Epitope Structure Research Articles

Development of an Immortalized Porcine Fibroblast Cell Panel With Different Swine Leukocyte Antigen Genotypes.

Immortalized cell lines are valuable resources to expand the molecular characterization of major histocompatibility complex genes and their presented antigens. We generated a panel of immortalized cell lines by transfecting human telomerase reverse transcriptase (hTERT) into primary fibroblast cells prepared from ear, fetal, and lung tissues of 10 pigs from five breeds and successfully cultured them for 30–45 passages. The cell growth characteristic of the immortalized fibroblasts was similar to that of primary fibroblast, which was unable to form colonies on soft agar. The genotypes of major swine leukocyte antigen (SLA) genes, including three classical class I (SLA-1, -2, and -3) and three class II genes (DQB1, DRB1, and DQA), were determined using high-resolution typing. A total of 58 alleles, including a novel allele for SLA-2, were identified. Each cell line was unique. A cell line derived from a National Institutes of Health miniature pig was homozygous across the six major SLA genes. The expression levels of SLA classical class I genes varied among the cell lines and were slightly upregulated in the immortalized compared to the primary cells based on semiquantitative reverse transcription polymerase chain reaction. The immortalized porcine fibroblast cell lines with diverse SLA haplotypes that were developed in this study have potential to be applied in studies regarding the molecular characteristics and genetic structure of SLA genes and epitope–major histocompatibility complex interactions in pigs.

Genome Canada precision medicine strategy for structured national implementation of epitope matching in renal transplantation

Advances in immunology support the understanding that precise structural epitopes on the antibody-accessible region of the HLA molecule determine antigenicity and challenge the need for identity across the full HLA molecule to minimize graft immunogenicity. Retrospective studies confirm that quantitative measurement of epitope-level mismatching between donor and recipient is an informative marker of graft rejection and survival and suggest that prospective allocation of donor organs based on this principle may improve graft survival. Here we describe the process for rigorous prospective evaluation of this hypothesis in a formal national proof-of-concept program for epitope-based matching. This encompasses broad societal consultation to engage the public, patients and providers; the development of clear allocation policies with strategies to support candidates who may be difficult to match; molecular and sequencing methods and web-based calculators enabling rapid epitope typing and recipient selection; precise immunological monitoring of the graft response; information systems permitting real-time monitoring of clinical outcomes; and assessment of health benefit and economic cost. The results of this objective evaluation can then be provided to payers and policy-makers for review, and adoption if of proven benefit.

Smaller Sized Hepatitis E Virus ORF2 Protein-Chitosan Nanoemulsion Conjugate Elicits Improved Immune Response

Comparative evaluation of immunomodulating efficiency of chitosan nanoemulsion and gold nanoparticles as an adjuvant for truncated HEV ORF2 structural proteins (26 kDa or 54 kDa) were analyzed. Hepatitis E virus (HEV) ORF2 structural protein has been reported to be immunogenic in nature. In the present study, we have used truncated forms of structural protein-containing C-terminus neutralization epitope as a vaccine candidate. A high-energy emulsification process synthesized chitosan nanoemulsions. HEV antigens were encapsulated in chitosan nanoemulsion (CNE26, CNE54). Swiss albino mice were immunized with these nanoconjugates, and their potency to stimulate humoral immune response as well as cell-mediated immune response was evaluated. The humoral response in mice immunized with CNE26 nanoconjugates was found to be 2.7 times that of IFA26 immunized group, while larger protein nanoconjugate (CNE54) and gold nanoconjugates displayed immune response comparable to that of IFA26 and IFA54. Furthermore, a slower rate of antibody formation was observed in the case of GNP26 immunized mice as compared to GNP54 ones, emphasizing the effect of the size of nanomaterials/nanoconjugates on the immune response. At the same time, the effect of surface functionalization and inherent immune modulation property of chitosan nanoemulsion was observed for CNE nanoconjugates. Both gold and chitosan nanoconjugates elicited cell-mediated immune response as well in mice. This study suggests that smaller-sized protein antigen-chitosan nanoemulsion conjugate has higher immunological potential and can be a better future vaccine candidate than gold nanoparticles.

