Exploring structural engineering approach to formulate and characterize next-generation adjuvants.

It is critical to emphasize the importance of vaccination as it protects us against harmful pathogens. Despite significant progress in vaccine development, there is an ongoing need to develop vaccines that are not only safe but also highly effective in protecting against severe infections. Subunit vaccines are generally safe, but they frequently fail to elicit strong immune responses. As a result, there is a need to improve vaccine effectiveness by combining them with adjuvants, which have the potential to boost the immune system many folds. The process of developing these adjuvants requires searching for molecules capable of activating the immune system, combining these promising compounds with an antigen, and then testing this combination using animal models before approving it for clinical use. Liposomal adjuvants work as delivery adjuvants and its activity depends on certain parameters such as surface charge, vesicle size, surface modification and route of administration. Self-assembly property of peptide adjuvants and discovery of hybrid peptides have widened the scope of peptides in vaccine formulations. Since most pathogenic molecules are not peptide based, phage display technique allows for screening peptide mimics for such pathogens that have potential as adjuvants. This chapter discusses about peptide and liposome-based adjuvants focusing on their properties imparting adjuvanticity along with the methods of formulating them. Methods of adjuvant characterization important for an adjuvant to be approved for clinical trials are also discussed. These include assays for cytotoxicity, T-lymphocyte proliferation, dendritic cell maturation, cytokine and antibody production, toll-like receptor dependent signaling and adjuvant half-life.

Extracellular Vesicle Molecular Profiling for Diagnostic Purposes: An Application of Phage Display Technology.

Phage display is a molecular biology cloning technique that allows the expression of genes of interest along with the phage surface protein. The technique described for the following method used a genomic library for the expression of peptides composed of 12 amino acids, with the objective of selecting peptides which presented specific affinity to the molecules of interest. As a target, purified extracellular vesicles from cell cultures of cells 5637 and RT4 were chosen, which in turn have enormous application and can help to understand the functioning of bladder cancer, allowing the development of new vaccines, drugs, therapies, and diagnoses.

Screening of sperm antigen epitopes by phage display technique and its preliminary clinical application.

BACKGROUND: At present, there is a lack of standardized preparation methods of sperm antigen for the detection of antisperm antibody (AsAb). To screen sperm antigen mimotopes from a phage display random peptide library and use them to establish an enzyme-linked immunosorbent assay (ELISA) for the detection of AsAb, immunoglobulins were extracted from the sera of rabbits with positive AsAb and negative AsAb, respectively, by the saturated ammonium sulfate method, and a phage display 12-mer peptide library was affinity panned by the extracted immunoglobins coated on the ELISA plate. Then, the obtained positive phage clones were identified by ELISA and sent for sequencing and peptides synthesis. Last, a diagnostic ELISA was established to detect clinical serum and seminal plasma samples. RESULTS: A total of sixty phage clones were chosen by affinity panning, and sixteen of them reacted positively with AsAb in indirect ELISA and sandwich ELISA. Following DNA sequencing and translation, the peptide sequences of the sixteen positive clones were obtained. By comparison in Blast database, four of sixteen positive clones were found to be closely related to male reproduction. Two (#1 and #25) of four mimotopes were synthesized, and an ELISA method was established using the two mimotopes as sperm specific antigens. One hundred and thirty-four serum samples and seventy-four seminal plasma samples from infertile couples were analyzed by the established ELISA with #1 and #25 mimotopes, respectively. The positive rates of AsAb in serum samples were 20.15% (27/134) for #1 and 11.19% (15/134) for #25, respectively, and the coincidence rate between them was 91.04% (122/134). The positive rates of AsAb in seminal plasma samples were 1.35% (1/74) for both #1 and #25, and the coincidence rate was 100%. CONCLUSION: Sperm antigen mimotopes can be obtained successfully by the phage display technique, and can be used as standard sperm specific antigens to establish an ELISA method for the detection of AsAb.

