Inhibition of avian influenza virus H9N2 infection by antiviral hexapeptides that target viral attachment to epithelial cells.

The H9N2 subtype of influenza A virus circulates frequently among poultry in Asian and North African countries causing economic loss in the poultry sector. The antigenic variations of the H9N2 virus were at the origin of its genetic evolution through the emergence of viral strains transmissible to humans and resistant to chemical antivirals, which require a strengthening of the fight means against this virus. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select new antiviral peptides that inhibit the infectivity of H9N2 virus. After three rounds of stringent selection and amplification, polyclonal phage-peptides directed against H9N2 virus were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to H9N2 virus by monoclonal phage ELISA. The DNA of 27 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences were deduced. Sixteen different phage-peptides were able to bind specifically the H9N2 virus, among them, 13 phage-peptides interacted with the hemagglutinin H9. Two selected peptides, P1 (LSRMPK) and P2 (FAPRWR) have shown antiviral activity in ovo and P1 was more protective in vivo then P2 when co-administered with the H9N2 virus. Mechanistically, these peptides prevent infection by inhibiting the attachment of the H9N2 virus to the cellular receptor. Molecular docking revealed that the peptides LSRMPK and FAPRWR bind to hemagglutinin protein H9, but interact differently with the receptor binding site (RBS). The present study demonstrated that the peptide P1 (LSRMPK) could be used as a new inhibitory molecule directed against the H9N2 virus.

Historical origins and zoonotic potential of avian influenza virus H9N2 in Tunisia revealed by Bayesian analysis and molecular characterization.

During 2009-2012, several outbreaks of avian influenza virus H9N2 were reported in Tunisian poultry. The circulating strains carried in their hemagglutinins the human-like marker 226L, which is known to be important for avian-to-human viral transmission. To investigate the origins and zoonotic potential of the Tunisian H9N2 viruses, five new isolates were identified during 2012-2016 and their whole genomes were sequenced. Bayesian-based phylogeny showed that the HA, NA, M and NP segments belong to the G1-like lineage. The PB1, PB2, PA and NS segments appeared to have undergone multiple intersubtype reassortments and to be only distantly related to all of the Eurasian lineages (G1-like, Y280-like and Korean-like). The spatiotemporal dynamic of virus spread revealed that the H9N2 virus was transferred to Tunisia from the UAE through Asian and European pathways. As indicated by Bayesian analysis of host traits, ducks and terrestrial birds played an important role in virus transmission to Tunisia. The subtype phylodynamics showed that the history of the PB1 and PB2 segments was marked by intersubtype reassortments with H4N6, H10N4 and H2N2 subtypes. Most of these transitions between locations, hosts and subtypes were statistically supported (BF > 3) and not influenced by sampling bias. Evidence of genetic evolution was observed in the predicted amino acid sequences of the viral proteins of recent Tunisian H9N2 viruses, which were characterized by the acquisition of new mutations involved in virus adaptation to avian and mammalian hosts and amantadine resistance. This study is the first comprehensive analysis of the evolutionary history of Tunisian H9N2 viruses and highlights the zoonotic risk associated with their circulation in poultry, indicating the need for continuous surveillance of their molecular evolution.

Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library.

Cutaneous leishmaniasis (CL) is a global problem caused by intracellular protozoan pathogens of the genus Leishmania for which there are no suitable vaccine or chemotherapy options. Thus, de novo identification of small molecules binding to the Leishmania parasites by direct screening is a promising and appropriate alternative strategy for the development of new drugs. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select binding peptides to metacyclic promastigotes from a highly virulent strain of Leishmania major (Zymodeme MON-25; MHOM/TN/94/GLC94). After four rounds of stringent selection and amplification, polyclonal and monoclonal phage-peptides directed against L. major metacyclic promastigotes were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to L. major by monoclonal phage ELISA. The DNA of 42 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences deduced. Six different peptide sequences were obtained with frequencies of occurrence ranging from 2.3% to 85.7%. The biological effect of the peptides was assessed in vitro on human monocytes infected with L. major metacyclic promastigotes, and in vivo on susceptible parasite-infected BALB/c mice. The development of cutaneous lesions in the right hind footpads of infected mice after 13 weeks post-infection showed a protection rate of 81.94% with the injected peptide P2. Moreover, Western blots revealed that the P2 peptide interacted with the major surface protease gp63, a protein of 63kDa molecular weight. Moreover, bioinformatics were used to predict the interaction between peptides and the major surface molecule of the L. major. The molecular docking showed that the P2 peptide has the minimum interaction energy and maximum shape complimentarity with the L. major gp63 active site. Our study demonstrated that the P2 peptide occurs at high frequency during the screening procedure, best inhibits L. major growth kinetics in vitro, and reduces cutaneous lesions in BALB/c mice, thus showing great promise in the development of new therapeutic molecules.