Immunogenic and Protective Features of the Recombinant Vaccinia Virus Strain Expressing Cassette of Genes of Marburg Virus Structural Proteins

The aim of the study was to create a highly immunogenic vaccine construct based on a recombinant variant of a replication-defective MVA strain of vaccinia virus, expressing virus-like particles that mimic natural infection with Marburg virus. Materials and methods. The recombinant virus was obtained through recombination between homologous viral DNA sequences and the insertion plasmid pDel2-GP-VP-Pat which carries transgenes of the structural proteins GP and VP40 of Marburg virus, flanked by fragments of MVA strain genome. Structure of the recombinant virus was confirmed in PCR and using sequencing, transgenes expression was analyzed by Western blotting, viruslike particles formation was recorded using electron microscopy. Evaluation of immunogenicity and protectivity was carried out using a guinea pig model. The antibody titer was determined in enzyme-linked immunosorbent assay. To assess T-cell response, the intracellular staining of cytokines was used, followed by analysis of samples on a flow cytometer. Results and discussion. On the basis of highly attenuated MVA strain of vaccinia virus a recombinant variant MVA-GP-VP40-MARV has been constructed, carrying a cassette of transgenes, GP and VP40, of Marburg virus in the region of deletion II of the genome. The expression of transgenes in MVA-permissive CER cells infected with recombinant MVA-GP-VP40-MARV strain and secretion of GP and VP40 proteins into culture medium have been demonstrated. Electron microscopy analysis has revealed the presence of Marburg virus-like particles in the culture medium of cells 12 hours after infection. Double vaccination of guinea pigs with MVA-GP-VP40-MARV strain at a dose of 108 PFU/animal induced the formation of antibodies to Marburg and vaccinia viruses, as well as 100 % protection against lethal Marburg virus infection (50 LD50). Using original TEpredict software, the structure of T-helper epitopes of GP protein has been predicted. Using the ICS method, the biological activity of these epitopes has been experimentally confirmed and it was shown that they provide the induction of a T-cell immune response as part of the MVA-GP-VP40-MARV vaccine construct.

Prokaryotic expression of the GapC protein of Streptococcus uberis and prediction, identification of its B-cell epitopes

The GapC protein of Streptococcus uberis located on the surface of bacteria is a protein with glyceraldehyde-3-phosphate dehydrogenase activity. It participates in cellular processes and exhibits a variety of biological activities. In addition, it has good antigenicity. The aim of this study was to predict the possible B-cell epitopes of the GapC protein and verify the immunogenicity of candidate epitope peptides. The gapC gene of S. uberis isolate RF5-1 was cloned into a recombinant expression plasmid pET-28a-GapC and inducibly expressed. The purified protein was used to immunize experimental rabbits to produce anti-GapC polyclonal antibodies. The three-dimensional structure and three-dimensional location of the GapC B-cell epitopes and the homology comparison of the GapC protein and its B-cell epitopes were carried out using bioinformatics softwares. The results showed that the 44-kDa GapC protein had a good immunological reactivity. Six linear and 3 conformational dominant B-cell epitopes against the GapC protein were selected and synthesized. Three dimensional analysis indicated that the selected peptides have better antigen epitope formation potential. Rabbit anti-GapC polyclonal antibodies were generated after immunized with the purified GapC protein, and the polyclonal antibodies were used to identify the epitope peptide by an indirect ELISA. The ELISA results showed that all of the 9 epitope peptides could react with anti-GapC polyclonal antibodies with varying titers. Among them, the epitope polypeptide 266AANDSYGYTEDPIVSSD282 reacted with the polyclonal antibodies significantly stronger than with other epitope peptides. This study laid an experimental foundation for in-depth understanding of the immunological properties and utilizing effective epitopes of the GapC protein of S. uberis.

Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM.

Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system. B lymphocytes in the cerebrospinal fluid (CSF) of patients with MS contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been epidemiologically linked to MS, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBV nuclear antigen 1 (EBNA1) and the central nervous system protein glial cell adhesion molecule (GlialCAM) and provide structural and in vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain Bcell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment enabled tracking of the development of the naive EBNA1-restricted antibody to a mature EBNA1-GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates disease in a mouse model of MS, and anti-EBNA1 and anti-GlialCAM antibodies are prevalent in patients with MS. Our results provide a mechanistic link for the association between MS and EBV and could guide the development of new MS therapies.

The polymorphism analysis and epitope predicted of Alphapapillomavirus 9 E6 in Sichuan, China

BackgroundThe Alphapapillomavirus 9 (α-9 HPV) is a member of the Alphapapillomavirus genus and Papillomaviridae family. These viruses are almost all carcinogenic HPV, which is closely related to 75% of invasive cervical cancer worldwide, and has a high prevalence in Sichuan. The carcinogenic function is mainly realized by its E6 oncoprotein.MethodsCell samples were collected by cervical scraped for HPV detecting and typing. HPV-16, HPV-31, HPV-33, HPV-52, HPV-58 5 α-9 genus HPV subtype positive samples were selected, their E6 gene was sequenced and analyzed. The positive selection sites of HPV E6 genes were estimated by PAML 4.8 server. The secondary and tertiary structure of E6 protein were predicted by PSIPred and Swiss-model. The T-cell antigen epitopes of E6 protein were predicted by IEDB.Resultsα-9 HPV has a high prevalence in Sichuan, China. From 2012 to 2017, 18,067 cell cervical samples were collected, and 3135 were detected with α-9 HPV infection. Among which, 250 cases HPV-16 E6, 96 cases HPV-31 E6, 216 cases HPV-33 E6, 288 cases HPV-52 E6 and 405 cases HPV-58 E6 were successfully amplified, 17, 6, 6, 13, and 4 non-synonymous nucleotide mutations were respectively detected in HPV-16, 31, 33, 52, and 58 E6, 7 positive selection sites of α-9 HPV E6 were selected out (D32E of HPV-16 E6, K35N, K93N and R145I of HPV-33 E6, K93R of HPV-52 E6, K93N and R145K of HPV-58 E6). The structure and antigen epitopes of E6 protein with amino acid substitution differ from those of wild-type E6 protein, especially for the mutation located in the E6 positive selection site.ConclusionsHPV E6 nucleotide non-synonymous mutation in the positive selection site influence the protein structure and decrease the antigen epitopes affinity of the E6 protein overall, making it more difficult for the HPV-infected cells to be detected by the immune system, and enhancing the HPV adaptability to the environment. Mutations influence the validity of HPV clinical diagnostic probes, the polymorphism analysis of α-9 HPV E6 enrich the data of HR-risk HPV in Sichuan China, and the detection probes designed with the polymorphism data in mind can improve the efficiency of clinical detection; Mutations influence epitopes affinity, the association of E6 polymorphism and epitope affinity can improve the design of therapeutic vaccine with good immunity and high generality antigen epitope; The above study all provide a good theoretical basis for the prevention and treatment of HPV-related diseases.

Digital gene expression analysis of NSCLC-patients reveals strong immune pressure, resulting in an immune escape under immunotherapy

BackgroundImmune checkpoint inhibitors (ICIs) are currently one of the most promising therapy options in the field of oncology. Although the first pivotal ICI trial results were published in 2011, few biomarkers exist to predict their therapy outcome. PD-L1 expression and tumor mutational burden (TMB) were proven to be sometimes-unreliable biomarkers. We have previously suggested the analysis of processing escapes, a qualitative measurement of epitope structure alterations under immune system pressure, to provide predictive information on ICI response. Here, we sought to further validate this approach and characterize interactions with different forms of immune pressure.MethodsWe identified a cohort consisting of 48 patients with advanced non-small cell lung cancer (NSCLC) treated with nivolumab as ICI monotherapy. Tumor samples were subjected to targeted amplicon-based sequencing using a panel of 22 cancer-associated genes covering 98 mutational hotspots. Altered antigen processing was predicted by NetChop, and MHC binding verified by NetMHC. The NanoString nCounter® platform was utilized to provide gene expression data of 770 immune-related genes. Patient data from 408 patients with NSCLC were retrieved from The Cancer Genome Atlas (TCGA) as a validation cohort.ResultsThe two immune escape mechanisms of PD-L1 expression (TPS score) (n = 18) and presence of altered antigen processing (n = 10) are mutually non-exclusive and can occur in the same patient (n = 6). Both mechanisms have exclusive influence on different genes and pathways, according to differential gene expression analysis and gene set enrichment analysis, respectively. Interestingly, gene expression patterns associated with altered processing were enriched in T cell and NK cell immune activity. Though both mechanisms influence different genes, they are similarly linked to increased immune activity.ConclusionPressure from the immune system will lay the foundations for escape mechanisms, leading to acquisition of resistance under therapy. Both PD-L1 expression and altered antigen processing are induced similarly by pronounced immunoactivity but in different context. The present data help to deepen our understanding of the underlying mechanisms behind those immune escapes.

