Phage-derived Peptides Research Articles

Genome characterization of the novel lytic phage vB_AbaAut_ChT04 and the antimicrobial activity of its lysin peptide against Acinetobacter baumannii isolates from different time periods.

Acinetobacter baumannii is an important opportunistic pathogen, usually associated with immunocompromised individuals with a prolonged period of stay in a hospital. Currently, the incidence of multi-drug resistant A. baumannii (MDR-AB) and extensively drug-resistant A. baumannii (XDR-AB) is increasing rapidly in Thailand, mirroring the trend worldwide. Novel therapeutic approaches for the treatment of A. baumannii infection using bacteriophages are being evaluated, and the use of phage-derived peptides is being tested as alternative approach to fighting infection. In this study, we isolated and determined the biological features of a lytic A. baumannii phage called vB_AbaAut_ChT04 (vChT04). The vChT04 phage was classified as a member of the family Autographiviridae of the class Caudoviricetes. It showed a short latent period (10 min) and a large burst size (280 PFU cell-1), and it was able to infect 52 out of 150 clinical MDR-AB strains tested (34.67%). Most of the phage-sensitive strains were A. baumannii strains that had been isolated during the same year that the phage was isolated. The phage showed activity across a broad pH (pH 5.0-8.0) and temperature (4-37°C) range. Whole-genome analysis revealed that the vChT04 genome comprises 41,158 bp with a 39.3% GC content and contains 48 open reading frames (ORFs), 28 of which were assigned putative functions based on homology to previously identified phage genes. Comparative genomic analysis demonstrated that vChT04 had the highest similarity to phage vB_AbaP_WU2001, which was isolated in the southern part of Thailand. An endolysin gene found in the vChT04 genome was used to synthesize an antimicrobial peptide (designated as PLysChT04) and its antimicrobial activity was evaluated using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. The MIC and MBC values of peptide PLysChT04 against MDR-AB and XDR-AB were 312.5-625 µg/mL, and it was able to inhibit both phage-susceptible and phage-resistant isolates collected over different time periods. PLysChT04 showed good efficacy in killing drug-resistant A. baumannii and other bacterial strains, and it is a promising candidate for development as an alternative therapeutic agent targeting A. baumannii infections.

Directions in the development of modern and promising antimicrobial agents

The use of antibiotics is becoming increasingly limited. This is mainly due to the development of resistance to pathogenic bacteria, and, over time, more and more bacteria will become resistant to antimicrobials. This problem inevitably leads to the conclusion that studies into alternative methods of combating pathogens, which are necessary to develop sufficiently reliable and effective therapies for bacterial infections, are indispensable. This review highlights some recent developments in conventional antibiotic and non-antibiotic treatment strategies. It has been shown that traditional antibacterial targets include derivatives of known antibiotic classes, new chemical classes with new targets, as well as unknown or undefined agents with unclear targets. Promising strategies for combating microbial pathogens have been identified, including new targets, namely, toxin secretion systems, biofilm formation, and adhesion mechanisms that affect quorum sensing of microbial populations. In addition, it is important to use new antimicrobial agents with other, non-antibiotic, mechanisms of action: phage and phage-derived peptides, microbiota-modulating therapies, and enhancing immune response.

Phage-derived peptides as new epitopes of breast cancer

Epidemiological data on death rates and incidence of Breast Cancer (BC) are still alarming, highlighting the need for new strategies of diagnosis and therapy. Our group has previously described a new antibody in Fab format, FabC4, for diagnostics, staging, and prognosis of BC. Objectives: To map the FabC4 epitopes and evaluate those for their diagnostic potential. Methods: A Phage Display-based assay against FabC4 was performed to identify and characterize peptides as new epitopes of BC. The selected peptides were also evaluated for their reactivity to patient sera through ELISA assay. We finally conducted molecular docking to assess the best conformation of FabC4-epitopes. Results: Four peptides-displaying phages differentiated sera samples from 50 patients with BC, benign disease, and from healthy women. The corresponding peptides were chemically synthesized (pA5, pA7, pC4 and pD6) and bound to FabC4. The peptide pD6 differentiated the neoplastic samples from benign and healthy sera. Molecular docking analyzes confirmed the interaction between FabC4 and the selected peptides. Conclusion: We have successfully mapped the FabC4 epitopes with diagnostic potential, opening new avenues for the understanding and treatment of BC.

