HLA-B evolutionary divergence is associated with poor outcomes after SARS-CoV-2 infection

Objectives Human leukocyte antigens (HLA) are highly diverse transmembrane proteins that present viral peptides to T cells and launch pathogen-specific immune responses. We aim to investigate the correlation between HLA evolutionary divergence (HED), a surrogate for the capacity to present different peptides, and the outcomes of SARS-CoV-2 infection in a cohort from the St. Louis Metropolitan area. Methods We enrolled adult patients with SARS-CoV-2 infection confirmed by RT-PCR who were hospitalized at two tertiary hospitals in St. Louis between March and July 2020. Genomic DNA was extracted from peripheral blood and genotyped by next-generation sequencing (NGS). HLA alleles were assigned based on key-exon sequences (G group) and limited to the 2-field resolution. HED was calculated by Grantham distance, which considers the difference in composition, polarity, and molecular volume between each pair of amino acids from maternal and paternal HLA. The HED score was obtained for HLA class I (HLA-A, -B, and -C) genotypes using the HLAdivR package in R. Clinical data were collected retrospectively from electronic medical records. A poor outcome was defined as an admission to the intensive care unit (ICU), a need for mechanical ventilation, or death. A favorable outcome was defined as the absence of the above poor outcomes. Results A total of 234 patients were enrolled in this study, 96 being females (41%). The median age and BMI were 66 years old and 28.30 kg/m2, respectively. African Americans comprised 71.4% of the cohort. Only 19 patients (8.1%) presented with no comorbidity;the rest had one or more comorbidities, with cardiovascular diseases being the most common. A total of 137 (58.5%) patients had poor outcomes from SARS-CoV-2 infection, while 97 (41.5%) patients had a favorable outcome. We detected a significant association between higher HLA-B HED and favorable outcomes, with each 1-point increase in HLA-B HED associated with 8% increased probability for the composite endpoint (OR 1.08, 95% CI=1.01-1.16, P = 0.04). The HED scores calculated for HLA-A or HLA-C were not significantly different between patients with favorable or poor outcomes. In a multivariate logistic regression analysis, increased HLA-B HED score, younger age, and no comorbidity were independently associated with favorable outcomes (P = 0.02, P = 0.01, and P = 0.05, respectively). Conclusion Our study shows a significant correlation between lower HLA-B HED scores and poor outcomes after SARS-CoV-2 infection. This finding suggests that maximizing the presentation of diverse SARS-CoV-2 peptides by HLA-B alleles may improve the clearance of SARS-CoV-2. Further studies are warranted to understand the functional and mechanistic implications of this finding.

Aptamers Targeting Membrane Proteins for Sensor and Diagnostic Applications.

Many biological processes (physiological or pathological) are relevant to membrane proteins (MPs), which account for almost 30% of the total of human proteins. As such, MPs can serve as predictive molecular biomarkers for disease diagnosis and prognosis. Indeed, cell surface MPs are an important class of attractive targets of the currently prescribed therapeutic drugs and diagnostic molecules used in disease detection. The oligonucleotides known as aptamers can be selected against a particular target with high affinity and selectivity by iterative rounds of in vitro library evolution, known as Systematic Evolution of Ligands by EXponential Enrichment (SELEX). As an alternative to antibodies, aptamers offer unique features like thermal stability, low-cost, reuse, ease of chemical modification, and compatibility with various detection techniques. Particularly, immobilized-aptamer sensing platforms have been under investigation for diagnostics and have demonstrated significant value compared to other analytical techniques. These "aptasensors" can be classified into several types based on their working principle, which are commonly electrochemical, optical, or mass-sensitive. In this review, we review the studies on aptamer-based MP-sensing technologies for diagnostic applications and have included new methodological variations undertaken in recent years.