Prediction of Epitope Peptides for PTK Gene of Acinetobacter baumannii

Background: Acinetobacter baumannii is a gramnegative bacilli acquiring both intrinsic and adaptive patterns of multi drug resistance and virulence. Immune-informatics approach holds promise to detect putative epitope peptides from vital virulence factors which can be further synthesized and evaluated for their immunological response.
 Aim: The aim of the study was to predict the immuno-dominant peptides from the ptk gene of A. baumannii. 
 Materials and Methods: Protein retrieval of the Ptk gene using CELLO V.2.5 was done with the evaluation of antigenicity and allergenicity of the predicted epitopes, using Vaxijen V2.0 server and AlgPred servers. Epitope structure prediction and validation by using RAMPAGE revealed the homology peptides. Molecular Docking of epitopes with HLA-alleles using ClusterPro server, and further identification of B cell epitope was performed by using Kolaskar and Tonganokar antigenicity method.
 Results: A total of 20 epitopes were predicted and 18 peptides were chosen based on antigenicity and stability analysis prediction. The structure predictions were carried out using pepfold server and based on Ramachandran plot analysis 10 epitopes were taken for further analysis.
 Conclusion: The present finding has detected and evaluated the desirable epitope as LFFSLIAQW using an immune-informatic approach. However, it needs further experimental validation for its immunological response using standard in-vitro studies.

In-silico design of a multi-epitope for developing sero-diagnosis detection of SARS-CoV-2 using spike glycoprotein and nucleocapsid antigens.

Graphical abstractCOVID-19 is a pandemic disease caused by novel corona virus, SARS-CoV-2, initially originated from China. In response to this serious life-threatening disease, designing and developing more accurate and sensitive tests are crucial. The aim of this study is designing a multi-epitope of spike and nucleocapsid antigens of COVID-19 virus by bioinformatics methods. The sequences of nucleotides obtained from the NCBI Nucleotide Database. Transmembrane structures of proteins were predicted by TMHMM Server and the prediction of signal peptide of proteins was performed by Signal P Server. B-cell epitopes’ prediction was performed by the online prediction server of IEDB server. Beta turn structure of linear epitopes was also performed using the IEDB server. Conformational epitope prediction was performed using the CBTOPE and eventually, eight antigenic epitopes with high physicochemical properties were selected, and then, all eight epitopes were blasted using the NCBI website. The analyses revealed that α-helices, extended strands, β-turns, and random coils were 28.59%, 23.25%, 3.38%, and 44.78% for S protein, 21.24%, 16.71%, 6.92%, and 55.13% for N Protein, respectively. The S and N protein three-dimensional structure was predicted using the prediction I-TASSER server. In the current study, bioinformatics tools were used to design a multi-epitope peptide based on the type of antigen and its physiochemical properties and SVM method (Machine Learning) to design multi-epitopes that have a high avidity against SARS-CoV-2 antibodies to detect infections by COVID-19.

Assembly of Glycochips with Mammalian GSLs Mimetics toward the On-site Detection of Biological Toxins.

