A Single Amino Acid at Position 158 in Hemagglutinin Affects the Antigenic Property of Eurasian Avian-like H1N1 Swine Influenza Viruses

Influenza viruses have been posing a great threat to public health and animal industry. The developed vaccines have been widely used to reduce the risk of potential pandemic; however, the ongoing antigenic drift makes influenza virus escape from host immune response and hampers vaccine efficacy. Until now, the genetic basis of antigenic variation remains largely unknown. In this study, we used A/swine/Guangxi/18/2011 (GX/18) and A/swine/Guangdong/104/2013 (GD/104) as models to explore the molecular determinant for antigenic variation of Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses (SIVs), and found that the GD/104 virus exhibited 32~64-fold lower antigenic cross-reactivity with antibodies against GX/18 virus. Therefore, we generated polyclonal antibodies against GX/18 or GD/104 virus and a monoclonal antibody (mAb), named mAb102-95, targeted to the hemagglutinin (HA) protein of GX/18 virus, and found that a single amino acid substitution at position 158 in HA protein substantially altered the antigenicity of virus. The reactivity of GX/18 virus containing G158E mutation with the mAb102-95 decreased 8-fold than that of the parental strain. Contrarily, the reactivity of GD/104 virus bearing E158G mutation with the mAb102-95 increased by 32 times as compared with that of the parental virus. Structural analysis showed that the amino acid mutation from G to E was accompanied with the R group changing from -H to -(CH ) -COOH. The induced steric effect and increased hydrophilicity of HA protein surface jointly contributed to the antigenic drift of EA H1N1 SIVs. Our study provides experimental evidence that G158E mutation in HA protein affects the antigenic property of EA H1N1 SIVs, and widens our horizon on the antigenic drift of influenza virus.

Skin prick test positivity to house dust mites (HDM) in patients with chronic urticaria

<p class="abstract"><strong>Background:</strong> The urticaria lasting for more than 6 weeks is termed chronic urticaria. The etiology of chronic urticaria and angioedema remains uncertain in most of the patients. Aeroallergens can induce or exacerbates chronic urticaria. The common aeroallergens are house dust mites (HDM), pollens, moulds, etc. House dust mites can trigger immunological process through ingestion, inhalation or inoculation. These mite allergens are resistant to high temperatures, and do not lose their antigenic property even on cooking. HDMs can also cause worsening of existing atopic dermatitis. Skin test with HDMs is well known to cause irritation due to their proteolytic enzymes.The study was undertaken with the objective to study the skin prick test positivity to house dust mites in patients with chronic urticaria<span lang="EN-IN">.</span></p><p class="abstract"><strong>Methods:</strong> The study was done on 56 consecutive patients of clinically diagnosed cases of chronic urticaria attending the OPD. The skin prick test was done according to the standard protocol.<strong></strong></p><p class="abstract"><strong>Results:</strong> We had a total of 56 patients with chronic urticaria, of which males were 30 and females were 26 with a male to female ratio of 1:0.8. Of the 56 patients with chronic urticaria skin prick test to HDM was seen in 8 (14.2%) patients. <em>D. pteronysinnus</em> 6 (10.7%) constituted the majority among the HDM positivity followed by <em>D. farinae</em> 2(3.5%). We also found skin prick test positivity to pollens, moulds and animal dander in 5, 4 and 2 patients respectively<span lang="EN-IN">. </span></p><p class="abstract"><strong>Conclusions:</strong> House dust mites can cause or trigger the urticarial symptoms and one should consider to do skin prick test to HDM in identifying the cause and thereby reliving the symptoms of urticaria on its avoidance<span lang="EN-IN">.</span></p>

SCREENING OF PUTATIVE THERAPEUTIC CANDIDATES IN SUPERBUG (STAPHYLOCOCCUS AUREUS): A SYSTEMATIC IN SILICO APPROACH

<p>ABSTRACT<br />Objective: Staphylococcus aureus, a superbug and antibiotic resistant pathogen, is one of the most infection causing organism, ranging from skin<br />allergies to severe lethal conditions. The prolonged use of different antibiotics and lack of optimal treatment over the antibiotic resistant species, led<br />to the identification of new, better and promising therapeutic candidates. <br />Methods: A systematic in silico filtration process was employed, which includes subtractive channels and reverse vaccinology techniques. <br />Results: Here, we report 12 possible drug targets and two vaccine candidates based on essentiality, non-human homolog, virulent and localization,<br />commonly in all the strains. Further characterization studies such as pathway analysis, chokepoint and structure prediction revealed, two proteins<br />as the best drug targets one being novel and the other druggable. Only one protein has shown the characteristic feature of vaccine candidate, having<br />antigenic property and an IgG binding domain. <br />Conclusion: Two best drug targets were commonly identified in all the strains of S. aureus namely UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--L-lysine<br />ligase (MurE) and cell division protein FtsA, whereas the best common vaccine candidate includes Peptidoglycan binding protein. The therapeutic candidates<br />reported in the present study might facilitate screening of new and better antimicrobial compounds, for an optimal treatment of S. aureus infections.<br />Keywords: Staphylococcus aureus, Drug target, Vaccine candidates, Subtractive proteomics, Reverse vaccinology.</p>