The analysis of VH and VL genes repertoires of Fab library built from peripheral B cells of human rabies virus vaccinated donors.

A human combinatorial Fab antibody library was generated from immune repertoire based on peripheral B cells of ten rabies virus vaccinated donors. The analysis of random Fab fragments from the unselected library presented some bias of V gene usage towards IGHV-genes and IGLV-gen families. The screening of the Fab library on rabies virus allowed specific human Fab antibody fragments characterized for their gene encoding sequences, binding and specificities to RV. Genetic analysis of selected Fabs indicated that the IGHV and IGLV differ from the germ-line sequence. At the level of nucleotide sequences, the IGHV and IGLV domains were found to share 74-92% and 90-96% homology with sequences encoded by the corresponding human germ-line genes respectively. IGHV domains are characterized most frequently by IGHV3 genes, and large proportions of the anti-RV heavy chain IGHV domains are obtained following a VDJ recombination process that uses IGHD3, IGHD2, IGHD1 and IGHD6 genes. IGHJ3 and IGHJ4 genes are predominantly used in RV-Fab. The IGLV domains are dominated by IGKV1, IGLV1 and IGLV3 genes. Numerous somatic hypermutations in the RV-specific IGHV are detected, but only limited amino acid replacement in most of the RV-specific IGLV particularly in those encoded by J proximal IGLV or IGKV genes are found. Furthermore, IGHV3-IGKV1, IGHV3-IGVL1, and IGHV3-IGLV3 germ-line family pairings are preferentially enriched after the screening on rabies virus.

Chemokine CCR3 ligands-binding peptides derived from a random phage-epitope library.

Eosinophils are major effectors cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The human chemokine receptor C-C receptor 3 (hCCR3) provides a mechanism for the recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop peptides antagonists of hCCR3-hCCL11 (human eotaxin) interactions, a random bacteriophage hexapeptide library was used to map structural features of hCCR3 by determining the epitopes of neutralizing anti-hCCR3 mAb 7B11. This mAb t is selective for hCCR3 and exhibit potent antagonist activity in receptor binding and functional assays. After three rounds of biopanning, four mAb7B11-binding peptides were identified from a 6-mer linear peptide library. The phage bearing the peptides showed specific binding to immobilized mAb 7B11 with over 94% of phages bound being competitively inhibited by free synthetic peptides. In FACScan analysis all selected phage peptides were able to strongly inhibit the binding of mAb 7B11 to hCCR3-transfected preB-300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody-hCCR3 interactions and to inhibit the binding of hCCL11 to hCCR3 transfectants. Chemically synthesized peptides CKGERF, FERKGK, SSMKVK and RHVSSQ, effectively competed for (125)I-hCCL11 binding to hCCR3 with IC(50) ranging from 3.5 to 9.7µM. Calcium release and chemotaxis of hCCR3 transfectants or human eosinophils were inhibited by all peptides in a dose-dependent manner. Furthermore, they showed inhibitory effects on chemotaxis of human eosinophils induced by hCCL11, hCCL5, hCCL7, hCCL8, and hCCL24. Specificities of all selected peptides were assessed with hCXCR1, hCXCR2, hCXCR3, and hCCR5 receptors. Peptides CKGERF and FERKGK showed inhibitory effects on eosinophil chemotaxis in a murine model of mCCL11-induced peritoneal eosinophilia. The development of peptides inhibiting the interactions between hCCR3 and its chemokine ligands will facilitate the development of small peptides antagonists with the hope of ameliorating chronic inflammatory diseases in humans.

Random phage-epitope library based identification of a peptide antagonist of Mac-1 ß2 integrin ligand binding.

The leukocyte ß2 integrin Mac-1 (CD11b/CD18) plays a pivotal role in inflammation and host defense. To develop peptide antagonists selectively inhibiting the function of Mac-1, we used a random constrained 6-mer (cys-6aa-cys) peptide library to map the structural features of CD11b, by determining the epitope of neutralizing monoclonal antibody mAb 44a (anti-CD11b). We have used a stringent phage display strategy, which resulted in the identification of one disulfide C-RLKEKH-C constrained peptide by direct biopanning of library on decreasing amounts of purified mAb 44a. The selected peptide mimics a discontinuous epitope, a peculiar shape on the CD11b-I-domain surface. Competitive ELISA experiments with different Mac-1 ligands showed that C-RLKEKH-C is able to bind to fibrinogen, iC3b, and C1q. Furthermore, the monomeric circular peptide C-RLKEKH-C, was effective in blocking the interaction between (125)I-fibrinogen and Mac-1 (IC(50)=3.35±0.1×10(-6)M), and inhibited the adhesion of human neutrophils to fibrinogen and iC3b. These data provide information about the relative location of amino acids on the I-domain surface using mAb 44a imprint of the CD11b protein. The derived mimotope may help in the design of future anti-inflammatory therapeutic agents that can act as specific therapeutic agents targeting PMNs mediated inflammation.

hCXCR1 and hCXCR2 antagonists derived from combinatorial peptide libraries.