According to our previously proposed scheme, each of three kinds of glycosphingolipid (GSL) derivatives, that is, lactosyl ceramide [Lac–Cer (1)] and gangliosides [GM1-Cer (2) and GT1b-Cer (3)], was installed onto the glass surface modified with Au nanoparticles. In the present study, we tried to apply microwave irradiation to promote their installing reactions. Otherwise, this procedure takes a lot of time as long as a conventional self-assembled monolayer (SAM) technique is applied. Using an advanced microwave reactor capable of adjusting ambient temperatures within a desired range, various GSL glycochips were prepared from the derivatives (1)–(3) under different microwave irradiation conditions. The overall assembling process was programed with an IC controller to finish in 1 h, and the derived GSL glycochips were evaluated in the analysis of three kinds of biological toxins [a Ricinus agglutinin (RCA120), botulinum toxin (BTX), and cholera toxin (CTX)] using a localized surface plasmon resonance (LSPR) biosensor. In the LSPR analysis, most of the irradiated GSL chips showed an enhanced response to the targeting toxin when they were irradiated under optimal temperature conditions. Lac–Cer chips showed the highest response to RCA120 (an agglutinin with β-D-Gal specificity) when the microwave irradiation was conducted at 30–35 °C. Compared to our former Lac–Cer glycochips with the conventional SAM condition, their response was enhanced by 3.6 times. Analogously, GT1b chips gained an approximately 4.1 times enhancement in their response to botulinum type C toxin (BTX/C) when the irradiation was conducted around at 45–60 °C. In the LSPR evaluation of the GM1-Cer glycochips using CTX, an optimal condition also appeared at around 30–35 °C. On the other hand, the microwave irradiation did not lead to a notable increase compared to the former GM1-Cer chips derived with the SAM technique. Judging from these experimental results, the microwave irradiation effectively promotes the installing process for all the three kinds of the GSL derivatives, while the optimal thermal condition becomes different from each other. Many bacterial and botanic proteinous toxins are composed of such carbohydrate binding domains or subunits that can discriminate both the key epitope structure and the dimension of glycoconjugates on the host cell surface. It is assumed that the optimal irradiation and thermal conditions are required to array these semi-synthetic GSL derivatives on the Au nanoparticles in a proper density and geometry for tight adhesion with each of the biological toxins.

Annotation of Potential Vaccine Targets and Design of a Multi-Epitope Subunit Vaccine against Yersinia pestis through Reverse Vaccinology and Validation through an Agent-Based Modeling Approach.

Yersinia pestis is responsible for plague and major pandemics in Asia and Europe. This bacterium has shown resistance to an array of drugs commonly used for the treatment of plague. Therefore, effective therapeutics measurements, such as designing a vaccine that can effectively and safely prevent Y. pestis infection, are of high interest. To fast-track vaccine development against Yersinia pestis, herein, proteome-wide vaccine target annotation was performed, and structural vaccinology-assisted epitopes were predicted. Among the total 3909 proteins, only 5 (rstB, YPO2385, hmuR, flaA1a, and psaB) were shortlisted as essential vaccine targets. These targets were then subjected to multi-epitope vaccine design using different linkers. EAAK, AAY, and GPGPG as linkers were used to link CTL, HTL, and B-cell epitopes, and an adjuvant (beta defensin) was also added at the N-terminal of the MEVC. Physiochemical characterization, such as determination of the instability index, theoretical pI, half-life, aliphatic index, stability profiling, antigenicity, allergenicity, and hydropathy of the ensemble, showed that the vaccine is highly stable, antigenic, and non-allergenic and produces multiple interactions with immune receptors upon docking. In addition, molecular dynamics simulation confirmed the stable binding and good dynamic properties of the vaccine–TLR complex. Furthermore, in silico and immune simulation of the developed MEVC for Y. pestis showed that the vaccine triggered strong immune response after several doses at different intervals. Neutralization of the antigen was observed at the third day of injection. Conclusively, the vaccine designed here for Y. pestis produces an immune response; however, further immunological testing is needed to unveil its real efficacy.

Heparan Sulfate Biosynthesis and Sulfation Profiles as Modulators of Cancer Signalling and Progression.