Attenuation of a Highly Pathogenic Porcine Deltacoronavirus Strain CZ2020 by a Serial Passage In Vitro

Porcine deltacoronavirus (PDCoV) is an emerging swine coronavirus that causes severe diarrhea to pigs of all ages, especially the suckling piglets under one-week-old. We previously isolated a highly pathogenic PDCoV strain, CZ2020, from a diarrheal piglet and have passaged it for over 100 passages. The adaptability of the CZ2020 increased gradually in vitro as the passage increased. Amino acid mutations were observed in pp1a, pp1ab, spike, envelop, and membrane proteins, and the spike protein accounts for 66.7% of all amino acid mutations. Then, the high passage strains, CZ2020-F80 and CZ2020-F100, were selected for evaluation of the pathogenicity in three-day-old piglets to examine whether these amino acid changes affected their virulence. At 2 days postchallenge (DPC), 2/5 piglets started to show typical diarrhea, and at 4 DPC, severe diarrhea was observed in the CZ2020-challenged piglets. Viral RNA could be detected at 1 DPC in rectal swabs and reached its highest at 4 DPC in the CZ2020-challenged group. CZ2020-F80- and CZ2020-F100-challenged groups have one piglet exhibiting mild diarrhea at 4 and 6 DPC, respectively. Compared with the CZ2020-challenged group, the piglets in CZ2020-F80- and F100-challenged groups had lower viral loads in rectal swabs, intestines, and other organs. No obvious histopathological lesions were observed in the intestines of CZ2020-F80- and F100-challenged piglets. Virulent PDCoV infection could also induce strong interferons and proinflammatory cytokines in vitro and in vivo. These data indicate that the strains, CZ2020-F80 and CZ2020-F100, were significantly attenuated via serial passaging in vitro and have the potential for developing attenuated vaccine candidates.

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution, new validation tools, and implications for the future.

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) "Resolution Revolution" made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps. After describing the history of the PDB and the Worldwide Protein Data Bank (wwPDB) partnership, which jointly manages both the PDB and EMDB archives, this review examines the origins of the resolution revolution and analyzes its impact on structural biology viewed through the lens of PDB holdings. Six areas of focus exemplifying the impact of 3DEM across the biosciences are discussed in detail (icosahedral viruses, ribosomes, integral membrane proteins, SARS-CoV-2 spike proteins, cryogenic electron tomography, and integrative structure determination combining 3DEM with complementary biophysical measurement techniques), followed by a review of 3DEM structure validation by the wwPDB that underscores the importance of community engagement.

RAMIHM generates fully human monoclonal antibodies by rapid mRNA immunization of humanized mice and BCR-seq.

As a clinical vaccine, lipid nanoparticle (LNP) mRNA has demonstrated potent and broad antibody responses, leading to speculation about its potential for antibody discovery. Here, we developed RAMIHM, a highly efficient strategy for developing fully human monoclonal antibodies that employs rapid mRNA immunization of humanized mice followed by single B cell sequencing (scBCR-seq). We immunized humanized transgenic mice with RAMIHM and generated 15 top-ranked clones from peripheral blood, plasma B, and memory B cell populations, demonstrating a high rate of antigen-specificity (93.3%). Two Omicron-specific neutralizing antibodies with high potency and one broad-spectrum neutralizing antibody were discovered. Furthermore, we extended the application of RAMIHM to cancer immunotherapy targets, including a single transmembrane protein CD22 and a multi-transmembrane G protein-coupled receptor target, GPRC5D, which is difficult for traditional protein immunization methods. RAMIHM-scBCR-seq is a broadly applicable platform for the rapid and efficient development of fully human monoclonal antibodies against an assortment of targets.

A Facile Method to Coat Nanoparticles with Lipid Bilayer Membrane: Hybrid Silica Nanoparticles Disguised as Biomembrane Vesicles by Particle Penetration of Concentrated Lipid Layers.

Natural membrane vesicles, including extracellular vesicles and enveloped viruses, participate in various events in vivo. To study and manipulate these events, biomembrane-coated nanoparticles inspired by natural membrane vesicles are developed. Herein, an efficient method is presented to prepare organic-inorganic hybrid materials in high yields that can accommodate various lipid compositions and particle sizes. To demonstrate this method, silica nanoparticles are passed through concentrated lipid layers prepared using density gradient centrifugation, followed by purification, to obtain lipid membrane-coated nanoparticles. Various lipids, including neutral, anionic, and cationic lipids, are used to prepare concentrated lipid layers. Single-particle analysis by imaging flow cytometry determines that silica nanoparticles are uniformly coated with a single lipid bilayer. Moreover, cellular uptake of silica nanoparticles is enhanced when covered with a lipid membrane containing cationic lipids. Finally, cell-free protein expression is applied to embed a membrane protein, namely the Spike protein of severe acute respiratory syndrome coronavirus 2, into the coating of the nanoparticles, with the correct orientation. Therefore, this method can be used to develop organic-inorganic hybrid nanomaterials with an inorganic core and a virus-like coating, serving as carriers for targeted delivery of cargos such as proteins, DNA, and drugs.

