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The filamentous bacteriophage fd bind a cell target with exquisite specificity through its few copies of display peptides, whereas nanoparticles functionalized with hundreds to thousands of synthetically generated phage display peptides exhibit variable and often-weak target binding. We hypothesise that some phage peptides in a hierarchical structure rather than in monomeric form recognise and bind their target. Here we show hierarchial forms of a brain-specific phage-derived peptide (herein as NanoLigand Carriers, NLCs) target cerebral endothelial cells through transferrin receptor and the receptor for advanced glycation-end products, cross the blood-brain-barrier and reach neurons and microglial cells. Through intravenous delivery of NLC-β-secretase 1 (BACE1) siRNA complexes we show effective BACE1 down-regulation in the brain without toxicity and inflammation. Therefore, NLCs act as safe multifunctional nanocarriers, overcome efficacy and specificity limitations in active targeting with nanoparticles bearing phage display peptides or cell-penetrating peptides and expand the receptor repertoire of the display peptide., Bacteriophages can bind targets with only a few copies of a display peptide while most nanoparticles with thousands achieve poor binding. Here the authors form hierarchical arrangements of phage peptides to delivery siRNA across the blood brain barrier.

We have exploited the properties of filamentous bacteriophage fd to deliver immunologically active lipids together with antigenic peptides. Filamentous bacteriophages resemble for size, capability to be permeable to blood vessels and high density antigen expression, a nature-made nanoparticle. In addition, their major coat protein pVIII, which is arranged to form a tubular shield surrounding the phage genome, has a high content of hydrophobic residues promoting lipid association. We conjugated bacteriophages to alpha-GalactosylCeramide (alpha-GalCer), a lipid antigen stimulating invariant Natural Killer T (iNKT) cells and capable of inducing their anti-tumoral activities. We found that bacteriophage fd/alpha-GalCer conjugates could repeatedly stimulate iNKT cells in vitro and in vivo, without inducing iNKT anergy. Moreover, co-delivery of alpha-GalactosylCeramide and a MHC class I restricted tumor associated antigenic determinant to APCs via bacteriophages strongly boosted adaptive CD8+ T cell response and efficiently delayed tumor progression. Co-delivery of a tumor antigen and iNKT-stimulatory lipid on the surface of filamentous bacteriophages is a novel approach to potentiate adaptive anti-cancer immune responses, overcoming the current limitations in the use of free alpha-GalactosylCeramide and may represent an attractive alternative to existing delivery methods, opening the path to a potential translational usage of this safe inexpensive and versatile tool.

The filamentous bacteriophage fd, codisplaying antigenic determinants and a single chain antibody fragment directed against the dendritic cell receptor DEC-205, is a promising vaccine candidate for its safety and its ability to elicit innate and adaptive immune response in absence of adjuvants. By using a system vaccinology approach based on RNA-Sequencing (RNA-Seq) analysis, we describe a relevant gene modulation in dendritic cells pulsed with anti-DEC-205 bacteriophages fd. RNA-Seq data analysis indicates that the bacteriophage fd virions are sensed as a pathogen by dendritic cells; they activate the danger receptors that trigger an innate immune response and thus confer a strong adjuvanticity that is needed to obtain a long-lasting adaptive immune response.

The fd bacteriophage is a filamentous virus consisting of a circular single-stranded DNA (ssDNA) wrapped by thousands of copies of a major coat protein subunit (the capsid). The coat protein subunits are mostly α-helical and curved, and are arranged in the capsid in consecutive pentamers related by a translation along the main viral axis and a rotation of ~36° (C5S2 symmetry). The DNA is right-handed and helical, but information on its structure and on its interface with the capsid is incomplete. We present here an approach for assigning the DNA nucleotides and studying its interactions with the capsid by magic-angle spinning solid-state NMR. Capsid contacts with the ssDNA are obtained using a two-dimensional (13)C-(13)C correlation experiment and a proton-mediated (31)P-(13)C polarization transfer experiment, both acquired on an aromatic-unlabeled phage sample. Our results allow us to map the residues that face the interior of the capsid and to show that the ssDNA-capsid interactions are sustained mainly by electrostatic interactions between the positively charged lysine side chains and the phosphate backbone. The use of natural abundance aromatic amino acids in the growth media facilitated the complete assignment of the four nucleotides and the observation of internucleotide contacts. Using chemical shift analysis, our study shows that structural features of the deoxyribose carbons reporting on the sugar pucker are strikingly similar to those observed recently for the Pf1 phage. However, the ssDNA-protein interface is different, and chemical shift markers of base pairing are different. This experimental approach can be utilized in other filamentous and icosahedral bacteriophages, and also in other biomolecular complexes involving structurally and functionally important DNA-protein interactions.

Filamentous phages have unique physical properties, such as uniform particle lengths, that are not found in other model systems of rod-like colloidal particles. Consequently, suspensions of such phages provided powerful model systems that have advanced our understanding of soft matter physics in general and liquid crystals in particular. We described some of these advances. In particular we briefly summarize how suspensions of filamentous phages have provided valuable insight into the field of colloidal liquid crystals. We also describe recent experiments on filamentous phages that have elucidated a robust pathway for assembly of 2D membrane-like materials. Finally, we outline unique structural properties of filamentous phages that have so far remained largely unexplored yet have the potential to further advance soft matter physics and material science.