RéSUMé: CONTEXTE: À ce jour, il n'existe pas de méthodes normalisées de préparation d'antigènes spermatiques pour la détection des anticorps anti-spermatozoïdes (ACAS). Dans le but d'élaborer un tel test ELISA (enzyme-linked immunosorbent assay), nous avons extrait de sérum de lapins des anticorps anti-spermatozoïdes humains via la technique du sulfate d'ammonium saturé et en ayant recours à une librairie phagique de peptides (12-mer). Les clones positifs ont été identifiés par ELISA, séquencés à façon et les peptides correspondants ont été synthétisés. In fine, un test ELISA diagnostic a été conçu pour être utilisé avec des échantillons cliniques de sérum et de plasmas séminaux. RéSULTATS: Au total, soixante clones de phages ont été sélectionnés, et seize d'entre eux se sont avérés interagir avec les ACAS en ELISA indirect comme en ELISA sandwich. Les séquences peptidiques de ces seize clones positifs ont été obtenues. Par comparaison avec les bases de données (Blast), quatre de ces seize clones positifs se sont révélés être étroitement liés à la reproduction masculine. Deux des quatre mimotopes (#1 et #25) ont été synthétisés, et un test ELISA a été généré en utilisant ces deux mimotopes comme antigènes spécifiques des spermatozoïdes. Cent trente-quatre échantillons de sérum et soixante-quatorze échantillons de plasma séminal de patients de couples infertiles ont alors été analysés avec ce test ELISA. Respectivement, les échantillons sériques se sont révélés positifs à 20,15% (27/134) pour le mimotope #1 et à 11,19% (15/134) pour le mimotope #25, avec un taux de coïncidence de 91,04% (122/134). Seul un échantillon de plasma séminal (1/74, soit 1, 35%) s'est révélé positif à la fois pour le mimotope #1 et #25 (coïncidence 100%). CONCLUSION: La technique « phage display¼ nous a permis d'identifier des mimotopes d'antigènes spermatiques qui ont pu être utilisés afin de générer un test ELISA pour la détection d'anticorps anti-spermatozoïdes.

Biotechnology Approach to Mineral Separation via Phage Flotation Collectors.

A long-standing challenge in the mining industry is the separation of mineral particles that have similar surface characteristics for which surfactant-based flotation collectors cannot discriminate. In Florida phosphate mining, this problem occurs in the separation of dolomite [CaMg(CO3)2] contaminants from the desired francolite mineral {a fluorapatite [Ca5(PO4)3(F,OH)]}. In this study, phage display techniques were used to select phage clones with specific binding affinity to francolite, which were then tested in a benchtop bubbler flotation apparatus for their ability to selectively float francolite particles from mixtures containing dolomite. Contact angles measured with the captive bubble technique were used to examine changes in the surface character of the mineral particles upon adsorption of the phage, which showed that the most selective phage led to an increase in the contact angle from 16 to 50°. Although this is below the level considered hydrophobic, the correlation between contact angles and increased flotation recovery suggests that the phage coat proteins are behaving as efficient bioamphiphiles for the attachment of the particles to air bubbles, demonstrating a new and environmentally friendly type of biocollector system. The chemical and physical characteristics of the phage "tail" peptides were evaluated to offer an explanation for the specificity of phage binding. We conclude with a discussion of the potential benefits of this biotechnology approach, even for commodity industries such as mining or other particle separation systems, when costs and sustainability are considered.

Nanobodies as efficient drug-carriers: Progress and trends in chemotherapy.

Nanobodies (Nb) have a promising future as a part of next generation chemodrug delivery systems. Nb, or VHH, are small (15 kDa) monomeric antibody fragments consisting of the antigen binding region of heavy chain antibodies. Heavy chain antibodies are naturally produced by camelids, however the structure of their VHH regions can be readily reproduced in industrial expression systems, such as bacteria or yeast. Due to their small size, high solubility, remarkable stability, manipulatable characteristics, excellent in vivo tissue penetration, conjugation advantages, and ease of production, Nb have many advantages when compared against their antibody precursors. In this review, we discuss the generation and selection of Nbs via phage display libraries for easy screening, and the conjugation techniques involved in creating target-specific nanocarriers. Furthermore, we provide a comprehensive overview of recent developments and perspectives in the field of Nb drug conjugates (NDCs) and Nb-based drug vehicles (NDv) with respect to antitumor therapeutics.