NucEnvDB: A Database of Nuclear Envelope Proteins and Their Interactions.

The nuclear envelope (NE) is a double-membrane system surrounding the nucleus of eukaryotic cells. A large number of proteins are localized in the NE, performing a wide variety of functions, from the bidirectional exchange of molecules between the cytoplasm and the nucleus to chromatin tethering, genome organization, regulation of signaling cascades, and many others. Despite its importance, several aspects of the NE, including its protein-protein interactions, remain understudied. In this work, we present NucEnvDB, a publicly available database of NE proteins and their interactions. Each database entry contains useful annotation including a description of its position in the NE, its interactions with other proteins, and cross-references to major biological repositories. In addition, the database provides users with a number of visualization and analysis tools, including the ability to construct and visualize protein-protein interaction networks and perform functional enrichment analysis for clusters of NE proteins and their interaction partners. The capabilities of NucEnvDB and its analysis tools are showcased by two informative case studies, exploring protein-protein interactions in Hutchinson-Gilford progeria and during SARS-CoV-2 infection at the level of the nuclear envelope.

Predicting the Assembly of the Transmembrane Domains of Viral Channel Forming Proteins and Peptide Drug Screening Using a Docking Approach.

A de novo assembly algorithm is provided to propose the assembly of bitopic transmembrane domains (TMDs) of membrane proteins. The algorithm is probed using, in particular, viral channel forming proteins (VCPs) such as M2 of influenza A virus, E protein of severe acute respiratory syndrome corona virus (SARS-CoV), 6K of Chikungunya virus (CHIKV), SH of human respiratory syncytial virus (hRSV), and Vpu of human immunodeficiency virus type 2 (HIV-2). The generation of the structures is based on screening a 7-dimensional space. Assembly of the TMDs can be achieved either by simultaneously docking the individual TMDs or via a sequential docking. Scoring based on estimated binding energies (EBEs) of the oligomeric structures is obtained by the tilt to decipher the handedness of the bundles. The bundles match especially well for all-atom models of M2 referring to an experimentally reported tetrameric bundle. Docking of helical poly-peptides to experimental structures of M2 and E protein identifies improving EBEs for positively charged (K,R,H) and aromatic amino acids (F,Y,W). Data are improved when using polypeptides for which the coordinates of the amino acids are adapted to the Cα coordinates of the respective experimentally derived structures of the TMDs of the target proteins.

Erratum: Remdesivir strongly binds to RNA-dependent RNA polymerase, membrane protein, and main protease of SARS-CoV-2: Indication from molecular modeling and simulations.

[This corrects the article DOI: 10.3389/fphar.2021.710778.].

Deterrent to COVID-19 infection - adopting a Srilankan university research study

This study provides a simple, widely available deterrence medication to minimize COVID-19 infection using by tea and coffee infusions. By mixing white with an equal amount of water, add a small amount of tea/coffee infusion. Dull brownish albumen-tannin complex, a soft precipitate is formed and sinks to the bottom. The infusion should reach the furthest points in the respiratory tract so that a maximum number of viral particles are trapped. It is necessary to hold the infusion rolling about for a little while, 20 seconds in the mouth before swallowing. Gargling with the infusion is better. Tannins in tea or coffee infusions will form complexes with the 4-5 types of viral surface proteins [spikes], rendering them ineffective. Protein-tannin astringent complexation is a fundamental chemical reaction and is bound to act, unlike specific antimetabolites or enzyme-mediated actions of antibiotics. In fact, tannins react to some extent with the cell lining of the mucous membranes of the mouth and stomach. Tannins also chelate iron [Fe] and other metal ions required for many of the metabolic reactions of micro-organisms [viruses?], depriving them of these nutrients and further retarding their propagation. Two gargles per day, 12 hours apart, are recommended to disable the virus and eventually kill it. The procedure disables free viruses before tissue invasion. Therefore, the earlier the gargle commences, the better. Suspected contacts should preferably have three daily eight-hourly gargles. Astringent activity is an added activity of tea and coffee, demonstrated in this study [using E. coli and Salmonella] to that of immune boosting action generally spoken about.