Early detection of Alzheimer’s disease by peptides from phage display screening

Alzheimer’s disease (AD) is the most common neurodegenerative disorder without effective treatment so far. As clinical trials show that early-stage patients are more likely to respond to potential interventions, various technologies have been used to search blood biomarkers for the early diagnosis of AD. Phage display could be used to select specific peptides against desired target and here, we established a peptide binding assay based on phage display peptide library to detect early-stage AD patients. We first selected peptides from phage display library against plasmas from AD patients (n = 10) and normal healthy controls (n = 10), respectively, in the discovery set. Then, we further characterized one AD-specific peptide (AD#1 peptide) and one control-specific peptide (Con#1 peptide), and evaluated their diagnostic performance in independent validation set (35 AD patients, 45 MCI, 45 controls and 20 PD patients). Our results show that both AD#1 peptide and Con#1 peptide could distinguish AD/MCI patients from controls and combination of these two peptides could greatly improve the diagnostic performance (AUC is above 0.80 in ROC curve analysis). In addition, we found that AD#1 peptide stained Aβ-treated primary astrocyte and bound to recombinant human YKL-40 protein in in-vitro assay. It supports that AD#1 peptide detects AD inflammation related cytokine. Thus, the detection assay based on phage-derived peptides may offer a novel blood biomarker test for the early diagnosis of AD.

In Vivo Translation of Peptide-Targeted Drug Delivery Systems Discovered by Phage Display

Therapeutic compounds with narrow therapeutic windows and significant systemic side effects benefit from targeted drug delivery strategies. Peptide-protein interactions are often exploited for targeting, with phage display a primary method to identify high-affinity peptide ligands that bind cell surface and matrix bound receptors preferentially expressed in target tissues. After isolating and sequencing high-binding phages, peptides are easily synthesized and chemically modified for incorporation into drug delivery systems, including peptide-drug conjugates, polymers, and nanoparticles. This review describes the phage display methodology to identify targeting peptide sequences, strategies to functionalize drug carriers with phage-derived peptides, specific examples of drug carriers with in vivo translation, and limitations and future applications of phage display to drug delivery.

Polypharmacy through Phage Display: Selection of Glucagon and GLP-1 Receptor Co-agonists from a Phage-Displayed Peptide Library

A promising emerging area for the treatment of obesity and diabetes is combinatorial hormone therapy, where single-molecule peptides are rationally designed to integrate the complementary actions of multiple endogenous metabolically-related hormones. We describe here a proof-of-concept study on developing unimolecular polypharmacy agents through the use of selection methods based on phage-displayed peptide libraries (PDL). Co-agonists of the glucagon (GCG) and GLP-1 receptors were identified from a PDL sequentially selected on GCGR- and GLP1R-overexpressing cells. After two or three rounds of selection, 7.5% of randomly picked clones were GLP1R/GCGR co-agonists, and a further 1.53% were agonists of a single receptor. The phages were sequenced and 35 corresponding peptides were synthesized. 18 peptides were potent co-agonists, 8 of whom showed EC50 ≤ 30 pM on each receptor, comparable to the best rationally designed co-agonists reported in the literature. Based on literature examples, two sequences were engineered to stabilize against dipeptidyl peptidase IV cleavage and prolong the in vivo half-life: the engineered peptides were comparably potent to the parent peptides on both receptors, highlighting the potential use of phage-derived peptides as therapeutic agents. The strategy described here appears of general value for the discovery of optimized polypharmacology paradigms across several metabolically-related hormones.