Generation of avian-derived anti-B7-H4 antibodies exerts a blockade effect on the immunosuppressive response.

For highly conserved mammalian protein, chicken is a suitable immune host to generate antibodies. Monoclonal antibodies have been successfully targeted with immunity checkpoint proteins as a means of cancer treatment; this treatment enhances tumor-specific immunity responses through immunoregulation. Studies have identified the importance of B7-H4 in immunoregulation and its use as a potential target for cancer treatment. High levels of B7-H4 expression are found in tumor tissues and are associated with adverse clinical and pathological characteristics. Using the phage display technique, this study isolated specific single-chain antibody fragments (scFvs) against B7-H4 from chickens. Our experiment proved that B7-H4 clearly induced the inhibition of T-cell activation. Therefore, use of anti-B7-H4 scFvs can effectively block the exhaustion of immunity cells and also stimulate and activate T-cells in peripheral blood mononuclear cells. Sequence analysis revealed that two isolated scFv S2 and S4 have the same VH complementarity-determining regions (CDRs) sequence. Molecule docking was employed to simulate the complex structures of scFv with B7-H4 to analyze the interaction. Our findings revealed that both scFvs employed CDR-H1 and CDR-H3 as main driving forces and had strong binding effects with the B7-H4. The affinity of scFv S2 was better because the CDR-L2 loop of the scFv S2 had three more hydrogen bond interactions with B7-H4. The results of this experiment suggest the usefulness of B7-H4 as a target for immunity checkpoints; the isolated B7-H4-specific chicken antibodies have the potential for use in future cancer immunotherapy applications.

Infectious bronchitis virus: Identification of Gallus gallus APN high-affinity ligands with antiviral effects.

Infectious bronchitis virus (IBV) is a coronavirus, causes infectious bronchitis (IB) with high morbidity and mortality, and gives rise to huge economic losses for the poultry industry. Aminopeptidase N (APN) may be one of the IBV functional receptors. In this study, Gallus gallus APN (gAPN) protein was screened by phage-displayed 12-mer peptide library. Two high-affinity peptides H (HDYLYYTFTGNP) and T (TKFSPPSFWYLH) to gAPN protein were selected for in depth characterization of their anti-IBV effects. In vitro, indirect ELISA showed that these two high-affinity ligands could bind IBV S1 antibodies. Quantitative real-time PCR (qRT-PCR) assay, virus yield reduction assay and indirect immunofluorescence assay results revealed 3.125-50 µg/ml of peptide H and 6.25-50 µg/ml of peptide T reduced IBV proliferation in chicken embryo kidney cells (CEKs). In vivo, high-affinity phage-vaccinated chickens were able to induce specific IBV S1 antibodies and IBV neutralizing antibodies. QRT-PCR results confirmed that high-affinity phages reduced virus proliferation in chicken tracheas, lungs and kidneys, and alleviated IBV-induced lesions. By multiple sequence alignment, motif 'YxYY' and 'FxPPxxWxLH' of high-affinity peptides were identified in IBV S1-NTD, while another motif 'YxFxGN' located in S2. These results indicated that high affinity peptides of gAPN could present an alternative approach to IB prevention or treatment.

Preclinical discovery and development of adalimumab for the treatment of rheumatoid arthritis.