Filamentous bacteriophage fd particles delivering antigenic determinants via DEC‐205 (fdsc‐αDEC) represent a powerful delivery system that induces CD8 + T‐cell responses even when administered in the absence of adjuvants or maturation stimuli for dendritic cells. In order to investigate the mechanisms of this activity, RNA‐Sequencing of fd‐pulsed dendritic cells was performed. A significant differential expression of genes involved in innate immunity, co‐stimulation and cytokine production was observed. In agreement with these findings, we demonstrate that induction of proinflammatory cytokines and type I interferon by fdsc‐αDEC was MYD88 mediated and TLR9 dependent. We also found that fdsc‐αDEC is delivered into LAMP‐1‐positive compartments and co‐localizes with TLR9. Thus, phage particles containing a single‐strand DNA genome rich in CpG motifs delivered via DEC‐205 are able to intercept and trigger the active TLR9 innate immune receptor into late endosome/lysosomes and to enhance the immunogenicity of the displayed antigenic determinants. These findings make fd bacteriophage a valuable tool for immunization without administering exogenous adjuvants.

A vibrational Raman optical activity (ROA) study of a range of different structural types of virus exemplified by filamentous bacteriophage fd, tobacco mosaic virus, satellite tobacco mosaic virus, bacteriophage MS2 and cowpea mosaic virus has revealed that, on account of its sensitivity to chirality, ROA is an incisive probe of their aqueous solution structures at the molecular level. Protein ROA bands are especially prominent from which, as we have shown by comparison with the ROA spectra of proteins with known structures and by using a pattern recognition program, the folds of the major coat protein subunits may be deduced. Information about amino acid side-chain conformations, exemplified here by the determination of the sign and magnitude of the torsion angle χ2,1 for tryptophan in fd, may also sometimes be obtained. By subtracting the ROA spectrum of the empty protein capsid (top component) of cowpea mosaic virus from those of the intact middle and bottom-upper components separated by means of a caesium chloride density gradient, the ROA spectrum of the viral RNA was obtained, which revealed that the RNA takes up an A-type single-stranded helical conformation and that the RNA conformations in the middle and bottom-upper components are very similar. This information is not available from the X-ray crystal structure of cowpea mosaic virus since no nucleic acid is visible.

The minor coat protein pIII at one end of the filamentous bacteriophage fd, mediates the infection of Escherichia coli cells displaying an F-pilus. pIII has three domains (D1, D2 and D3), terminating with a short hydrophobic segment at the C-terminal end. Domain D2 binds to the tip of F-pilus, which is followed by retraction of the pilus and penetration of the E. coli cell membrane, the latter involving an interaction between domain D1 and the TolA protein in the membrane. Surface residues on the D2 domain of pIII were replaced systematically with alanine. Mutant virions were screened for D2-pilus interaction in vivo by measuring the release of infectious virions from E. coli F + cells infected with the mutants. A competitive ELISA was developed to measure in vitro the ability of mutant phages to bind to purified pili. This allowed the identification of amino acid residues involved in binding to F and to EDP208 pili. These residues were found to cluster on the outer rim of the 3D structure of the D2 domain, unexpectedly identifying this as the F-pilus binding region on the pIII protein.

For early detection of many diseases, it is critical to be able to diagnose small amounts of biomarkers in blood or serum. One of the most widely used sensing assays is the enzyme-linked immunosorbent assay (ELISA), which typically uses detection monoclonal antibodies conjugated to enzymes to produce colorimetric signals. To increase the overall sensitivities of these sensors, we demonstrate the use of a dually modified version of filamentous bacteriophage Fd that produces significantly higher colorimetric signals in ELISAs than what can be achieved using antibodies alone. Because only a few proteins at the tip of the micron-long bacteriophage are involved in antigen binding, the approximately 4000 other coat proteins can be augmented—by either chemical functionalization or genetic engineering—with hundreds to thousands of functional groups. In this article, we demonstrate the use of bacteriophage that bear a large genomic fusion that allows them to bind specific antibodies on coat protein 3 (p3) and multiple biotin groups on coat protein 8 (p8) to bind to avidin-conjugated enzymes. In direct ELISAs, the anti-rTNFα (recombinant human tumor necrosis factor alpha)-conjugated bacteriophage show approximately 3- to 4-fold gains in signal over that of anti-rTNFα, demonstrating their use as a platform for highly sensitive protein detection.

Residual dipolar couplings arise from small degrees of alignment of molecules in a magnetic field and have proven to provide valuable structural information. Colloidal suspensions of rod-shaped viruses and bacteriophages constitute a frequently employed medium for imparting such alignment onto biomolecules. The stability and behavior of the liquid crystalline phases with respect to solution conditions such as pH, ionic strength, and temperature vary, and characterization should benefit practical applications as well as theoretical understanding. In this Communication we describe the pH dependence of the cholesteric liquid crystalline phase of the filamentous bacteriophage fd and demonstrate that the alignment tensor of the solute protein is modulated by pH. We also report the interesting observation that the relative sign of the residual dipolar coupling changes at low pH values. In addition, we demonstrate that the degree of alignment inversely scales with the lengths of the phage particles for phages with identical mass and charge per unit length.