Heparan Sulfate Proteoglycans (HSPGs) are important cell surface and Extracellular Matrix (ECM) maestros involved in the orchestration of multiple cellular events in physiology and pathology. These glycoconjugates bind to various bioactive proteins via their Heparan Sulfate (HS) chains, but also through the protein backbone, and function as scaffolds for protein-protein interactions, modulating extracellular ligand gradients, cell signalling networks and cell-cell/cell-ECM interactions. The structural features of HS chains, including length and sulfation patterns, are crucial for the biological roles displayed by HSPGs, as these features determine HS chains binding affinities and selectivity. The large HS structural diversity results from a tightly controlled biosynthetic pathway that is differently regulated in different organs, stages of development and pathologies, including cancer. This review addresses the regulatory mechanisms underlying HS biosynthesis, with a particular focus on the catalytic activity of the enzymes responsible for HS glycan sequences and sulfation motifs, namely D-Glucuronyl C5-Epimerase, N- and O-Sulfotransferases. Moreover, we provide insights on the impact of different HS structural epitopes over HSPG-protein interactions and cell signalling, as well as on the effects of deregulated expression of HS modifying enzymes in the development and progression of cancer. Finally, we discuss the clinical potential of HS biosynthetic enzymes as novel targets for therapy, and highlight the importance of developing new HS-based tools for better patients’ stratification and cancer treatment.

APC-Targeted DNA Vaccination Against Reticulocyte-Binding Protein Homolog 5 Induces Plasmodium falciparum-Specific Neutralizing Antibodies and T Cell Responses.

Targeted delivery of antigen to antigen presenting cells (APCs) is an efficient way to induce robust antigen-specific immune responses. Here, we present a novel DNA vaccine that targets the Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5), a leading blood-stage antigen of the human malaria pathogen, to APCs. The vaccine is designed as bivalent homodimers where each chain is composed of an amino-terminal single chain fragment variable (scFv) targeting unit specific for major histocompatibility complex class II (MHCII) expressed on APCs, and a carboxyl-terminal antigenic unit genetically linked by the dimerization unit. This vaccine format, named “Vaccibody”, has previously been successfully applied for antigens from other infectious diseases including influenza and HIV, as well as for tumor antigens. Recently, the crystal structure and key functional antibody epitopes for the truncated version of PfRH5 (PfRH5ΔNL) were characterized, suggesting PfRH5ΔNL to be a promising candidate for next-generation PfRH5 vaccine design. In this study, we explored the APC-targeting strategy for a PfRH5ΔNL-containing DNA vaccine. BALB/c mice immunized with the targeted vaccine induced higher PfRH5-specific IgG1 antibody responses than those vaccinated with a non-targeted vaccine or antigen alone. The APC-targeted vaccine also efficiently induced rapid IFN-γ and IL-4 T cell responses. Furthermore, the vaccine-induced PfRH5-specific IgG showed inhibition of growth of the P. falciparum 3D7 clone parasite in vitro. Finally, sera obtained after vaccination with this targeted vaccine competed for the same epitopes as PfRH5-specific mAbs from vaccinated humans. Robust humoral responses were also induced by a similar P. vivax Duffy-binding protein (PvDBP)-containing targeted DNA vaccine. Our data highlight a novel targeted vaccine platform for the development of vaccines against blood-stage malaria.

Elucidating the Structural and Minimal Protective Epitope of the Serogroup X Meningococcal Capsular Polysaccharide.

Despite the considerable progress toward the eradication of meningococcal disease with the introduction of glycoconjugate vaccines, previously unremarkable serogroup X has emerged in recent years, recording several outbreaks throughout the African continent. Different serogroup X polysaccharide-based vaccines have been tested in preclinical trials, establishing the principles for further improvement. To elucidate the antigenic determinants of the MenX capsular polysaccharide, we generated a monoclonal antibody, and its bactericidal nature was confirmed using the rabbit serum bactericidal assay. The antibody was tested by the inhibition enzyme-linked immunosorbent assay and surface plasmon resonance against a set of oligosaccharide fragments of different lengths. The epitope was shown to be contained within five to six α-(1–4) phosphodiester mannosamine repeating units. The molecular interactions between the protective monoclonal antibody and the MenX capsular polysaccharide fragment were further detailed at the atomic level by saturation transfer difference nuclear magnetic resonance (NMR) spectroscopy. The NMR results were used for validation of the in silico docking analysis between the X-ray crystal structure of the antibody (Fab fragment) and the modeled hexamer oligosaccharide. The antibody recognizes the MenX fragment by binding all six repeating units of the oligosaccharide via hydrogen bonding, salt bridges, and hydrophobic interactions. In vivo studies demonstrated that conjugates containing five to six repeating units can produce high functional antibody levels. These results provide an insight into the molecular basis of MenX vaccine-induced protection and highlight the requirements for the epitope-based vaccine design.

