route to design improved mAb-based next-generation EVD therapeutics.

Tailored multivalent peptide targeting the B-subunit pentamer of cholera toxin inhibits its intestinal toxicity by inducing aberrant transport of the toxin in cells

Cholera toxin (Ctx) is a major virulence factor produced by Vibrio cholerae that can cause gastrointestinal diseases, including severe watery diarrhea and dehydration, in humans. Ctx binds to target cells through multivalent interactions between its B-subunit pentamer and the receptor ganglioside GM1 present on the cell surface. Here, we identified a series of tetravalent peptides that specifically bind to the receptor-binding region of the B-subunit pentamer using affinity-based screening of multivalent random-peptide libraries. These tetravalent peptides efficiently inhibited not only the cell-elongation phenotype but also the elevated cAMP levels, both of which are induced by Ctx treatment in CHO cells or a human colon carcinoma cell line (Caco-2 cells), respectively. Importantly, one of these peptides, NRR-tet, which was highly efficient in these two activities, markedly inhibited fluid accumulation in the mouse ileum caused by the direct injection of Ctx. In consistent, NRR-tet reduced the extensive Ctx-induced damage of the intestinal villi. After NRR-tet bound to Ctx, the complex was incorporated into the cultured epithelial cells and accumulated in the recycling endosome, affecting the retrograde transport of Ctx from the endosome to the Golgi, which is an essential process for Ctx to exert its toxicity in cells. Thus, NRR-tet may be a novel type of therapeutic agent against cholera, which induces the aberrant transport of Ctx in the intestinal epithelial cells, detoxifying the toxin.

Molecular characterization and evolutionary dynamics of influenza A(H1N1) strains isolated from 2015 to 2017 in North India

Background and Objectives: The influenza A(H1N1) virus is known for large outbreaks, epidemics and pandemics world- wide owing to its genome plasticity which evolves constantly. In the year 2015 and then in 2017, India witnessed an upsurge in cases. Materials and Methods: The study was carried out in this period (2015-2017) with samples from 5 states across north India. The hemagglutinin 1 (HA1) and non-structural 1 (NS1) gene segments of the viral genome were characterised by phylogenetic analysis, selection pressure analysis, prediction of potential glycosylation sites and phylodynamic analysis of the study strains. Results: The study strains belonged to genogroup 6B. A total of 12 mutations were observed, half of which were located on the key receptor binding region of the HA1 protein. Established virulence markers D222G, S183P were observed in 2017 samples. Acquisition of an extra glycosylation site was observed in few strains from 2017 and 2016. Selection pressure analysis found the average dN/dS (v) ratio of 0.2106 and few codon sites in particular showed significant evidence of being under negative selection. Conclusion: The genogroup 6B continues to be the dominant circulating strain in Indian subcontinent region however the presence of pathogenic mutations in the 2017 strains from north India underlines the importance of continued molecular surveillance.

Global distribution, cross-species transmission, and receptor binding of canine parvovirus-2: Risks and implications for humans

For canine parvovirus -2 (CPV-2), a zoonotic virus capable of cross-species transmission in animals, the amino acid changes of capsid protein VP2 are key factors when binding to other species' transferrin receptors (TfR). CPV-2 variants can spread from felines and canines, for example, to Carnivora, Artiodactyla, and Pholidota species, and CPV-2c variants are essential to spread from Carnivora to Artiodactyla and Pholidota species in particular. In our study, a CPV-2a variant maintained a relatively stable trend, and the proportion of CPV-2c gradually rose from 1980 to 2021. The VP2 amino acid sequence analysis showed that five amino acid mutations at 426E/D, 305H/D, and 297S may be necessary for the virus to bind to different host receptors. Meanwhile, receptor-binding loop regions and amino acid sites 87 L, 93 N, 232I, and 305Y were associated with CPV-2 cross-species transmission. The homology of TfRs in different hosts infected with CPV-2 ranged from 77.2 % to 99.0 %, and from pig to feline, canine, and humans was 80.7 %, 80.4 %, and 77.2 %, respectively. The amino acid residues of TfRs involved in the viral binding in those hosts are highly conserved, which suggests that CPV-2 may be capable of pig-to-human transmission. Our analysis of the origin, evolutionary trend, cross-species transmission dynamics, and genetic characteristics of CPV-2 when binding to host receptors provides a theoretical basis for further research on CPV-2's mechanism of cross-species transmission and for establishing an early warning and monitoring mechanism for the possible threat of CPV-2 to animal–human public security.