The gene 5 protein of filamentous bacteriophage fd is a single-stranded DNA-binding protein that binds non-specifically to all single-stranded nucleic acid sequences, but in addition is capable of specific binding to the sequence d(GT5G4CT4C) and the RNA equivalent r(GU5G4CU4C), the latter interaction being important for translational repression. We show that this sequence preference arises from the formation of a tetraplex structure held together by a central block of G-quartets, the structure of which persists in the complex with gene 5 protein. Binding of gene 5 protein to the tetraplex leads to formation of a ∼170 kDa nucleoprotein complex consisting of four oligonucleotide strands and eight gene 5 protein dimers, with a radius of gyration of 45 A and an overall maximum dimension of 120–130 A. A model of the complex is presented that is consistent with the data obtained. It is proposed that the G-quartet may act as a nucleation site for binding gene 5 protein to adjacent single-stranded regions, suggesting a novel mechanism for translational repression.

Gene 3 protein (pIII), a minor coat protein at one end of the filamentous bacteriophage fd, is involved in initiating the infection by the virus of Escherichia coli cells that display an F-pilus. Infection is thought to start with the adsorption of the D2 domain of pIII to the tip of the pilus, retraction of the pilus, and penetration of the E. coli cell membrane mediated by an interaction between the D1 domain of pIII and the Tol protein complex in the membrane. A subgene encoding the pIII-D1D2 di-domain was created, and the subgene was successfully overexpressed in E. coli cells. Domains D1 and D2 were separated after limited proteolysis of a modified pIII-D1D2 (designated pIII-D1D2.trp) into which two tryptic cleavage sites were introduced at appropriate points. The purified pIII-D1D2 di-domain and pIII-D2 domain were able to bind to the F-pilus, competing with the wild-type pIII and delaying infection by the intact filamentous phage. The pIII-D1 domain was unable to bind to the F-pilus by this criterion. This provides conclusive evidence that the pIII-D2 domain is responsible for the adsorption to the tip of the F-pilus and can achieve this in the absence of domain D1, opening the way to identifying the molecular basis of the interaction of pIII-D2 with the pilus.

Structures of protein subunits of supramolecular assemblies can be determined from the orientations of bonds relative to an axis collinear with a stationary magnetic field or an axis about which rotational diffusion occurs, such as the length of a rod-shaped particle. As an example, here we demonstrate that equivalent angular constraints can be measured from solid-state NMR spectra of the filamentous bacteriophage fd whether made from oriented samples that are stationary or unoriented samples undergoing magic angle spinning as long as uniaxial rotational diffusion along the long axis of the virus particles is present. This is a general NMR approach and is applicable to other biological supramolecular assemblies such as membranes.

The genome of the filamentous bacteriophage fd has been engineered so that small peptides can be inserted into the exposed N-terminal segment of pVIII, the major protein of the virus capsid. Most small peptides can be displayed on all 2700 copies of pVIII (a recombinant virion), but larger peptides can be displayed only in virions in which modified and wild-type proteins are intermingled (hybrid virions). Peptides displayed in this way are highly immunogenic and capable of interacting with receptors and other ligands. The physical accessibility of the displayed peptides was tested by examining their susceptibility to digestion with proteinases. Potential cleavage sites in peptides displayed on recombinant or hybrid virions were in general found to be accessible to trypsin and chymotrypsin; and the density of incorporation of peptides in the virion had no effect on the susceptibility to cleavage. However, peptide bonds towards the C-terminal end of an insert, located approximately 47 residues or fewer from the C-terminus of the coat protein, were protected from digestion, presumably because of their proximity to the bulk viral surface. These results have important implications for the design and optimization of peptide display systems using filamentous bacteriophages.

Peptides displayed on the surface of filamentous bacteriophage fd are able to induce humoral as well as cell-mediated immune responses, which makes phage particles an attractive antigen delivery system to design new vaccines. The immune response induced by phage-displayed peptides can be enhanced by targeting phage particles to the professional antigen presenting cells, utilizing a single-chain antibody fragment that binds dendritic cell receptor DEC-205. Here, we review recent advances in the use of filamentous phage fd as a platform for peptide vaccines, with a special focus on the use of phage fd as an antigen delivery platform for peptide vaccines in Alzheimer’s Disease and cancer.

The principal neutralizing determinant of the human immunodeficiency virus type 1 (HIV-1) is an intra-chain disulphide-bridged loop, designated V3, in the third hypervariable region of the surface glycoprotein gp 120. Peptide sequences from the V3 loop of gp120 from HIV-1 strain MN (HIV-1 MN ) were engineered into the N-terminal region of the major coat protein of filamentous bacteriophage fd, leading to their display in multiple copies on the surface of the bacteriophage virion. Peptides displayed in this way were shown to be remarkably effective structural mimics of the natural epitope. They were recognised by human HIV antisera and evoked high titres of antibodies in mice, which cross-reacted with other strains of HIV and were capable of neutralizing the virus. In addition, antibody production could be stimulated by simultaneous inoculation with T-cell epitopes similarly displayed on filamentous bacteriophage. The bacteriophage display system offers a powerful means of studying the immunological recognition of proteins and is a promising vaccine model.