The kinase insert domain-containing receptor is an angiogenesis-associated antigen recognized by human cytotoxic T lymphocytes

Antigen-specific cancer immunotherapy directed toward tumor-nourishing angiogenic blood vessels holds the promise of high efficacy, low toxicity, and ease of application. To evaluate whether the human angiogenic kinase insert domain-containing receptor (KDR) can serve as a target for cellular immunotherapy, 19 peptide sequences with HLA-A*0201 motifs were selected by computer-based algorithms. Five peptides (KDR82-90, KDR288-297, KDR766-774, KDR1093-1101, KDR1035-1044) stimulated specific cytotoxic T lymphocytes (CTLs) from peripheral-blood mononuclear cells (PBMCs) of 3 HLA-A*0201 donors. The decapeptide KDR288-297 was efficient in sensitizing target cells for recognition by a CTL clone at a concentration of 10 nM. More important, KDR288-297 - specific CTLs lysed target cells transfected with HLA-A2/KDR cDNAs and a range of HLA-matched KDR+ angiogenic endothelial cells (aECs) and also recognized CD34+ endothelial progenitor cells. The specificity of CTLs was further confirmed by tetramer assay and cold-target inhibition assay. In addition, ex vivo exposure of aECs to the inflammatory cytokines enhanced CTL reactivity, which is in keeping with up-regulated KDR and HLA class 1 expression. In Matrigel assays, recognition of aECs by specific CTLs triggered an antivascular effect. These findings provide the first proof of the antigenic property of KDR protein and may be useful for devising new immunotherapeutic approaches to human cancers.

Biopolymer Polyelectrolyte Complex – Hydroxyapatite Composites for Bone Tissue Engineering

ABSTRACTThe excellent biocompatibility, biofunctionality, and non-antigenic property make chitosan an ideal material for tissue regeneration. In addition to that its hydrophilic surface promotes cell adhesion, proliferation, and differentiation, and evokes a minimal foreign body reaction on implantation. In spite of these favorable properties, the inadequate mechanical strength and loosening of structural integrity under wet conditions, limit its application for bone tissue engineering. To improve the suitability of chitosan for bone tissue engineering, we have biomimetically synthesized composites of chitosan, polygalacturonic acid and hydroxyapatite. Polygalacturonic acid (PgA) is biocompatible, biodegradable and electrostatically complementary to chitosan. The strong interactions between negatively charged carboxylate groups of PgA and positively charged amino groups of chitosan lead to complex formation. This biopolymer complex provides improved mechanical strength and better structural integrity under wet condition. In this study, we have investigated the applicability of chitosan-PgA-hydroxyapatite composites for bone tissue engineering.

Identification of Erythroid Cell Stimulating Factor as a Unique Protein, Quiescin Q6: Role in Erythropoiesis.

We demonstrated earlier the presence of an erythroid cell stimulating factor (ESF) in mouse serum that enhanced the erythropoietin-dependent erythroid cell proliferation in vitro. Employing a highly specific monoclonal antibody generated in our laboratory, we showed further that ESF had an essential role in normal erythropoiesis in vivo [Blood98, 296a, 2001, Exp Hematol.31, Supp. 1, 165, 2003]. In order to clone and express this protein, ESF was purified from mouse serum and on Western blotting using the monoclonal anti-ESF antibody the purified protein was stained as bands of ~62/64 kDa. These bands were analyzed by Mass Spectrometry. The data were processed using Protein Lynx 2.1 (Waters) and the resulting peak list was submitted for database searching on Mascot. One of the peptide sequences had a highest similarity with Quiescin Q6 (Q6). This protein was then BLAST searched for mouse DNA sequences in the NCBI database for nucleotides corresponding to the mouse Q6. We designed the primers to reclone Q6 ORF (Open reading frame) having restriction sites NCO1 and ECOR1. Amplified product was sequenced to confirm DNA fragment. Fragment was cloned in pGEMT vector and subsequently re-cloned in expression vector pET30a(+). After each cloning step, the orientation of the insert was analyzed using restriction enzyme digestion. His-tagged Q6 protein was over-expressed in E.coli BL21 Codon Plus (DE3)-RP cells. Western blot analysis using anti-ESF antibody identified 62/64 kDa-bands of Q6 in the soluble fraction of the bacterial extract. Further characteristics of this recombinant protein and its CFU-E enhancing properties are being investigated and will be reported. Thus we have identified ESF to be a unique protein, quiescin Q6 and successfully cloned and expressed it. Expressed protein is similar to ESF as regards its antigenic property.