Cry Toxins Use Multiple ATP-Binding Cassette Transporter Subfamily C Members as Low-Efficiency Receptors in Bombyx mori.

Recent studies have suggested that ABC transporters are the main receptors of Cry toxins. However, the receptors of many Cry toxins have not been identified. In this study, we used a heterologous cell expression system to identify Bombyx mori ABC transporter subfamily C members (BmABCCs) that function as receptors for five Cry toxins active in Lepidopteran insects: Cry1Aa, Cry1Ca, Cry1Da, Cry8Ca, and Cry9Aa. All five Cry toxins can use multiple ABCCs as low-efficiency receptors, which induce cytotoxicity only at high concentrations. Surface plasmon resonance analysis revealed that the KD values between the toxins and BmABCC1 and BmABCC4 were 10-5 to 10-9 M, suggesting binding affinities 8- to 10,000-fold lower than those between Cry1Aa and BmABCC2, which are susceptibility-determining receptors for Cry1Aa. Bioassays in BmABCC-knockout silkworm strains showed that these low-efficiency receptors are not involved in sensitivity to Cry toxins. The findings suggest that each family of Cry toxins uses multiple BmABCCs as low-efficiency receptors in the insect midgut based on the promiscuous binding of their receptor-binding regions. Each Cry toxin seems to have evolved to utilize one or several ABC transporters as susceptibility-determining receptors.

Antigenic commonality and divergence of hemagglutinin-esterase-fusion protein among influenza D virus lineages revealed using epitope mapping.

Influenza D virus (IDV) is one of the causative agents of bovine respiratory disease complex, which is the most common and economically burdensome disease affecting the cattle industry, and the need for an IDV vaccine has been proposed to enhance disease control. IDVs are classified into five genetic lineages based on the coding sequences of the hemagglutinin-esterase-fusion (HEF) protein, an envelope glycoprotein, which is the main target of protective antibodies against IDV infection. Herein, we prepared a panel of monoclonal antibodies (mAbs) against the HEF protein of viruses of various lineages to investigate the antigenic characteristics of IDVs and found that the mAbs could be largely separated into three groups. The first, second, and third groups demonstrated lineage-specific reactivity, cross-reactivity to viruses of multiple but not all lineages, and cross-reactivity to viruses of all lineages, respectively. Analyzing the escape mutant viruses from virus-neutralizing mAbs revealed that the receptor-binding region of the HEF molecule harbors virus-neutralizing epitopes that are conserved across multiple lineage viruses. In contrast, the apex region of the molecule possessed epitopes unique to each lineage virus. Furthermore, reverse genetics-generated recombinant viruses with point mutations revealed that amino acids within positions 210-214 of the HEF protein determined the antigenic specificity of each lineage virus. Taken together, this study reveals considerable antigenic variation among IDV lineages, although they are presumed to form a single serotype in terms of HEF antigenicity. Characterization of the antigenic epitope structure of HEF may contribute to selecting and creating effective vaccine viruses against IDV.IMPORTANCEInfluenza D viruses (IDVs) are suggested to create cross-reactive single serotypes in hemagglutinin-esterase-fusion (HEF) antigenicity, as indicated by serological analyses among distinct HEF lineage viruses. This is supported by the high identities of HEF gene sequences among strains, unlike the hemagglutinin (HA) genes of the influenza A virus that exhibit HA subtypes. Herein, we analyzed HEF antigenicity using a monoclonal antibody panel prepared from several virus lineages and found the existence of lineage-conserved and lineage-specific epitopes in HEF molecules. These findings confirm the HEF commonality and divergence among IDVs and provide useful information for constructing a vaccine containing a recombinant IDV virus with an engineered HEF gene, thereby leading to broad immunogenicity.

N123I mutation in the ALV-J receptor-binding domain region enhances viral replication ability by increasing the binding affinity with chNHE1.

The subgroup J avian leukosis virus (ALV-J), a retrovirus, uses its gp85 protein to bind to the receptor, the chicken sodium hydrogen exchanger isoform 1 (chNHE1), facilitating viral invasion. ALV-J is the main epidemic subgroup and shows noteworthy mutations within the receptor-binding domain (RBD) region of gp85, especially in ALV-J layer strains in China. However, the implications of these mutations on viral replication and transmission remain elusive. In this study, the ALV-J layer strain JL08CH3-1 exhibited a more robust replication ability than the prototype strain HPRS103, which is related to variations in the gp85 protein. Notably, the gp85 of JL08CH3-1 demonstrated a heightened binding capacity to chNHE1 compared to HPRS103-gp85 binding. Furthermore, we showed that the specific N123I mutation within gp85 contributed to the enhanced binding capacity of the gp85 protein to chNHE1. Structural analysis indicated that the N123I mutation primarily enhanced the stability of gp85, expanded the interaction interface, and increased the number of hydrogen bonds at the interaction interface to increase the binding capacity between gp85 and chNHE1. We found that the N123I mutation not only improved the viral replication ability of ALV-J but also promoted viral shedding in vivo. These comprehensive data underscore the notion that the N123I mutation increases receptor binding and intensifies viral replication.