The adsorption protein g3p and another minor coat protein, g6p, are located at one end of the filamentous bacteriophage fd [Grant, R.A., Lin, T.C., Konigsberg, W.E. & Webster, R.E. (1981) J. Biol. Chem. 256, 539-546]. Both proteins, representing the proximal tip, were detached as an entity by a technique that allowed for gentle solubilization. Disrupting the phage particle with the detergent sodium deoxycholate and chloroform dissociates the major coat protein g8p, frees the phage DNA, but leaves g3p and g6p associated with each other. The g3p-g6p complex, which we termed the adsorption complex, and an oligomeric form of g3p with lower molecular mass were isolated and purified by gel-filtration chromatography in the presence of deoxycholate. These different oligomeric structures of g3p showed a different mobility in non-denaturing polyacrylamide-gel electrophoresis. Both forms were also found in non-denaturing polyacrylamide-gel electrophoresis from deoxycholate- and Triton-X-100-solubilized phage without prior chromatographic separation. The two oligomeric forms of g3p are composed of two g3p polypeptide chains in the case of the low-molecular-mass species, and four g3p and four g6p polypeptide chains for the adsorption complex.

Filamentous bacteriophages have been used in numerous applications for the display of antibodies and random peptide libraries. Here we describe the introduction of a 13 amino acid sequence LASIFEAQKIEWR (designated BT, which is biotinylated in vivo by E. coli) into the N termini of four of the five structural proteins of the filamentous bacteriophage fd (Proteins 3, 7, 8 and 9). The in vivo and in vitro biotinylation of the various phages were compared. The production of multifunctional phages and their application as affinity reagents are demonstrated.

The efficacy of a new vaccine-delivery vector, based on the filamentous bacteriophage fd displaying a single-chain antibody fragment known to bind the mouse DC surface molecule DEC-205, is reported. We demonstrate both in vitro and in vivo an enhanced receptor-mediated uptake of phage particles expressing the anti-DEC-205 fragment by DCs. We also report that DCs targeted by fd virions in the absence of other stimuli produce IFN-α and IL-6, and acquire a mature phenotype. Moreover, DC-targeting with fd particles double-displaying the anti-DEC-205 fragment on the pIII protein and the OVA 257-264 antigenic determinant on the pVIII protein induced potent inhibition of the growth of the B16-OVA tumor in vivo. This protection was much stronger than other immunization strategies and similar to that induced by adoptively transferred DCs. Since targeting DEC-205 in the absence of DC activation/maturation agents has previously been described to result in tolerance, the ability of fd bacteriophages to induce a strong tumor-specific immune response by targeting DCs through DEC-205 is unexpected, and further validates the potential employment of this safe, versatile and inexpensive delivery system for vaccine formulation. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

We describe here two systems for encoding foreign amino acid sequences in the exposed N-terminal segment of the major coat protein of bacteriophage fd. Small peptides can be encoded directly; larger peptides are encoded in hybrid bacteriophage particles, in which the capsid is formed from a mixture of wild-type and modified coat proteins. In both cases, the peptides are present in multiple copies per phage particle. Peptides that represent the circumsporozoite protein, the major surface antigen of the sporozoites of the malaria parasite, Plasmodium falciparum, were inserted in this way and found to be highly immunogenic. These systems should prove to be valuable in displaying specific or random peptides as antigens, and could lead to cheap and effective vaccines. They will also allow rapid screening of peptides as potential agents of other biological effects, with important applications in biomolecular design.

We have modified the genome of the filamentous bacteriophage fd and also constructed a number of new vectors for the purpose of displaying peptides on the surface of the virion. These vectors facilitate the directional cloning of DNA encoding a peptide of interest at or near the N terminus of the major coat protein, the product of the bacteriophage gene VIII, and the construction of hybrid capsids in which the modified coat protein is interspersed with wild-type coat protein subunits.

Virions of filamentous bacteriophage fd are capable of displaying multiple copies of peptide epitopes and generating powerful immune responses to them. To investigate the antigen processing mechanisms in human B cell lines used as antigen presenting cells, the major coat protein (pVIII) in intact virions was fluorescently labeled, and its localization in various intracellular compartments was followed using confocal microscopy. We show that the virions were taken up and processed to yield peptides that reach both the major histocompatibility complex (MHC) class II compartment and the endoplasmic reticulum. Moreover, when exposed to bacteriophages displaying a cytotoxic T lymphocyte (CTL) epitope from the reverse transcriptase of human immunodeficiency virus type-1 (HIV-1), B cells were lysed by specific cytotoxic lymphocytes. This confirms that filamentous bacteriophage virions are capable of being taken up and processed efficiently by MHC class I and class II pathways, even in nonprofessional antigen presenting cells. These remarkable features explain, at least in part, the unexpected ability of virions displaying foreign T-cell epitopes to prime strong T-helper-dependent CTL responses. These findings have important implications for the development of peptide-based vaccines, using filamentous bacteriophage virions as scaffolds.