Detection of methicillin-resistant Staphylococcus aureus using phage amplification combined with matrix-assisted laser desorption/ionization mass spectrometry

Antibiotic resistance continues to contribute significantly to morbidity and mortality across the world. Developing new tests for antibiotic-resistant bacteria is a core action to combat resistant infections. We describe a method that uses phage amplification detection (PAD) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to rapidly identify Staphylococcus aureus and determine phenotypic susceptibility to cefoxitin. Samples tested for S. aureus are incubated together with bacteriophage in the presence and absence of cefoxitin and subjected to rapid trypsin digestion followed by MALDI-MS analysis. Tryptic peptides derived from amplified phage proteins can be detected by MALDI-MS, as validated by time-of-flight (TOF)/TOF analysis of each peptide combined with database searching. Methicillin-resistant S. aureus show significant phage amplification in the presence of cefoxitin, while methicillin-sensitive S. aureus show no phage amplification relative to a no-antibiotic control. We also show that PAD methodology can be implemented on an FDA-approved commercial MALDI-MS bacterial identification system to identify S. aureus and determine antibiotic susceptibility. The novelty of this assay includes the use of phage-derived tryptic peptides as detected by MALDI-MS to monitor the results of PAD on an instrument common to many modern microbiology laboratories.

Induction of anti-EGFR immune response with mimotopes identified from a phage display peptide library by panitumumab.

The epidermal growth factor receptor (EGFR) is overexpressed in several epithelial tumors. Anti-EGFR humanized monoclonal antibodies, cetuximab and panitumumab, in combination with chemotherapy have improved the prognosis for patients with wild-type RAS tumors. To identify mimotopes of EGFR and develop mimotope-based EGFR vaccines, we screened a phage display peptide library with panitumumab. Two EGFR mimotopes P19 and P26, which could be recognized by panitumumab specifically, were isolated. To enhance the immune responses, we generated recombinant proteins of P19 or P26 fused to a heat-shock cognate protein 70 (Hsc70), and evaluated the efficacy of Hsc70-P19 and Hsc70-P26 as vaccines in vivo. Immunization with Hsc70-P19 or Hsc70-P26 fusion protein stimulated the immune system to produce specific antibodies against peptides as well as EGFR. Moreover, antibodies elicited against mimotopes could induce antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and inhibit the proliferation of EGFR-overexpressing A431 cells. Treatment with Hsc70-P19 and Hsc70-P26 significantly reduced tumor growth in BALB/c transplantable lung cancer models. Although there was no sequence homology between the phage-derived peptides and EGFR by alignments, both peptides mimic the conformational structure of EGFR binding to panitumumab. In conclusion, the mimotopes we identified from phage display peptide library could be promising candidate vaccines for active anti-EGFR immunotherapy against cancers.

Recognition Dynamics of p53 and MDM2: Implications for Peptide Design.

Peptides that inhibit MDM2 and attenuate MDM2-p53 interactions, thus activating p53, are currently being pursued as anticancer drug leads for tumors harboring wild type p53. The thermodynamic determinants of peptide-MDM2 interactions have been extensively studied. However, a detailed understanding of the dynamics that underlie these interactions is largely missing. In this study, we explore the kinetics of the binding of a set of peptides using Brownian dynamics simulations. We systematically investigate the effect of peptide C-terminal substitutions (Ser, Ala, Asn, Pro) of a Q16ETFSDLWKLLP27 p53-based peptide and a M1PRFMDYWEGLN12 12/1 phage-derived peptide on their interaction dynamics with MDM2. The substitutions modulate peptide residence times around the MDM2 protein. In particular, the highest affinity peptide, Q16ETFSDLWKLLS27, has the longest residence time (t ∼ 25 μs) around MDM2, suggesting its potentially important contribution to binding affinity. The binding of the p53-based peptides appears to be kinetically driven while that of the phage-derived series appears to be thermodynamically driven. The phage-derived peptides were found to adopt distinctly different modes of interaction with the MDM2 protein compared to their p53-based counterparts. The p53-based peptides approach the N-terminal region of the MDM2 protein with the peptide C-terminal end oriented toward the protein, while the M1PRFMDYWEGLN12-based peptides adopt the reverse orientation. To probe the determinants of this switch in orientation, a designed mutant of the phage-derived peptide, R3E (M1PEFMDYWEGLN12), was simulated and found to adopt the orientation adopted by the p53-based peptides and also to result in almost a 5-fold increase in the peptide residence time (∼120 μs) relative to the p53-based peptides. On this basis, we suggest that the R3E mutant phage-derived peptide has a higher affinity for MDM2 than the p53-based peptides and would therefore, competitively inhibit MDM2-p53. The study, therefore, provides a novel computational framework for kinetics-based lead optimization for anticancer drug development strategies.