The human CXCL8 plays important roles in inflammation by activation of neutrophils through the hCXCR1 and hCXCR2 receptors. The role of hCXCR1 and hCXCR2 in the pathogenesis of inflammatory responses has encouraged the development of antagonists of these receptors. In this study, we used phage display peptide libraries to identify peptides antagonists that block the interactions between hCXCL8 and hCXCR1/2. Two linear hexapeptides (MSRAKE and CAKELR) and two disulfide-constrained hexapeptides (CLRSGRFC and CLPWKENC) were recovered by panning phage libraries on hCXCR1- and hCXCR2-transfected murine pre-B cells after specific elution with hCXCL8. Sequence analysis revealed homology between the linear hexapeptides and the N-terminal domain (1-SAKELR-6), whereas the constrained peptides are composed of non-contiguous amino acids mimicking spatial structure on the surface of folded C-terminal portion of hCXCL8 (50-CLDPKENWVQRVVEKFLKRAENS-72). The synthetic linear and structurally constrained peptides competed for (125)I-hCXCL8 binding to hCXCR1 and hCXCR2 (IC(50) comprised between 10 and 100µM). Furthermore, they inhibited the intracellular calcium flux and the migration of hCXCR1/hCXCR2 transfectants; and desensitized hCXCR1 and hCXCR2 receptors on neutrophils, reducing their chemotactic responses induced by ELR-CXC chemokines (hCXCL8, hCXCL1, hCXCL2, hCXCL3, and hCXCL5). To better characterize the residues required for hCXCL8 binding, we identified three linear peptides MLRQTR, HASILP and KKEPWI specific to hCXCL8. These peptides similarly displaced the binding of (125)I-hCXCL8 to hCXCR1 (IC(50) ranging from 8.5 to 10µM) in a dose-dependent manner, inhibited hCXCL8 induced increases in the intracellular calcium, and migration of hCXCR1- and hCXCR2-transfected cells. The identified peptides could be used as antagonists of hCXCL8-induced activities related to its interaction with hCXCR1 and hCXCR2 receptors and may help in the design of new anti-inflammatory therapeutic molecules.

Peptide mimotopes of rabies virus glycoprotein with immunogenic activity.

A random constrained hexapeptide phage display library (Cys-6aa-Cys) was screened with purified neutralizing human anti-rabies virus IgG antibodies (hRABVIgG) to identify peptides that correspond to or mimic natural epitopes on rabies virus glycoprotein (RABVG) and to investigate their immunogenicities in vivo. After four rounds of biopanning, 20 phage clones randomly selected for their specificity to hRABVIgG, effectively blocked the binding of the inactive rabies virus (RABV) to hRABVIgG. The phage clones were sequenced and the deduced amino acid sequences were derived (C-KRDSTW-C; C-KYLWSK-C; C-KYWLSR-C; C-KYWWSK-C; C-KYAWSR-C; C-KYSMSK-C). Alignments to the amino acid sequence of RABVG showed good match with the antigenic site III (at 330-338 aa), indicating that the hRABVIgG antibodies most likely recognize preferentially this antigenic site. The selected mimotopes were able to inhibit the interactions of the hRABVIgG antibodies with RABV in a dose-dependent manner. Subcutaneous administration of phageKRDSTW expressing the RABVG site III mimotope induced an RABVG-specific IgG response in BALB/c mice. The results indicated that peptide mimotopes when displayed on phages, are accessible to the mice immune system to trigger a humoral response and to induce IgG production. The RABVG site III mimotope (C-KRDSTW-C) would provide a new and promising concept for the development of rabies vaccine.

Isolation and characterization of human neutralizing antibodies to rabies virus derived from a recombinant immune antibody library.