A look at the C3 and C5 proteins in the Covid‐19

Because of the crucial role played by proteins C3 and C5 in the infection by the SARS-COV-2 virus, new complement system inhibition treatments have emerged as a possible first line of defense against the worst symptoms developed during Covid-19 disease. C3, C5, Complemento, Inhibición, SARS-CoV-2 INTRODUCTION In December 2019, China informed the World Health Organization (WHO) about the spread of a new pathogen in the Chinese city of Wuhan, this pathogen was named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (Abd El-Aziz and Stockand, 2020), causing the Covid-19 disease. Once a person is infected with the SARSCoV-2 virus, the spike homotrimer glycoprotein of the virus connects to the integral membrane protein ACE2 of human cells (Lan et al., 2020;Rothan and Byrareddy, 2020), many of these receptors are found in the lung epithelium layer, making them one of the main sites of infection of the SARS-CoV-2 virus (Connors and Levy, 2020). The activation of the complement system is vital to the control of viruses, fungi, bacteria, and other pathogens, this activation triggers the opsonization of the pathogens and the elimination of them (Merle et al., 2015;Sarma and Ward, 2011);the activation is sequential: a catalytic cascade is produced through the generation of complex enzymes and proteins, in one step an enzyme or protein of this cascade can generate many active molecules for the next step in the activation of the complement system (Merle et al., 2015).

21st ESPD Annual Meeting, 20-22 May 2022

The results of polymerase chain reaction tests for SARS-CoV-2 were positive for 4 (%9,3) patients, and the results of SARS-CoV-2 immunoglobulin G tests were positive for 39 (%90,7) patients. 21 (%48,8) of the patients had urticarial, 12 (%27,9) of them maculopapular, 5 (%11,6) patients livedoid and 1 (%2,3) patient had a skin rash compatible with pseudopernio. For patients not achieving the primary endpoints at Week 16, 33.3% and 57.8% of 135 patients initially treated with tralokinumab and transferred to open-label treatment at Week 16 achieved IGA 0/1 or EASI-75 at Week 52. With regard to skin-related QoL measured with Children's Dermatology Life Quality Index (CDLQI), 5 patients suffered from moderate QoL impairment whereas 42 individuals revealed only mild and 39 patients no QoL reduction. [Extracted from the article] Copyright of Pediatric Dermatology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Analysis of Glycosylation and Disulfide Bonding of Wild-Type SARS-CoV-2 Spike Glycoprotein

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E, and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to purify the Golgi-modified fraction of a wild-type SARS-CoV-2 S glycoprotein trimer and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteo-lytic cleavage and to retain a prefusion conformation, more of the wild-type S glyco-protein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on other characterized S trimer preparations, is predominantly modified in the Golgi compartment by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutrali-zation by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi process -ing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions. IMPORTANCE The SARS-CoV-2 coronavirus, which causes COVID-19, uses its spike glycoprotein to enter host cells. The viral spike glycoprotein is the main target of host neutralizing antibodies that help to control SARS-CoV-2 infection and are important for the protection provided by vaccines. The SARS-CoV-2 spike glyco-protein consists of a trimer of two subunits covered with a coat of carbohydrates (sugars). Here, we describe the disulfide bonds that assist the SARS-CoV-2 spike glycoprotein to assume the correct shape and the composition of the sugar moieties on the glycoprotein surface. We also evaluate the consequences of natural virus variation in O-linked sugar addition and in the cysteine residues involved in disulfide bond formation. This information can expedite the improvement of vac-cines and for COVID-19.

New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition.