Dual-functioning phage-derived peptides encourage human bone marrow cell-specific attachment to mineralized biomaterials

Cell instructive mineralized biomaterials are a promising alternative to conventional auto-, allo-, and xenograft therapies for the reconstruction of critical sized defects. Extracellular matrix proteins, peptide domains, and functional motifs demonstrating cell and mineral binding activity have been used to improve cell attachment. However, these strategies vary in their tissue regeneration outcomes due to lack of specificity to both regenerative cell populations and the material substrates. In order to mediate cell-specific interactions on apatite surfaces, we identified peptide sequences with high affinity towards apatite (VTKHLNQISQSY, VTK) and clonally derived human bone marrow stromal cells (DPIYALSWSGMA, DPI) using phage display. The primary aims of this study were to measure apatite binding affinity, human bone marrow stromal cell (hBMSC) adhesion strength, and peptide specificity to hBMSCs when the apatite and cell-specific peptides are combined into a dual-functioning peptide. To assess binding affinity to hydroxyapatite (HA), binding isotherms were constructed and peptide binding affinity (K1) determined. HBMSC, MC3T3 and mouse dermal fibroblast (MDF) adhesion strength on biomimetic apatite functionalized with single- and dual-functioning peptide sequences were evaluated using a centrifugation assay. DPI-VTK had the highest binding strength towards hBMSCs (p < 0.01). DPI-VTK, while promoting strong initial attachment to hBMSCs, did not encourage strong adhesions to MC3T3s or fibroblasts (p < 0.01). Taken together, phage display is a promising strategy to identify preferential cell and material binding peptide sequences that can tether specific cell populations onto specific biomaterial chemistries.

Rapid Detection of Pathogenic Bacteria and Screening of Phage-Derived Peptides Using Microcantilevers

We report the use of an array of microcantilevers to measure the specific binding of Salmonella to peptides derived from phage display libraries. Selectivity of these phage-derived peptides for Salmonella spp. and other pathogens ( Listeria monocytogenes and Escherichia coli ) are compared with a commercially available anti- Salmonella antibody and the antimicrobial peptide alamethicin. A Langmuir isotherm model was applied to determine the binding affinity constants of the peptides to the pathogens. One particular peptide, MSal 020417, demonstrated a higher binding affinity to Salmonella spp. than the commercially available antibody and is able to distinguish among eight Salmonella serovars on a microcantilever. A multiplexed screening system to quickly determine the binding affinities of various peptides to a particular pathogen highly improves the efficiency of the peptide screening process. Combined with phage-derived peptides, this microcantilever-based technique provides a novel biosensor to rapidly and accurately detect pathogens and holds potential to be further developed as a screening method to identify pathogen-specific recognition elements.

Selection of ceramic fluorapatite‐binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography

Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA-specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N-terminal sequence was found in two selected peptides: F4-2 (KPRSMLH) and F5-4 (KPRSVSG). The peptide F5-4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5-4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage-derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins.