Introduction: Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by progressive joint disorders with significant pain and stiffness. In the past, RA was a difficult -to-treat ailment, but nowadays with the advent of biologics and better treatment strategies, disease remission is an achievable goal. Tumor necrosis factor α (TNFα) inhibitors were the first category of biologics to emerge with adalimumab being the first fully human TNFα.Areas covered: the authors provide an overview of the historical events that led to the discovery of TNFα inhibitors and more specifically the drug adalimumab. Several key trials are presented regarding the safety of the drug as well as its successful journey, but there is also a narrative description of the drug's future after patent expiration.Expert opinion: Adalimumab is a fully human TNFα inhibitor with a fairly rapid onset of action. It has a generally good safety and efficacy profile. Clinicians must be aware of the possible side effects and treat them in a timely manner or discontinue the drug where appropriate. Due to the success of the bio-originator adalimumab, a multitude of biosimilars have emerged but not, thus far, for all of the indications of the bio-originator.

Screening specific binding peptides of Mycobacterium tuberculosis PPE17 protein by Phage display technique / 公共卫生与预防医学

Objective The specific binding peptide of Mycobacterium tuberculosis PPE17 protein was screened by phage display technique. Methods PPE17 gene was amplified from Mycobacterium tuberculosis genome, cloned into pET28a, expressed in E. coli BL21, purified by Ni2+ column, and identified by SDS-PAGE and Western blot. The purified PPE17 protein was coated into an ELISA plate and screened by phage 7 peptide library. After three rounds of panning, phage plaques were randomly selected for sequencing. DNAMAN was used to analyze and compare the amino acid sequences of the polypeptide encoded by the positive clones. Results PPE17 gene was successfully constructed and expressed, and soluble protein with molecular weight of about 37kD was obtained. From the third round of eluents, 20 plaque were randomly selected. The sequencing results could be translated into 8 polypeptide molecules, among which the polypeptide sequence repeated for 6 times was LKWGHVY. Conclusion The specific binding peptide of PPE17 protein is screened by phage display technology, which is expected to be a small molecular diagnostic reagent for the identification of this antigen.

Phage-based Electrochemical Sensors: A Review.

Phages based electrochemical sensors have received much attention due to their high specificity, sensitivity and simplicity. Phages or bacteriophages provide natural affinity to their host bacteria cells and can serve as the recognition element for electrochemical sensors. It can also act as a tool for bacteria infection and lysis followed by detection of the released cell contents, such as enzymes and ions. In addition, possible detection of the other desired targets, such as antibodies have been demonstrated with phage display techniques. In this paper, the recent development of phage-based electrochemical sensors has been reviewed in terms of the different immobilization protocols and electrochemical detection techniques.

Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique.

Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.

Creation of mouse TNFR2-selective agonistic TNF mutants using a phage display technique.

Tumor necrosis factor-α (TNF), which is an immuno-modulatory cytokine, has been suggested to cause inflammatory responses as well as protection against tissue dysfunction by binding two types of TNF receptor (TNFR1/TNFR2). However, the physiological effects of TNFR2-specific activation remain unclear. We therefore aimed to generate a TNF mutant with full TNFR2-selective agonist activity as a functional analytical tool. In this study, we utilized a phage display technique to create mouse TNFR2 (mTNFR2)-selective TNF mutants that bind specifically to mTNFR2 and show full bioactivity compared with wild-type TNF. A new phage library displaying TNF mutants was created, in which nine amino acid residues at the predicted receptor-binding site were randomized. From this library, an agonistic TNF mutant exhibiting high binding selectivity and bioactivity to mTNFR2 was isolated. We propose that this TNF mutant would be a powerful tool with which to elucidate the functional roles of mTNFR2.

Putative phage-display epitopes of the porcine epidemic diarrhea virus S1 protein and their anti-viral activity.