Abstract B045: Novel Small Molecule Inhibitors Targeting Proinflammatory Cytokines in Metastatic Breast Cancer

Abstract Breast cancer is currently the most commonly diagnosed and second leading cause of cancer-related deaths among women in the Unites States. For the past several years, research has focused on the role of inflammatory cytokines such as interleukin-6 (IL-6) and their role in cancer. Increasing evidence suggests that breast cancer patients with high levels of proinflammatory modulators have an overall worse prognosis. Research performed by our lab and others has also shown that oncostatin M (OSM) promotes breast cancer progression and metastasis by inducing circulating tumor cell (CTC) numbers, and metastasis to distant sites in vivo. While efforts have been made to develop FDA approved therapeutics for OSM, previous studies have all utilized monoclonal antibodies and none have survived clinical trials. For the first time, our research group has developed a stepwise approach towards designing and testing novel small molecule inhibitors (SMIs) against OSM. To determine viable candidates, in silico computational screening of compound libraries towards OSM revealed top potential compounds that interact with the receptor binding region and inhibit biological function. These top candidate inhibitors were then analyzed for direct interaction with cytokines using fluorescent quenching and chemical shift perturbation NMR (CSP-NMR). In vitro efficacy of each SMI was then analyzed by measuring inhibition of all signaling cascades activated by OSM using enzyme-linked immunosorbent assays (ELISA) and immunoblot assays. From these in silico and in vitro methods, SMIs were then structurally modified with effective functional groups to increase efficacy of binding interaction and reduce toxicity for living systems. Finally, our most promising small molecule inhibitor that has been sufficiently tested for efficacy was examined in vivo utilizing a novel estrogen receptor-positive (ER+) mouse model of metastatic breast cancer, and treatment with our SMI proved to decrease tumor burden and metastasis, particularly to the lung when compared to vehicle control. In conclusion, our lab has generated the first ever SMI aimed to inhibit OSM-mediated cancer progression. Our continued efforts aim to develop a novel FDA approved therapeutic to improve outcomes for breast cancer patients. Citation Format: Cody L Wolf, Clyde Pruett, Darren Lighter, Cooper McGrath, Andrea Feci, Joseph Tuccinardi, Simion Dinca, Ken Tawara, Matthew King, Lisa Warner, Don Warner, Cheryl L. Jorcyk. Novel Small Molecule Inhibitors Targeting Proinflammatory Cytokines in Metastatic Breast Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B045.

Lectin Microarray Analysis of Antibodies to SARS-CoV-2 Elicited by COVID-19 mRNA Vaccine BNT162b2

The outstanding clinical efficacy of mRNA vaccines to the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has shown a new direction in vaccine design and development. Accumulation of knowledge on neutralizing antibodies induced by the mRNA vaccines is necessary to ensure the future development of effective and safe mRNA vaccines, not limited to Coronavirus Disease 2019 (COVID-19). Here we performed lectin microarray analysis of vaccine-induced immunoglobulin (Ig) G along with total IgG derived from volunteers who were vaccinated with BNT162b2 (Pfizer/BioNTech) mRNA COVID-19 vaccine. Two types of vaccine-induced IgG to the spike and the receptor binding region (RBD) protein of the SARS-CoV-2 were isolated for comparative analysis with total IgG, revealing that distinct differences in glycan profiles that exceeded the differences among individuals, even from an extremely low amount of Ig such as 30 ng of Ig. In particular, the anti-RBD IgG, which demonstrated a uniform glycosylation pattern as the IgG group, showed an increase in fucose and a decrease in sialic acid compared to the total IgG, indicating similar glycan changes as previously reported. The lectin microarray, which can sensitively detect alternations and characteristics in glycan structures, might be used for large-scale analysis to assess the relationship between vaccine side effects and effector activity in the future.