Physicochemical and structural characterization, epitope mapping and vaccine potential investigation of a new protein containing Tetratrico Peptide Repeats of Acinetobacter baumannii: An in-silico and in-vivo approach

Acinetobacter baumannii is an opportunistic multidrug-resistant pathogen that causes a significant mortality rate. The proteins containing Tetratrico Peptide Repeats (TPRs) are involved in the pathogenicity and virulence of bacteria and have different roles such as transfer of bacterial virulence factors to host cells, binding to the host cells and inhibition of phagolysosomal maturation. So, in this study, physicochemical properties of a new protein containing TPRs in A. baumannii which was named PcTPRs1 by this study were characterized and its 3D structure was predicted by in-silico tools. The protein B and T cell epitopes were mapped and its vaccine potential was in-silico and in-vivo investigated. Domain analysis indicated that the protein contains the Flp pilus assembly protein TadD domain which has three TPRs. The helix is dominant in the protein structure, and this protein is an outer membrane antigen which, is extremely conserved among A. baumannii strains; thus, has good properties to be applied as a recombinant vaccine. The best-predicted and refined model was applied in ligand-binding sites and conformational epitopes prediction. Based on epitope mapping results, several epitopes were characterized which could stimulate both immune systems. BLAST results showed the introduced epitopes are completely conserved among A. baumannii strains. The in-vivo analysis indicates that a 101 amino acid fragment of the protein which contains the best selected epitope, can produce a good protectivity against A. baumannii as well as the whole TPR protein and thus could be investigated as an effective subunit and potential vaccines.

Antigenic Structural Similarity as a Predictor for Antibody Cross-Reactivity

Antibodies are an essential component of the adaptive immune system that function to neutralize foreign invaders, such as bacterial and parasitic pathogens. However, B-cell epitopes remain difficult to predict due to their general indistinguishability from other protein regions. Epitope prediction tools in the past have largely relied on amino acid sequence similarity; however, implementing three-dimensional protein structure analyses into the epitope prediction algorithms has been shown to increase detection accuracy. Furthermore, structural comparisons between antigenic proteins for their potential to bind cross-reactive antibodies have not been explored extensively in the literature. Recent studies have pointed to the utility of looking at shared epitope structures in predicting antibody crossreactivity, which may shed light on cross-immunity between infectious pathogens and autoimmune diseases induced after infection. Thus, herein, the potential impact of including structural similarity comparisons in detecting shared epitopes is discussed. With the large amount of structural information being determined by three-dimensional computational protein modelling methods, the ability to perform these analyses is becoming more feasible.

In silico approach to design a multi-epitopic vaccine candidate targeting the non-mutational immunogenic regions in envelope protein and surface glycoprotein of SARS-CoV-2

The novel corona virus (COVID-19) is a causative agent for severe acute respiratory syndrome (SARS-CoV-2) and responsible for the current human pandemic situation which has caused global social and economic commotion. The currently available vaccines use whole viruses whereas there is scope for peptide based vaccines. Thus, the global raise in statistics of this infection at an alarming rate evoked us to determine a novel and effective vaccine candidate against SARS-CoV-2. To find the potential vaccine candidate targets, immunoinformatics approaches were used to analyze the mutations in the envelope protein and surface glycoprotein and determine the conserved region; further specific T-cell epitopes VSLVKPSFY, SLVKPSFYV, RVKNLNSSR, SEETGTLIV, LVKPSFYVY, LTDEMIAQY, YLQPRTFLL, RLFRKSNLK, SPRRARSVA, AEIRASANL, TLLALHRSY, YSRVKNLNS and FELLHAPAT and B-cells epitopes TLAILTALRLCAYCCN and AGTITSGWTFGAGAAL were identified. The 3 D structure of epitope was predicted, refined and validated. The molecular docking analysis of multi-epitope vaccine candidates with TLR receptors, predicted effective binding. Overall, using bioinformatics approach this multi-epitopic target facilitates the proof of concept for SARS-CoV-2 conserved epitopic vaccine design. Communicated by Ramaswamy H. Sarma