The fourth enzymatic reaction in the de novo pyrimidine biosynthesis, the oxidation of dihydroorotate to orotate, is catalyzed by dihydroorotate dehydrogenase (DHODH). Enzymes belonging to the DHODH Class II are membrane-bound proteins that use ubiquinones as their electron acceptors. We have designed this study to understand the interaction of an N-terminally truncated human DHODH (HsΔ29DHODH) and the DHODH from Escherichia coli (EcDHODH) with ubiquinone (Q10) in supported lipid membranes using neutron reflectometry (NR). NR has allowed us to determine in situ, under solution conditions, how the enzymes bind to lipid membranes and to unambiguously resolve the location of Q10. Q10 is exclusively located at the center of all of the lipid bilayers investigated, and upon binding, both of the DHODHs penetrate into the hydrophobic region of the outer lipid leaflet towards the Q10. We therefore show that the interaction between the soluble enzymes and the membrane-embedded Q10 is mediated by enzyme penetration. We can also show that EcDHODH binds more efficiently to the surface of simple bilayers consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine, and tetraoleoyl cardiolipin than HsΔ29DHODH, but does not penetrate into the lipids to the same degree. Our results also highlight the importance of Q10, as well as lipid composition, on enzyme binding.

Guest Editors Introductory Words

[...]the study of molecular events behind the entry of the COVID-19 into the host cells revealed a number of co-operating proteins among which a key role plays the transmembrane protease serine 2 (TMPRSS2), which is needed to cleave the spike protein and assist in membrane fusion, the expression of which is increased by androgens via androgen receptor activation (Stárka and Dušková, this issue, Knížatová et al., this issue). All these actions are genomic, but recent research of the role of androgens revealed that the latter possess also rapid, non-genomic response, as demonstrated that they are not inhibited by both transcription and translation inhibitors (actinomycin, cycloxeximide) as well as androgen receptors blockers (flutamide). Besides classical androgens testosterone, dihydrotestosterone and dehydroepiandrosterone, a particular function has the betaepimer of dihydrotestosterone (5ß-DHT), completely inactive to intracellular androgen receptors (Perusquía, this issue). [...]low androgen levels as well as hyperandrogenemia are risk factors for development and severity of COVID-19 disease.

Role of membrane proteins in bacterial resistance to antimicrobial peptides.

Several natural antimicrobial peptides (AMPs), including the novel semisynthetic lipoglycopeptide antibiotics telavancin, dalbavancin, and oritavancin, have been approved for clinical use to address the growing problem of multiple antibiotic-resistant Gram-positive bacterial infections. Nevertheless, the efficacy of these antibiotics has already been compromised. The SARS-CoV-2 pandemic led to the increased clinical use of all antibiotics, further promoting the development of bacterial resistance. Therefore, it is critical to gain a deeper understanding of the role of resistance mechanisms to minimize the consequential risks of long-term antibiotic use and misuse. Here, we summarize for the first time the current knowledge of resistance mechanisms that have been shown to cause resistance to clinically used AMPs, with particular focus on membrane proteins that have been reported to interfere with the activity of AMPs by affecting the binding of AMPs to bacteria.

Remdesivir Strongly Binds to RNA-Dependent RNA Polymerase, Membrane Protein, and Main Protease of SARS-CoV-2: Indication From Molecular Modeling and Simulations.

Development of new drugs is a time-taking and expensive process. Comprehensive efforts are being made globally toward the search of therapeutics against SARS-CoV-2. Several drugs such as remdesivir, favipiravir, ritonavir, and lopinavir have been included in the treatment regimen and shown effective results in several cases. Among the existing broad-spectrum antiviral drugs, remdesivir is found to be more effective against SARS-CoV-2. Remdesivir has broad-spectrum antiviral action against many single-stranded RNA viruses including pathogenic SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). In this study, we proposed that remdesivir strongly binds to membrane protein (Mprotein), RNA-dependent RNA polymerase (RDRP), and main protease (Mprotease) of SARS-CoV-2. It might show antiviral activity by inhibiting more than one target. It has been found that remdesivir binds to Mprotease, Mprotein, and RDRP with -7.8, -7.4, and -7.1 kcal/mol, respectively. The structure dynamics study suggested that binding of remdesivir leads to unfolding of RDRP. It has been found that strong binding of remdesivir to Mprotein leads to decrease in structural deviations and gyrations. Additionally, the average solvent-accessible surface area of Mprotein decreases from 127.17 to 112.12 nm2, respectively. Furthermore, the eigenvalues and the trace of the covariance matrix were found to be low in case of Mprotease-remdesivir, Mprotein-remdesivir, and RDRP-remdesivir. Binding of remdesivir to Mprotease, Mprotein, and RDRP reduces the average motions in protein due to its strong binding. The MMPBSA calculations also suggested that remdesivir has strong binding affinity with Mprotein, Mprotease, and RDRP. The detailed analysis suggested that remdesivir has more than one target of SARS-CoV-2.