Clinical characteristics of outpatients with influenza-B-associated pneumonia and molecular evolution of influenza B virus in Beijing, China, during the 2021–2022 influenza season

We analyzed the clinical characteristics of outpatients with influenza-B-associated pneumonia during the 2021–2022 influenza season and analyzed the molecular epidemiology and evolution of influenza B virus. The presence of influenza B virus was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Electronic medical records were used to collect and analyze data of outpatients. The HA and NA genes were phylogenetically analyzed using ClustalW 2.10 and MEGA 11.0. Out of 1569 outpatients who tested positive for influenza B virus, 11.7% (184/1569) developed pneumonia, and of these, 19.0% (35/184) had underlying diseases. Fever, cough, and sore throat were the most common symptoms. Among the complications, acute respiratory distress syndrome (ARDS), acute kidney injury (AKI), and shock accounted for 2.7% (5/184), 4.9% (9/184), and 1.6% (3/184), respectively. Of the outpatients, 2.7% (5/184) were admitted to the hospital, and 0.5% (1/184) of them died. All of the strains from Beijing were identified as belonging to the B/Victoria lineage. The HA and NA gene sequences of 41 influenza B viruses showed high similarity to each other, and all of them belonged to clade 1A.3. Compared with the vaccine strain B/Washington/02/2019, all of the isolates contained N150K, G181E, and S194D mutations. S194D, E195K, and K200R mutations were detected in the 190 helix of the receptor binding region of HA. Co-mutations of H122Q, A127T, P144L, N150K, G181E, S194D, and K200R in HA and D53N, N59S, and G233E in NA were detected in 78.0% (32/41) of the isolates, and 56.3% (18/32) of these were from outpatients with influenza-B-associated pneumonia. Influenza outpatients with underlying diseases were more likely to develop pneumonia. No significant differences were observed in clinical symptoms or laboratory results between outpatients with and without pneumonia, so testing for influenza virus seems to be a good choice. The observed amino acid variations suggest that current vaccines might not provide effective protection.

Screening and Identification of Anti-Idiotypic Nanobody Capable of Broad-Spectrum Recognition of the Toxin Binding Region of Lepidopteran Cadherins and Mimicking Domain II of Cry2Aa Toxin.

The widespread use of Bacillus thuringiensis toxins as insecticides has brought about resistance problems. Anti-idiotypic nanobody approaches provide new strategies for resistance management and toxin evolution. In this study, the monoclonal antibody generated against the receptor binding region Domain II of Cry2Aa toxin was used as a target to screen materials with insecticidal activity. After four rounds of screening, anti-idiotypic nanobody 1C12 was obtained from the natural alpaca nanobody phage display library. To better analyze the activity of 1C12, soluble 1C12 was expressed by the Escherichia coli BL21 (DE3). The results showed that 1C12 not only binds the midgut brush border membrane vesicles (BBMV) of two lepidopteran species and cadherin CR9-CR11 of three lepidopteran species but also inhibits Cry2Aa toxins from binding to CR9-CR11. The insect bioassay showed that soluble 1C12 caused 25.65% and 23.61% larvae mortality of Helicoverpa armigera and Plutella xylostella, respectively. Although 1C12 has low insecticidal activity, soluble 1C12 possesses the ability to screen a broad-spectrum recognition of the toxin binding region of lepidopteran cadherins and can be used for the identification of the toxin binding region of other lepidopteran cadherins and the subsequent evolution of Cry2Aa toxin. The present study demonstrates a new strategy to screen for the production of novel insecticides.

Pre-existing cross-reactive neutralizing activity against SARS-CoV-2 and seasonal coronaviruses prior to the COVID-19 pandemic (2014-2019) with limited immunity against recent emerging SARS-CoV-2 variants, Vietnam

ObjectivesSARS-CoV-2 transmission and epidemic potential is related to the population's immunity levels. As such, assessing different regions’ preexisting immune responses to SARS-CoV-2 is important to understand the transmission potential of emerging SARS-CoV-2 variants. DesignIn 975 serum samples from Vietnam (2014 to 2019), anti-SARS-CoV-2 Immunoglobulin G levels were determined by enzyme-linked immunosorbent assay. Plaque reduction neutralization test (PRNT) was performed using Wuhan strain and variants of concern (VOCs). Cross-reactivity was confirmed by analyzing B-cell receptor (BCR) repertoire sequences and identifying BCR repertoire sequences-derived T-cell epitopes. ResultsOverall, 20.9% (n = 76/364) and 9.2% (n = 7) demonstrated SARS-CoV-2 neutralizing activity (PRNT50) against the Wuhan and Alpha strain, respectively. Neutralizing activity against Beta, Gamma, and Delta strains was absent (PRNT50<5) in all samples. Cross-reactive epitopes against SARS-CoV-2 and other coronavirus spike proteins were detected in the N-terminal domain, S2, and receptor-binding domain regions. ConclusionsFollowing BCR and major histocompatibility complex analysis, T-cell receptor-recognized epitope motif (TREM) among pathogenic coronaviruses and coronaviruses spike proteins were the top TREM peptide, suggesting that pre-existing immunity against SARS-CoV-2 in Vietnam was due to exposure to common cold coronaviruses. With limited immunity against emerging VOCs, further monitoring, and control of the epidemic, along with COVID-19 vaccine programs against VOCs, are necessary.