Although much effort has been expended on evaluating recombinant proteins and synthetic peptides as immunogens, they have generally proved incapable of inducing an efficient cytotoxic T-cell (CTL) response. Filamentous bacteriophage fd can display multiple copies of foreign peptides in the N-terminal region of its major coat protein pVIII, 2,700 copies of which make up the virus capsid. Here we show that fd virions displaying peptide RT2 (ILKEPVHGV), corresponding to residues 309–317 of the reverse transcriptase (RTase) of HIV-1, are able to prime a CTL response specific for this HIV-1 epitope in human cell lines. Successful priming also requires a T-helper epitope, pep23 (KDSWTVNDIQKLVGK), corresponding to residues 249–263 of HIV-1 RTase. Supplying this by displaying it on either the same or a separate bacteriophage virion led to activation of antigen-specific CD4+ T cells. Likewise, HLA-A2 transgenic mice immunized with bacteriophage virions displaying peptide RT2 were shown to mount an effective, specific anti-HIV-RT2 CTL response. This unexpected ability to elicit a designated cytolytic T-cell response, in addition to a B-cell response, has important implications for access to the class I major histocompatibility complex (MHC) loading compartment and the development of recombinant vaccines.

At pH6 added filamentous bacteriophage fd is compatible with many of the detergents used to solubilize membrane proteins for solution NMR studies of membrane proteins and, therefore, serves as an alignment media. In combination with strained polyacrylamide gel alignment, Dipolar Waves can be used to directly assess the secondary structure and a lambda-map extracts the order tensors for de novo structure calculation of membrane proteins without distance restraints.

Solid-state NMR spectroscopy was used to analyze the conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd. Both one and two-dimensional solid-state NMR spectra of magnetically aligned samples of fd bacteriophage reveal that an increase in temperature and a single site substitution (Tyr21 to Met, Y21M) reduce the conformational heterogeneity observed throughout wild-type pVIII. The NMR results are consistent with previous studies indicating that conformational flexibility in the hinge-bend segment that links the amphipathic and hydrophobic helices in the membrane-bound form of the protein plays an essential role during phage assembly, which involves a major change in the tertiary, but not secondary, structure of the coat protein.

The filamentous bacteriophage fd is a member of the Ff class of Inovirus, which includes phages f1 and M13. Ultraviolet resonance Raman (UVRR) spectra of fd have been obtained using excitation wavelengths of 257, 244, 238, and 229 nm. Excitation at 257 nm selectively enhances Raman markers of the packaged single-stranded (ss) DNA genome, while excitation at the shorter wavelengths favors the detection of Raman signals from coat protein aromatics, particularly tryptophan (W26) and tyrosine residues (Y21 and Y24) of the viral coat subunit (pVIII). The principal findings are the following: (1) Distinctive markers of dA, dC, dG, and dT residues of the packaged genome are identified in UVRR spectra of fd excited at 257 and 244 nm, despite the low DNA mass composition (12%) of the virion. (2) Raman bands of the bases of packaged ssDNA show extraordinary resonance Raman hypochromism. Raman intensity losses as large as 80% of the parent DNA nucleotide intensities are observed. This is interpreted as evidence of extensive short-range interactions involving bases of the packaged genome. (3) Conversely, Raman bands of tryptophan and tyrosine residues of the coat protein generally exhibit strong hyperchromism. Typically, Raman markers of the aromatic amino acids are about 3-fold more intense in the UVRR spectrum of fd than in spectra of the free amino acids. The very high Raman cross sections for residues Y21, Y24, and W26 are indicative of unusual hydrophobic environments in the viral assembly. (4) UVRR band shifts that accompany the transfer of fd from H2O to D2O solution indicate that bases of the packaged ssDNA are readily exchanged by the solvent. Similarly, the indole N1H group of W26 is accessible to solvent, as shown by N1H --> N1D exchange in D2O solution. (5) The UVRR markers of the packaged fd genome confirm the conclusion reached previously from off-resonance Raman studies that fd DNA nucleosides favor the C3'-endo/anti conformation, rather than the C2'-endo/anti conformation that is characteristic of the lowest energy structure of DNA. We conclude that nucleoside conformations of the packaged fd genome are influenced by the specific organization of ssDNA and coat protein subunits in the native virion assembly.

We have developed a new system for producing hybrid virions of filamentous bacteriophage fd simultaneously displaying two different peptides by infecting cells harbouring a plasmid containing a modified gene VIII with an engineered bacteriophage carrying a second and different copy of a modified gene VIII. The simultaneous display of different peptides has many potential applications in exploring the immune response and studying protein-protein interaction.

Publisher Summary This chapter describes spectroscopic methods and preliminary results from structural studies of the epitope (NANP) 3 displayed close to the N-terminus of the major coat protein in hybrid phage. The main antigenic determinant of the circumsporozoite (CS) protein contains several repetitions of the tetrapeptide NANP, and proteins containing several units of this epitope are used to raise a high titer of antibodies. When the epitope is displayed on the surface of the filamentous bacteriophage fd, an even higher immunogenic response is observed. The chapter describes the use of multidimensional solution nuclear magnetic resonance (NMR) to study the three-dimensional structure of the epitope in isolated coat proteins solubilized in micelles. The solid-state NMR is applied to intact phage particles. The spontaneous orientation of the virion in the presence of strong magnetic fields enables the epitope to be characterized on the surface of the bacteriophage. The results obtained by solution and solid-state NMR spectroscopy show that the epitope is folded in the context of the phage coat protein and its structure in micelles, and in the virion, particles can be determined by NMR spectroscopy.