New bioactive motifs and their use in functionalized self-assembling peptides for NSC differentiation and neural tissue engineering

Developing functionalized biomaterials for enhancing transplanted cell engraftment in vivo and stimulating the regeneration of injured tissues requires a multi-disciplinary approach customized for the tissue to be regenerated. In particular, nervous tissue engineering may take a great advantage from the discovery of novel functional motifs fostering transplanted stem cell engraftment and nervous fiber regeneration. Using phage display technology we have discovered new peptide sequences that bind to murine neural stem cell (NSC)-derived neural precursor cells (NPCs), and promote their viability and differentiation in vitro when linked to LDLK12 self-assembling peptide (SAPeptide). We characterized the newly functionalized LDLK12 SAPeptides via atomic force microscopy, circular dichroism and rheology, obtaining nanostructured hydrogels that support human and murine NSC proliferation and differentiation in vitro. One functionalized SAPeptide (Ac-FAQ), showing the highest stem cell viability and neural differentiation in vitro, was finally tested in acute contusive spinal cord injury in rats, where it fostered nervous tissue regrowth and improved locomotor recovery. Interestingly, animals treated with the non-functionalized LDLK12 had an axon sprouting/regeneration intermediate between Ac-FAQ-treated animals and controls. These results suggest that hydrogels functionalized with phage-derived peptides may constitute promising biomimetic scaffolds for in vitro NSC differentiation, as well as regenerative therapy of the injured nervous system. Moreover, this multi-disciplinary approach can be used to customize SAPeptides for other specific tissue engineering applications.

Evolution of Potent and Stable Placental-Growth-Factor-1-Targeting CovX-Bodies from Phage Display Peptide Discovery

Novel phage-derived peptides are the first reported molecules specifically targeting human placental growth factor 1 (PlGF-1). Phage data enabled peptide modifications that decreased IC(50) values in PlGF-1/VEGFR-1 competition ELISA from 100 to 1 μM. Peptides exhibiting enhanced potency were bioconjugated to the CovX antibody scaffold 1 (CVX-2000), generating bivalent CovX-Bodies with 2 nM K(D) against PlGF-1. In vitro and in vivo peptide cleavage mapping studies enabled the identification of proteolytic hotspots that were subsequently chemically modified. These changes decreased IC(50) to 0.4 nM and increased compound stability from 5% remaining at 6 h after injection to 35% remaining at 24 h with a β phase half-life of 75 h in mice. In cynomolgus monkey, a 78 h β half-life was observed for lead compound 2. The pharmacological properties of 2 are currently being explored.

Peptides panned from a phage-displayed random peptide library are useful for the detection of Bacillus anthracis surrogates B. cereus 4342 and B. anthracis Sterne

Recent use of Bacillus anthracis as a bioweapon has highlighted the need for a sensitive monitoring system. Current bacterial detection tests use antibodies as bio-molecular recognition elements which have limitations with regard to time, specificity and sensitivity, creating the need for new and improved cost-effective high-affinity detection probes. In this study, we screened a commercially available bacteriophage-displayed random peptide library using Bacillus cereus 4342 cells as bait to identify peptides that could be used for detection of Bacillus. The method enabled us to identify two 12-amino acid consensus peptide sequences that specifically bind to B. cereus 4342 and B. anthracis Sterne, the nonpathogenic surrogates of B. anthracis strain. The two Bacillus-binding peptides (named BBP-1 and BBP-2) were synthesized with biotin tag to confirm their binding by four independent detection assays. Dot-blot analysis revealed that the peptides bind specifically to B. cereus 4342 and B. anthracis Sterne. Quantitative analysis of this interaction by ELISA and fluorometry demonstrated a detection sensitivity of 10 2 colony forming U/ml (CFU/ml) by both assays. When the peptides were used in combination with Qdots, the sensitivity was enhanced further by enabling detection of even a single bacterium by fluorescence microscopy. Immunoblot analysis and protein sequencing showed that BBP-1 and BBP-2 bound to the S-layer protein of B. anthracis Sterne. Overall, our findings validate the usefulness of synthetic versions of phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity bioprobes for various microbial detection platforms.