A human immune Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from rabies virus hyperimmune volunteers on phagemid vector. The size of the constructed Fab library was 2 x 10(7) Escherichia coli transformants. After four rounds of panning on whole inactivated rabies virus (PV-11), phage clones displaying rabies virus-specific human Fab were selected. The specificity of soluble Fab antibody fragments, derived from positive phage clones was verified by ELISA. Among 20 specific Fab clones, the genetic sequence of 6 of them (FabRV01, FabRV02, FabRV03, FabRV04, FabRV05, and FabRV06) was analyzed. The variable heavy (VH) and variable light (VL) domains were found to share 90% and 93% homology with sequences encoded by the corresponding human germline genes, respectively. The soluble Fab fragments, expressed in Escherichia coli were purified by a single step Nickel-NTA affinity chromatography via a hexa-histidine tag and their binding specificities to rabies virus were confirmed. Three of the Fab antibodies, FabRV01, FabRV02 and FabRV03, showed binding characteristics to rabies virus glycoprotein antigenic site III with affinities in the K(D) range 7 x 10(-9) to 5 x 10(-8)M. The Fab fragments showed dose-dependent neutralization properties for the challenge virus standard (CVS-11).

Identification of biologically active peptides that inhibit binding of human CXCL8 to its receptors from a random phage-epitope library.

A random bacteriophage peptide library was used to map structural features of human (h)CXCR1 and hCXCR2 by determining the epitopes of neutralizing mAb 5A12 anti-hCXCR1 and mAb 6C6 anti-hCXCR2. After three rounds of biopanning, five mAb5 A12- and four mAb 6C6-binding peptides were identified from a 6-mer peptide library. Consensus sequences (S/T)(1)(F/A/N/D)(2)(I/M)(3)W(4)D(5)F(6) and F/L/M)(1)W(2)(D/N/L)(3)D(4)F(5)W(6) were deduced from sequences of these peptides. They correspond to a highly conserved N-domain sequence (9)MWDF(12) of hCXCR1 and (13)DFW(15) of hCXCR2. The phage bearing the peptides showed specific binding to immobilized mAb 5A12 or mAb 6CC, and over 86% of phages bound were competitively inhibited by free synthetic peptides. In FACScan analysis, all selected phage peptides were able to strongly inhibit the binding of mAb 5A12 and mAb 6C6 to hCXCR1- and hCXCR2-transfected preB 300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody-CXCR1/2 interactions and to inhibit the binding of hCXCL8 to hCXCR1 and hCXCR2 transfectants. Peptides 5A12/2 (SAMWDF) and 6C6/1 (FWDDFW) competed effectively for (125)I-hCXCL8 binding to hCXCR1 and hCXCR2 with IC(50), respectively, equal to 10 muM and 5.4 muM. Calcium release and chemotaxis of hCXCR1/2 transfectants or human neutrophils were inhibited by all peptides in a dose-dependent manner. Furthermore, the peptide 6C6/1 FWDDFW showed inhibitory effects on chemotaxis of human netrophils induced by hCXCR2 chemokines such as hCXCL1-3 and hCXCL5. Specificities of peptides 5A12/2 and 6C6/1 were assessed with hCXCR3, hCXCR4, hCXCR5, hCCR3, and hCCR5 receptors. In vivo, peptides 5A12/2 and p6C6/1 blockade hCXCL8-induced neutrophil recruitment in skin inflammation in rabbits. Taken together, these data demonstrate that phage-display analysis provides information about the relative location of amino acids on the N-domain surfaces of hCXCR1 and hCXCR2 proteins using antibody imprints of the receptor-surface structure. The derived mimotopes could be used as inhibitors of hCXCL8-induced activities related to its interaction with the N-domain of hCXCR1 and hCXCR2.

Comparison of pp65 antigenemia, quantitative PCR and DNA hybrid capture for detection of cytomegalovirus in transplant recipients and AIDS patients.

The cytomegalovirus (CMV) antigenemia assay has been used frequently for rapid diagnosis of CMV infection, and antigenemia threshold values are recommended for triggering preemptive therapy. Hybrid capture of CMV's DNA and quantitative polymerase chain reaction (qPCR) are increasingly being adopted for early detection of CMV. The performance of the antigenemia assay, qPCR in plasma and hybrid capture in leukocytes were compared in 110 immunocompromised patients (38 bone-marrow transplants, 50 renal transplants and 22 AIDS patients). The most sensitive test was hybrid capture for transplants, while antigenemia and the qPCR showed similar performance for patients with AIDS. QPCR and hybrid capture thresholds requiring antiviral therapy were calculated using a receiver-operating-characteristic curve for antigenemia values corresponding to 2 positive cells for bone-marrow transplants and to 10 positive cells for renal transplants and AIDS patients. These threshold values varied with the group of patients considered, with corresponding sensitivities higher than 86% and specificities higher than 76% for hybrid capture, and sensitivities higher than 61% and specificities higher than 75% for qPCR in plasma. Hybrid capture in leukocytes can substitute for antigenemia in the case of transplants, and qPCR in plasma can substitute for it in the case of AIDS patients.