We report the display of human ciliary neurotrophic factor (hCNTF), a survival factor for neuronal cells belonging to the alpha-helical cytokine superfamily, on the surface of the filamentous bacteriophage fd. The hCNTF cDNA was fused to a DNA sequence encoding the C-terminal domain of pIII, a minor coat protein exposed at one end of fd. Gene fusions were cloned into a plasmid containing the ColE1 plasmid and fd origins of replication, and were packaged into phagemid particles upon superinfection with M13KO7 helper phage. The resulting fusion phage bound specifically to anti-CNTF antibodies and to the recombinant soluble CNTF alpha-receptor. Moreover, phage-displayed hCNTF was found to possess biological activity at concentrations comparable to those of the soluble cytokine. These results demonstrate that CNTF can be displayed on phage in a correctly folded and functionally active form. Binding of fusion phage to immobilized CNTF alpha-receptor and subsequent elution at low pH resulted in affinity purification of CNTF-displaying virions. Utilization of this technology should enable the selection of high-affinity variants from libraries of CNTF mutants displayed on phage.

The virion of the filamentous bacteriophage fd (fl and M13 are very similar strains) is a flexible rod about 1 μm long and 6 nm in diameter, comprising a tubular sheath of approx. 2700 copies of the major coat protein subunit surrounding a DNA core. The DNA is a single-stranded circular molecule of 6408 nucleotides embodying 10 genes; these genes are tightly packed and, in some instances, overlapping, apart from a short region (the intergenic space) which encodes no protein component but which contains a double-stranded helical hairpin loop responsible for initiating assembly of the virion. There are a few copies (about 5) of each of two minor coat proteins at the two ends of the virion: gVIIp and gIXp at the end where assembly is initiated, and glllp and gVIp at the end where the process is terminated [for general reviews, see Model & Russe1,1988; Russel, 1991)].

A major challenge in vaccine design is to identify antigen presentation and delivery systems capable of rapidly stimulating both the humoral and cellular components of the immune system to elicit a strong and sustained immunity against different viral isolates. Approaches to achieve this end involve live attenuated and inactivated virions, viral vectors, DNA, and protein subunits. This review reports the state of current antigen delivery, and focuses on two innovative systems recently established at our labs. These systems are the filamentous bacteriophage fd and an icosahedral scaffold formed by the acyltransferase component (E2 protein) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.

Filamentous bacteriophages are particularly efficient for the expression and display of combinatorial random peptides. Two phage proteins are often employed for peptide display: the infectivity protein, PIII, and the major coat protein, PVIII. The use of PVIII typically requires the expression of two pVIII genes: the wild-type and the recombinant pVIII gene, to generate mosaic phages. ‘Type 88’ vectors contain two pVIII genes in one phage genome. In this study a novel ‘type 88’ expression vector has been rationally designed and constructed. Two factors were taken into account: the insertion site and the genetic stability of the second pVIII gene. It was found that selective deletion of recombinant genes was encountered when inserts were cloned into either of the two non-coding regions of the phage genome. The deletions were independent of recA yet required a functional F-episome. Transcription was also found to be a positive factor for deletion. Taking the above into account led to the generation of a novel vector, designated fth1, which can be used to express recombinant peptides as pVIII chimeric proteins in mosaic bacteriophages. The fth1 vector is not only genetically stable but also of high copy number and produces high titers of recombinant phages.

The filamentous bacteriophage fd and its major coat protein are being studied by nuclear magnetic resonance (NMR) spectroscopy. 31P NMR shows that the chemical shielding tensor of the DNA phosphates of fd in solution is only slightly reduced in magnitude by motional averaging, indicating that DNA-protein interactions substantially immobilize the DNA packaged in the virus. There is no evidence of chemical interactions between the DNA backbone and the coat protein, since experiments on solid virus show the 31P resonances to have the same principle elements of its chemical shielding tensor as DNA. 1H and 13C NMR spectra of fd virus in solution indicate that the coat proteins are held rigidly in the structure except for some aliphatic side chains that undergo relatively rapid rotations. The presence of limited mobility in the viral coat proteins is substantiated by finding large quadrupole splittings in 2H NMR of deuterium labeled virions. The structure of the coat protein in a lipid environment differs significantly from that found for the assembled virus. Data from 1H and 13C NMR chemical shifts, amide proton exchange rates, and 13C relaxation measurements show that the coat protein in sodium dodecyl sulfate micelles has a native folded structure that varies from that of a typical globular protein or the coat protein in the virus by having a partially flexible backbone and some rapidly rotating aromatic rings.