High-Throughput Discovery of Synthetic Surfaces That Support Proliferation of Pluripotent Cells

Synthetic materials that promote the growth or differentiation of cells have advanced the fields of tissue engineering and regenerative medicine. Most functional biomaterials are based on a handful of peptide sequences derived from protein ligands for cell surface receptors. Because few proteins possess short peptide sequences that alone can engage cell surface receptors, the repertoire of receptors that can be targeted with this approach is limited. Materials that bind diverse classes of receptors, however, may be needed to guide cell growth and differentiation. To provide access to such new materials, we utilized phage display to identify novel peptides that bind to the surface of pluripotent cells. Using human embryonal carcinoma (EC) cells as bait, approximately 3 x 10(4) potential cell-binding phage clones were isolated. The pool was narrowed using an enzyme-linked immunoassay: 370 clones were tested, and seven cell-binding peptides were identified. Of these, six sequences possess EC cell-binding ability. Specifically, when displayed by self-assembled monolayers (SAMs) of alkanethiols on gold, they mediate cell adhesion. The corresponding soluble peptides block this adhesion, indicating that the identified peptide sequences are specific. They also are functional. Synthetic surfaces displaying phage-derived peptides support growth of undifferentiated human embryonic stem (ES) cells. When these cells were cultured on SAMs presenting the sequence TVKHRPDALHPQ or LTTAPKLPKVTR in a chemically defined medium (mTeSR), they expressed markers of pluripotency at levels similar to those of cells cultured on Matrigel. Our results indicate that this screening strategy is a productive avenue for the generation of materials that control the growth and differentiation of cells.

Multiple Novel Classes of APRIL-specific Receptor-blocking Peptides Isolated by Phage Display

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) ligand superfamily and has a proliferative effect on both normal and tumor cells. The TNF family receptors (B-cell maturation antigen (BCMA), transmembrane activator and CAML-interactor (TACI), and BAFF receptor-3 (BR3)) for APRIL and the closely related ligand, B-cell activating factor of the TNF family (BAFF), bind these ligands through a highly conserved six residue DXL motif ((F/Y/W)-D-X-L-(V/T)-(R/G)). Panning peptide phage display libraries led to the identification of several novel classes of APRIL-binding peptides, which could be grouped by their common sequence motifs. Interestingly, only one of these ten classes consisted of peptides containing the DXL motif. Nevertheless, all classes of peptides prevented APRIL, but not BAFF, from binding BCMA, their shared receptor. Synthetic peptides based on selected sequences inhibited APRIL binding to BCMA with IC50 values of 0.49–27 μM. An X-ray crystallographic structure of APRIL bound to one of the phage-derived peptides showed that the peptide, lacking the DXL motif, was nevertheless bound in the DXL pocket on APRIL. Our results demonstrate that even though a focused, highly conserved motif is required for APRIL–receptor interaction, remarkably, many novel and distinct classes of peptides are also capable of binding APRIL at the ligand receptor interface.

Phage-Displayed Peptide-Q Dot Nanocrystal Combo for High-Sensitivity Bacterial Detection in Plasma.