Porcine epidemic diarrhea virus (PEDV) is a pathogen of swine that causes severe diarrhea and dehydration resulting in substantial morbidity and mortality in newborn piglets. Phage display is a technique with wide application, in particular, the identification of key antigen epitopes for the development of therapeutic and diagnostic reagents and vaccines. To identify antigen epitopes with specificity for PEDV, a monoclonal antibody (MAb-5E12) against the immunodominant region of the PEDV Spike protein (S1) was used as the target for biopanning a 12-mer phage display, random peptide library. After multiple rounds of biopanning and stringent washing, three phage-displayed peptides, designated L, W and H, were identified that recognize MAb-5E12. Sequence analysis showed that the one or more of the peptides exhibited partial sequence similarity to the native S1 sequence 'MQYVYTPTYYML' (designated peptide M) at position 201-212. In combination with software analysis for the prediction of B cell epitopes, aa 201-212 exhibited characteristics of a linear epitope on the PEDV S1 protein. In contrast to peptide M, a consensus motif 'PxxY' was identified on both peptides L and W, and on the S1 protein, but not on peptide H. Peptide M and the MAb-5E12-recognizing peptides L and W significantly inhibited the adsorption of PEDV on the cell surface as monitored through plaque-reduction assays. Furthermore, data from real-time PCR and indirect immunofluorescence assays were consistent with the ability of peptides M, L and W to block viral protein expression and thereby function as antiviral agents for PEDV.

Identification of conformational antigenic epitopes and dominant amino acids of buffalo ß-lactoglobulin.

Major allergen ß-lactoglobulin exists in many mammalian types of milk except human breast. Buffalo milk also contains this major allergen but the detailed information on its epitopes is not available. The aim of this work was to map and characterize its conformational antigenic epitopes. Sixty mimotopes of buffalo ß-lactoglobulin were produced by biopanning of phage display peptide library and then 2 mimotopes, specific for sera from rabbit 1 and 2, respectively, were predicted to be conformational epitope candidates by the use of DNAStar and web tool of MIMOX. On the basis of bioinformation analysis, 5 conserved amino acid residues PL-ENK were identified in 2 conformational epitope sequences and 7 conformational epitopes were derived from 2 mimotopes by molecular modeling. The result showed that these conformational epitopes were located in the 2 regions on buffalo ß-lactoglobulin and composed of 5 hydrophilic and 2 hydrophobic amino acids.

Screening and identification of recombinant anti-idiotypic antibody against monoclonal antibody MGb1 / 免疫学杂志

Objective T9 provide candidate molecules for developing recombinant anti-idiotypic antibody (anti-Id) vaccine of gastric carcinoma by selection of recombinant anti-Id to monoclonal antibody ( McAb) MGb1 directed against the cancer with phage display technique.Methods Balb/c mice were immunized with MGb1 and the mRNA was isolated from the spleens of the immunized mice. The VL and VH cDNAs of the antibody were amplified separately by RT-PCR and assembled into ScFv DNAs with a linker DNA. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage antibody ScFv library. After four rounds of biopanning to the library with MGb1, the MGb1-positive clones were selected from the enriched phages by ELISA. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA. Results The VL and VH cDNAs was about 320 bp and 340 bp, respectively. The ScFv DNA were about 750 bp. After four rounds panning to the phage antibody ScFv library with MGb1, 18 MGb1-positive phage clones displayed anti-Id ScFv were selected from 50 pre-selected phage clones, among which 4 clones displayed β or γ type anti-Id ScFv. Conclusion The phagedisplayed anti-Id ScFvs to McAb MGb1 are successfully selected by recombinant phage antibody technique, which might lay a foundation for screening the anti-Id ScFv possessing the characteristics of inducing anti-gastric carcinoma immunity.

SCREENING AND CHARACTERIZATION OF HUMAN PHAGE ANTIBODY TO HEPATITIS C VIRUS E2 ANTIGEN / 解放军医学杂志

To screen and characterize human phage antibody to hepatitis C virus E2 antigen. The recombinant phages were panned by HCV E2 antigen which was coated in a microwell plate. After three rounds of biopanning, 56 clones specific to HCV E2 antigen were obtained. The specificity of scFv was determined by ELISA method and cross reaction with BSA and competitive inhibition assay. HCV E2 phage antibody had a specific combination character with recombinant hepatitis C virus E2 antigen. The DNA sequence data showed that the scFv coding gene was 771bp in size