Omicron and Delta variant prevalence detection and identification during the fourth COVID-19 wave in Mexico using wastewater-based epidemiology

ObjectivesTo identify the SARS-CoV-2 variants Delta and Omicron during the fourth wave of the COVID-19 pandemic in Mexico using samples taken from 19 locations in 18 out of the 32 states. MethodsThe genetic material concentration was done with PEG/NaCl precipitation, SARS-CoV-2 presence was confirmed by reverse transcriptase-quantitative polymerase chain reaction assay, the variant detection was carried out using a commercial mutation detection panel kit, and variant/mutation confirmation was done by amplicon sequencing of receptor-binding domain target region. The study used 41 samples. ResultsThe Delta variant was confirmed in two samples during August 2021 (Querétaro and CDMX) and in three samples during November 2021 (Aguascalientes, Ciudad Juárez campuses, and Nuevo Leon). In December 2021, another sample with the Delta variant was confirmed in Nuevo Leon. Between January to March 2022 only the presence of Omicron was confirmed, (variant BA.1). Additionally, in this period six samples were identified with the status “Variant Not Determined”. ConclusionTo our knowledge, this study is one of the first to identify Omicron and Delta variants with polymerase chain reaction in Mexico and Latin America and its distribution across the country with 56% Mexican states making it a viable alternative for variant detection without conducting a large quantity of sequencing of clinical tests.

Natural immunity to malaria preferentially targets the endothelial protein C receptor-binding regions of PfEMP1s.

Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an "adult-like" response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This "adult-like" response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant.

Dissecting the Molecular Basis of Host Leucine-Rich Repeat Containing 15 Mediated Interaction with Receptor Binding Domain of SARS-CoV-2 Spike Protein: A Computational Approach.

The detection of leucine-rich repeat containing 15 (LRRC15) as a connecting link with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the possibility of its involvement in differential restriction activity of SARS-CoV-2 pathways. However, the structure-function mechanism of LRRC15 involving the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and their mode of interaction is largely unknown. Using state-of-the-art AlphaFold2 and all-atom molecular dynamics simulations, our findings provide evidences of alternative binding modes of RBD with LRR units of LRRC15 having varied affinities. Contribution of both the receptor binding regions in RBD, including receptor binding motif in accommodating the LRR domain, towards the C-terminal region, emphasizes its differential role in modulating host cell receptiveness for SARS-CoV-2, the innate immune system, as well as antiviral tone. However, further experimental validations are necessary for unravelling the unknown mechanism and distinctive features of this host receptor in the COVID-19 pandemic, involving both the transmembrane as well as cytoplasmic domain.

Discovery and pharmacophoric characterization of chemokine network inhibitors using phage-display, saturation mutagenesis and computational modelling

CC and CXC-chemokines are the primary drivers of chemotaxis in inflammation, but chemokine network redundancy thwarts pharmacological intervention. Tick evasins promiscuously bind CC and CXC-chemokines, overcoming redundancy. Here we show that short peptides that promiscuously bind both chemokine classes can be identified from evasins by phage-display screening performed with multiple chemokines in parallel. We identify two conserved motifs within these peptides and show using saturation-mutagenesis phage-display and chemotaxis studies of an exemplar peptide that an anionic patch in the first motif and hydrophobic, aromatic and cysteine residues in the second are functionally necessary. AlphaFold2-Multimer modelling suggests that the peptide occludes distinct receptor-binding regions in CC and in CXC-chemokines, with the first and second motifs contributing ionic and hydrophobic interactions respectively. Our results indicate that peptides with broad-spectrum anti-chemokine activity and therapeutic potential may be identified from evasins, and the pharmacophore characterised by phage display, saturation mutagenesis and computational modelling.