Abstract 3153Poster Board III-90 IntroductionDespite improved phlebotomy practices, refrigeration of red cells, freezing of plasma and improved materials for transfusion product collection and storage, bacterial contamination of transfusion products is still a longstanding problem. Current bacterial detection tests relevant to transfusion medicine, especially for stored platelets have limitations with regard to time, specificity and sensitivity. There is a need for new and improved cost-effective high-affinity detection probes to fill this gap. In this study, using plasma spiked with Bacillus cereus 4342 and B. anthracis-Sterne as an experimental system, we identified peptides from a bacteriophage-displayed random peptide library that selectively bind and detect the Bacillus strains. By labeling the peptides with Q dot-liquid nanocrystals, the detection sensitivity of the peptides was further enhanced. MethodsA commercially available bacteriophage-displayed random peptide library was screened using B. cereus 4342 as bait, using appropriate controls under stringent conditions. The screening and subsequent sequencing of the phage DNA identified two phages each containing a coding sequence for 12-amino acid peptide that are selectively capable of binding to the Bacillus. Based on the nucleic acid sequence, the two synthetic peptides with biotin tag were prepared for detection assays and to enhance the detection sensitivity further, the peptides were labeled with streptavidin-conjugated fluorescent quantum-dots (Q dots). Fluorescence was measured either by a plate reader or using a fluorescence microscope. ResultsThe two synthetic peptides selectively bound to the Bacillus strains in both dot blot and ELISA assays. The membrane-based dot blot assay demonstrated an assay sensitivity of 103 colony forming units/ml (CFU/ml), whereas ELISA demonstrated a sensitivity of 102 CFU/ml detection limit. Fluorometry analysis of spiked plasma samples revealed that the two peptides were able to bind to B. cereus 4342 and B. anthracis Sterne and detect these bacteria in plasma at 102 CFU/ml concentrations. The peptide-Qdot combo even detected a single bacterium in fluorescence microscopy. ConclusionOverall, the results reported here validate the usefulness of affinity-selected recombinant filamentous phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity detection probes for bacteria in various platforms and settings relevant to the blood safety and transfusion medicine.The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. DisclosuresNo relevant conflicts of interest to declare.

Mimotopes of Apical Membrane Antigen 1: Structures of Phage-Derived Peptides Recognized by the Inhibitory Monoclonal Antibody 4G2dc1 and Design of a More Active Analogue

Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum is an integral membrane protein that plays a key role in merozoite invasion of host erythrocytes. A monoclonal antibody, 4G2dc1, recognizes correctly folded AMA1 and blocks merozoite invasion. Phage display was used to identify peptides that bind to 4G2dc1 and mimic an important epitope of AMA1. Three of the highest-affinity binders--J1, J3, and J7--were chosen for antigenicity and immunogenicity studies. J1 and J7 were found to be true antigen mimics since both peptides generated inhibitory antibodies in rabbits (J. L. Casey et al., Infect. Immun. 72:1126-1134, 2004). In the present study, the solution structures of all three mimotopes were investigated by nuclear magnetic resonance spectroscopy. J1 adopted a well-defined region of structure, which can be attributed in part to the interactions of Trp11 with surrounding residues. In contrast, J3 and J7 did not adopt an ordered conformation over the majority of residues, although they share a region of local structure across their consensus sequence. Since J1 was the most structured of the peptides, it provided a template for the design of a constrained analogue, J1cc, which shares a structure similar to that of J1 and has a disulfide-stabilized conformation around the Trp11 region. J1cc binds with greater affinity to 4G2dc1 than does J1. These peptide structures provide the foundation for a better understanding of the complex conformational nature of inhibitory epitopes on AMA1. With its greater conformational stability and higher affinity for AMA1, J1cc may be a better in vitro correlate of immunity than the peptides identified by phage display.

Antibodies to malaria peptide mimics inhibit Plasmodium falciparum invasion of erythrocytes.

Apical membrane antigen 1 (AMA1) is expressed on the surfaces of Plasmodium falciparum merozoites and is thought to play an important role in the invasion of erythrocytes by malaria parasites. To select for peptides that mimic conformational B-cell epitopes on AMA1, we screened a phage display library of >10(8) individual peptides for peptides bound by a monoclonal anti-AMA1 antibody, 4G2dc1, known to inhibit P. falciparum invasion of erythrocytes. The most reactive peptides, J1, J3, and J7, elicited antibody responses in rabbits that recognized the peptide immunogen and both recombinant and parasite AMA1. Human antibodies in plasma samples from individuals exposed to chronic malaria reacted with J1 and J7 peptides and were isolated using immobilized peptide immunoadsorbents. Both rabbit and human antibodies specific for J1 and J7 peptides were able to inhibit the invasion of erythrocytes by P. falciparum merozoites. This is the first example of phage-derived peptides that mimic an important epitope of a blood-stage malaria vaccine candidate, inducing and isolating functional protective antibodies. Our data support the use of J1 and J7 peptide mimics as in vitro correlates of protective immunity in future AMA1 vaccine trials.