A restriction fragment carrying the major coat protein gene (gene VIII) was excised from the DNA of the class I filamentous bacteriophage fd, which infects Escherichia coli. This fragment was cloned into the expression plasmid pKK223-3, where it came under the control of the tac promoter, generating plasmid pKf8P. Bacteriophage fd gene VIII was similarly cloned into the plasmid pEMBL9+, enabling it to be subjected to site-directed mutagenesis. By this means the positively charged lysine residue at position 48, one of four positively charged residues near the C terminus of the protein, was turned into a negatively charged glutamic acid residue. The mutated fd gene VIII was cloned back from the pEMBL plasmid into the expression plasmid pKK223-3, creating plasmid pKE48. In the presence of the inducer isopropyl-β- d -thiogalactoside, the wild-type and mutated coat protein genes were strongly expressed in E. coli TG1 cells transformed with plasmids pKf8P and pKE48, respectively, and the product procoat proteins underwent processing and insertion into the E. coli cell inner membrane. A net positive charge of only 2 on the side-chains in the C-terminal region is evidently sufficient for this initial stage of the virus assembly process. However, the mutated coat protein could not encapsidate the DNA of bacteriophage R252, an fd bacteriophage carrying an amber mutation in its own gene VIII, when tested on non-suppressor strains of E. coli. On the other hand, elongated hybrid bacteriophage particles could be generated whose capsids contained mixtures of wild-type (K48) and mutant (E48) subunits. This suggests that the defect in assembly may occur at the initiation rather than the elongation step(s) in virus assembly. Other mutations of lysine-48 that removed or reversed the positive charge at this position in the C-terminal region of the coat protein were also found to lead to the production of commensurately longer bacteriophage particles. Taken together, these results indicate direct electrostatic interaction between the DNA and the coat protein in the capsid and support a model of non-specific binding between DNA and coat protein subunits with a stoicheiometry that can be varied during assembly.

This study reports on various parameters which affect the binding stoichiometry for complexes of bacteriophage T4 gene 32 protein (P32) and single stranded polynucleotides (determined by UV absorbance and fluorescence quenching) and presents results of a quantitative electron spin resonance assay to determine physiologically effective binding affinity differences of nucleic acid binding proteins. The assay employs macromolecular spin probes (spin-labeled nucleic acids) which are used to determine the fraction of saturation in competition experiments with unlabeled nucleic acids. It was found that the fraction of complexed spin-labeled polynucleotides can be directly monitored by ESR with a two-component analysis approach when ligands such as poly(L-lysine), gene 5 protein (P5) of filamentous bacteriophage fd, and gene 32 protein (P32) of bacteriophage T4 are used. The ESR data unequivocally show that: 1) the binding stoichiometry for poly(L-lysine), P5 and P32 is nucleotide/lysine, 4 nucleotides/P5 monomer, and 10 nucleotides/P32 monomer, respectively; and 2) under physiologically relevant buffer conditions the relative affinity of P32 in the cooperative binding mode for polythymidylic acid is about 4 times greater than for polydeoxyinosinic acid and about 12 times greater than for polyinosinic acid, and the relative affinity of P32 for polydeoxyinosinic acid is about 3 times greater than for polyinosinic acid.

Equilibrium properties and kinetics of the disassembly of coliphage fd induced by guanidine hydrochloride and sodium dodecyl sulphate were investigated by means of uv absorption, circular dichroism, and sedimentation. In both cases the phase disassembled in an all-or-none fashion, and the concentrations of the midtransition were 6.4 M and 0.03%, respectively, at 25°. The dissociation of the minor coat protein and the loss of infectivity were ascertained to take place in the disassembly. The DNA and major coat protein molecules released in guanidine hydrochloride solutions were both in nearly random coil conformations. In the sodium dodecyl sulphate disassembly the dissociated DNA molecule exhibited the same uv absorption as in the intact phage, and the dissociated major coat protein molecule remained in an approximately 50% α-helical conformation.

The three-dimensional structure of part of the coat protein in the filamentous bacteriophage fd is described by nuclear magnetic resonance (n.m.r.). Residues 40 to 45 are in a somewhat distorted alpha-helix. This n.m.r. approach for determining protein structure relies on the spectral manifestations of chemical shift and heteronuclear dipolar couplings in a symmetrical assembly of protein subunits oriented parallel to the applied magnetic field. The angles between individual peptide linkages and the filament axis of the virion constitute the basic source of structural information. These angles are directly related to x, y, z co-ordinates for describing the protein structure.

DNA cloning vectors were developed which utilize the replication origin (ori) of bacteriophage fd for their propagation. These vectors depend on the expression of viral gene 2 that was inserted into phage lambda, which in turn was integrated into the host genome. The constitutive expression of gene 2 in the host cells is sufficient for the propagation of at least 100 pfd plasmids per cell. In addition to the fd ori, the pfd vectors carry various antibiotic-resistance genes and unique restriction sites. Some of these vectors have no homologies to commonly used pBR plasmids or to lambda DNA. The nucleotide sequence of the vectors can be deduced from published sequences. Large DNA inserts can be stably propagated in pfd vectors; these are more stable than similar DNA fragments cloned in intact genomes of filamentous bacteriophage. Inclusion of phage sequences required for efficient phage packaging and infection with a helper phage resulted in formation of phage particles containing single-stranded plasmid genomes. Growth at 42 degrees C without selective pressure results in loss of pfd plasmids.