The citrullinated/native index of autoantibodies against hnRNP-DL predicts an individual \u201cwindow of treatment success\u201d in RA patients

BackgroundThere is a need for biomarker to identify patients “at risk” for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers.MethodsUsing protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry.ResultsHnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP-2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. The citrullinated/native index of autoantibodies against hnRNP-DL (CNDL-Index) was identified as a new value for an “individual window of treatment success” in early RA and for the detection of RF IgM/α-CCP-2 seronegative RA patients (24–46%). Negative CNDL-index was found in SLE patients, risk-RA and early RA cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CNDL-values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients.ConclusionCNDL-index defines people at risk to develop RA and the “window of treatment success” thereby closing the sensitivity gap in RA.

Structure prediction and biological activity analysis of dybowskin-1ST antimicrobial peptide in Rana dybowskii

The evolution, structure and antigenic epitopes prediction of Rana dybowskii antimicrobial peptide dybowskin-1ST were carried out using bioinformatics software available online. Its antibacterial mechanism and structural properties were analyzed, and its activity was verified by applying wound healing assay in mice and bacteriostatic assay in vitro. This provides the theoretical basis for the improvement of parental peptide and the development of novel derivative peptides. The software MEGA_X were used to conduct homology alignment and to construct a phylogenetic tree. The online software ProtParam, ProtScale, PeptideCutter, signal, TMHMM Server were respectively used to predict the physicochemical parameters, hydrophilia/hydrophobicity, shear sites, signal peptides, and transmembrane domains of dybowskin-1ST. The online software SOPMA, Jpred4, DNAstar Protean were used to predict the secondary structure of dybowskin-1ST, and SWISS-MODEL, I-TASSER were used to predict the tertiary structure. ABCpred and SYFPEITHI were respectively used to predict its B-and T-cell epitopes. The effect of dybowskin-1ST on the wound healing was observed on experimental mice. Kirby-Bauer method and dilution method were used to determine the bacteriostatic activity of dybowskin-1ST. The dybowskin-1ST consists of 59 amino acid residues, of which leucine accounts for 16.9%, with a molecular formula of C₃₁₈H₅₁₀N₈₀O₉₃S₂. Its theoretical isoelectric point is 5.10 and the charge is -2. The dybowskin-1ST and dybowskin-1CDYa are closely related phylogenetically. The secondary structure of dybowskin-1ST predicted by the three methods were similar, which consisted of α-helix (44.07%), extended strand (16.95%), β-turns (3.39%), and random coil (35.39%). The prediction of tertiary structure showed that dybowskin-1ST was mainly composed of α-helix, and it was regarded as a hydrophilic protein with signal peptide sequence. Subcellular localization analysis showed that the probability of secreting the mitochondrial targeted peptides was 0.944. Dybowskin-1ST is an extracellular protein with no transmembrane structure region, but contains seven phosphorylation sites, three T-cell epitopes and eight B-cell epitopes. The dybowskin-1ST promoted wound healing and effectively inhibited the growth of Escherichia coli and Staphylococcus aureus. However, it had limited antibacterial activity against fungi and drug-resistant bacteria. Although the structure of dybowskin-1ST is rich in α-helix, the verification experiments showed that its antibacterial ability needs to be enhanced. The reason may be that it is a negatively charged and hydrophilic protein, and amino acid modification with the aim of increasing the number of positive charges and changing the hydrophobicity may be used to obtain derived peptides with enhanced activity.