Klebsiella pneumoniae enolase-like membrane protein interacts with human plasminogen.

Many models assessing the risk of sepsis utilize the knowledge of the constituents of the plasminogen system, as it is proven that some species of bacteria can activate plasminogen, as a result of interactions with bacterial outer membrane proteins. However, much is yet to be discovered about this interaction since there is little information regarding some bacterial species. This study is aimed to check if Klebsiella pneumoniae, one of the major factors of nosocomial pneumonia and a factor for severe sepsis, has the ability to bind to human plasminogen. The strain used in this study, PCM 2713, acted as a typical representative of the species. With use of various methods, including: electron microscopy, 2-dimensional electrophoresis, immunoblotting and peptide fragmentation fingerprinting, it is shown that Klebsiella pneumoniae binds to human plasminogen, among others, due to plasminogen-bacterial enolase-like protein interaction, occurring on the outer membrane of the bacterium. Moreover, the study reveals, that other proteins, such as: phosphoglucomutase, and phosphoenolpyruvate carboxykinase act as putative plasminogen-binding factors. These information may virtually act as a foundation for future studies investigating: the: pathogenicity of Klebsiella pneumoniae and means for prevention from the outcomes of Klebsiella-derived sepsis.

Mini-review: antibody therapeutics targeting G protein-coupled receptors and ion channels.

Antibodies are now well established as therapeutics with many additional advantages over small molecules and peptides relative to their selectivity, bioavailability, half-life and effector function. Major classes of membrane-associated protein targets include G protein-coupled receptors (GPCRs) and ion channels that are linked to a wide range of disease indications across all therapeutic areas. This mini-review summarizes the antibody target landscape for both GPCRs and ion channels as well as current progress in the respective research and development pipelines with some example case studies highlighted from clinical studies, including those being evaluated for the treatment of symptoms in COVID-19 infection.

Immunoinformatic approach to design a multiepitope vaccine targeting non-mutational hotspot regions of structural and non-structural proteins of the SARS CoV2.

BACKGROUND: The rapid Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV2) outbreak caused severe pandemic infection worldwide. The high mortality and morbidity rate of SARS CoV2 is due to the unavailability of vaccination and mutation in this virus. The present article aims to design a potential vaccine construct VTC3 targeting the non-mutational region of structural and non-structural proteins of SARS CoV2. METHODS: In this study, vaccines were designed using subtractive proteomics and reverse vaccinology. To target the virus adhesion and evasion, 10 different structural and non-structural proteins have been selected. Shortlisted proteins have been screened for B cell, T cell and IFN gamma interacting epitopes. 3D structure of vaccine construct was modeled and evaluated for its physicochemical properties, immunogenicity, allergenicity, toxicity and antigenicity. The finalized construct was implemented for docking and molecular dynamics simulation (MDS) with different toll-like receptors (TLRs) and human leukocyte antigen (HLA). The binding energy and dissociation construct of the vaccine with HLA and TLR was also calculated. Mutational sensitivity profiling of the designed vaccine was performed, and mutations were reconfirmed from the experimental database. Antibody production, clonal selection, antigen processing, immune response and memory generation in host cells after injection of the vaccine was also monitored using immune simulation. RESULTS: Subtractive proteomics identified seven (structural and non-structural) proteins of this virus that have a role in cell adhesion and infection. The different epitopes were predicted, and only extracellular epitopes were selected that do not have similarity and cross-reactivity with the host cell. Finalized epitopes of all proteins with minimum allergenicity and toxicity were joined using linkers to designed different vaccine constructs. Docking different constructs with different TLRs and HLA demonstrated a stable and reliable binding affinity of VTC3 with the TLRs and HLAs. MDS analysis further confirms the interaction of VTC3 with HLA and TLR1/2 complex. The VTC3 has a favorable binding affinity and dissociation constant with HLA and TLR. The VTC3 does not have similarities with the human microbiome, and most of the interacting residues of VTC3 do not have mutations. The immune simulation result showed that VTC3 induces a strong immune response. The present study designs a multiepitope vaccine targeting the non-mutational region of structural and non-structural proteins of the SARS CoV2 using an immunoinformatic approach, which needs to be experimentally validated.