Bacteriophage fd is a class I filamentous virus (others are M13 and f1) that comprises a circular, single-stranded DNA molecule enclosed in a cylindrical protein sheath to form a flexible particle approximately 890 nm long and 7 nm in diameter. The viral DNA contains 6,408 nucleotides incorporating 10 genes, and the protein sheath is composed of about 2,700 major coat protein subunits in a shingled helical array, the symmetry of which is defined by a fivefold rotational axis combined with a twofold screw axis of pitch 3.2 nm. The DNA extends throughout the length of the particle but is not base-paired and has a symmetry different from that of the protein helix. How the DNA is packed remains unclear but the number (2.4) of nucleotides packaged per major coat protein subunit is certainly not integral, in contrast with, say, the packaging of RNA in tobacco mosaic virus. The coat protein subunit is 50 amino-acid residues in length and, in the virus particle, adopts a largely alpha-helical conformation, with the long axis of the helix aligned close to the long axis of the filament. This protein is arranged with its negatively charged N-terminal region on the outside of the filament and its positively charged C-terminal region on the inside abutting the DNA. We report here that positive charge on one of the four lysine side chains in the latter region has a direct effect on DNA packaging, because when this charge is absent, elongated particles are produced with lengths that can be correlated with the residual positive charge in the C-terminal region of the coat protein subunit.

Natural abundance 13 C and high field 1 H NMR spectroscopy are used to characterize the major coat protein of the filamentous bacteriophage fd in sodium dodecyl sulfate micelles. Chemical shift dispersion of protein resonances, slow and differential exchange rates of amide protons, and relaxation parameters of the alpha carbons of the protein indicate that the detergent solubilized coat protein has a stable native conformation. The structure of the coat protein in micelles differs from that found for typical globular proteins in solution in that parts of the peptide backbone exhibit rapid segmental motion.

The molecular structure of the single-stranded fd DNA inside its filamentous virion has been stabilized by the photochemical reaction with a psoralen derivative and examined in the electron microscope. The results support the notion that the 6389 nucleotide-long DNA molecule is folded back on itself inside the 1 μm-long protein coat. At one end of the virion, there exists a DNA hairpin region 200±50 base-pairs long. This “end hairpin” is mapped on the fd genome to the site of the replication origin. The most stable in vitro hairpin of fd DNA has been mapped previously to this same site. This unique duplex region of fd DNA may play an important role in the formation of specific protein-DNA complexes which are crucial to stages of the fd life cycle: the adsorption of the phage to the bacteria, the initiation of replication of the single-stranded DNA, and the assembly of newly synthesized DNA strands into the filamentous virions.

31P NMR experiments on the filamentous bacteriophage fd are used to characterize the viral DNA. Because fd is a 16.4 X 10(6) dalton rod-shaped particle, methods of high-resolution solid-state NMR including cross polarization, proton decoupling, and magic angle sample spinning are utilized. The 31P chemical shielding tensor of solid fd is indistinguishable from that of single-stranded or double-stranded DNA in the absence of proteins; therefore the 31P chemical shift does not show evidence of structural changes in DNA upon incorporation into the virus. fd in solution has a very broad 31P resonance line width. The line width is due to static chemical shift anisotropy that is not motionally averaged, as shown by the generation of sidebands with magic angle sample spinning and a linear dependence of line width on magnetic field strength. These results indicate that DNA packaged inside fd is immobilized by the coat proteins.

The lattice parameters determined from X-ray diffraction patterns of fd show that the phage particles crystallize with a minimum interparticle distance of 56 A, and that the phage structure repeats regularly every 32·2 A along its length. The distribution of intensity on the diffraction pattern shows features typical of diffraction from a supercoiled α-helix. This suggests that a large fraction of the phage protein is in the form of α-helix, with the α-helix axis at a small angle to the phage axis. Electron micrographs of phosphotungstic acid-stained phage show a dark central line which can be interpreted in terms of a two-strand structure. A model for fd is proposed, analogous to a circle of string pulled taut from opposite sides of its circumference, where the string represents a single-stranded DNA ring enclosed in a tube of α-helices.

Physically homogeneous preparations of DNA can be isolated from fd. This DNA is resistant to hydrolysis by exonuclease I. The kinetics of pancreatic DNase action on the DNA as followed by sedimentation and viscosity in suitable solvents show that a single hit (biological inactivation) leads to an expansion of the DNA structure but no change in molecular weight. The native DNA molecule is resistant to a variety of agents which destroy secondary structure. These results are taken to indicate that the DNA has a stable ring topology.

Two non-pathogenic scaffolds (represented by the filamentous bacteriophage fd and the dihydrolipoyl acetyltransferase E2 protein of the Bacillus stearothermophilus pyruvate dehydrogenase (PDH) complex) able to deliver human immunodeficiency virus (HIV)-1 antigenic determinants, were designed in our laboratories and investigated in controlled assay conditions. Based on a modification of the phage display technology, we developed an innovative concept for a safe and inexpensive vaccine in which conserved antigenic determinants of HIV-1 reverse transcriptase (RTase) were inserted into the N-terminal region of the major pVIII coat protein of bacteriophage df virions. Analogously, we developed another antigen delivery system based on the E2 component from the PDH complex and capable of displaying large intact proteins on the surface of an icosahedral lattice. Our data show that both of these systems can deliver B and T epitopes to their respective presentation compartments in target cells and trigger a humoral response as well as a potent helper and cytolytic response in vitro and in vivo.