Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry.

Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion1-4. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies5 and peptides6 or that address late steps of the viral replication cycle7.

Head-to-Head Comparison of Soluble vs. Qß VLP Circumsporozoite Protein Vaccines Reveals Selective Enhancement of NANP Repeat Responses.

Circumsporozoite protein (CSP) of Plasmodium falciparum is a promising malaria vaccine target. RTS,S, the most advanced malaria vaccine candidate consists of the central NANP repeat and carboxy-terminal region of CSP displayed on a hepatitis B virus-like particle (VLP). To build upon the success of RTS,S, we produced a near full-length Plasmodium falciparum CSP that also includes the conserved amino-terminal region of CSP. We recently showed that this soluble CSP, combined with a synthetic Toll-like-receptor-4 (TLR4) agonist in stable oil-in-water emulsion (GLA/SE), induces a potent and protective immune response in mice against transgenic parasite challenge. Here we have investigated whether the immunogenicity of soluble CSP could be further augmented by presentation on a VLP. Bacteriophage Qß VLPs can be readily produced in E.coli, they have a diameter of 25 nm and contain packaged E. coli RNA which serves as a built in adjuvant through the activation of TLR7/8. CSP was chemically conjugated to Qß and the CSP-Qß vaccine immunogenicity and efficacy were compared to adjuvanted soluble CSP in the C57Bl/6 mouse model. When formulated with adjuvants lacking a TLR4 agonist (Alum, SE and Montanide) the Qß-CSP induced higher anti-NANP repeat titers, higher levels of cytophilic IgG2b/c antibodies and a trend towards higher protection against transgenic parasite challenge as compared to soluble CSP formulated in the same adjuvant. The VLP and soluble CSP immunogenicity difference was most pronounced at low antigen dose, and within the CSP molecule, the titers against the NANP repeats were preferentially enhanced by Qß presentation. While a TLR4 agonist enhanced the immunogenicity of soluble CSP to levels comparable to the VLP vaccine, the TLR4 agonist did not further improve the immunogenicity of the Qß-CSP vaccine. The data presented here pave the way for further improvement in the Qß conjugation chemistry and evaluation of both the Qß-CSP and soluble CSP vaccines in the non-human primate model.

Inactivation of F-specific bacteriophages during flocculation with polyaluminum chloride - a mechanistic study.

Bacteriophages are often used as surrogates for enteric viruses in spiking experiments to determine the efficiencies of virus removal of certain water treatment measures, like e.g. flocculation or filtration steps. Such spiking experiments with bacteriophages are indispensable if the natural virus concentrations in the raw water of water treatment plants are too low to allow the determination of elimination levels over several orders of magnitude. In order to obtain reliable results from such spiking tests, it is essential that bacteriophages behave comparable to viruses and remain stable during the experiments. To test this, the influence of flocculation parameters on the bacteriophages MS2, Qß and phiX174 was examined. Notably, the F-specific phages MS2 and Qß were found to be inactivated in flocculation processes with polyaluminum chloride (PACl). In contrast, other aluminum coagulants like AlCl3 or Al2(SO4)3 did not show a comparable effect on MS2 in this study. In experiments testing the influence of different PACl species on MS2 and Qß inactivation during flocculation, it could be shown that cationic dissolved PACl species (Al13) interacted with the MS2 surface and hereby reduced the surviving phage fraction to c/c0 values below 1*10(-4) even at very low PACl concentrations of 7 µmol Al/L. Other inactivation mechanisms like the irreversible adsorption of phages to the floc structure or the damage of phage surfaces due to entrapment into the floc during coagulation and floc formation do not seem to contribute to the low surviving fraction found for both F-specific bacteriophages. Furthermore, no influence of phage agglomeration or pH drops during the flocculation process on phage inactivation could be observed. The somatic coliphage phiX174 in contrast did not show sensitivity to chemical stress and in accordance only slight interaction between Al13 and the phage surface was observed. Consequently, F-specific phages like MS2 should not be used as surrogate for viruses in flocculation experiments with PACl to determine the removal rates of viruses, as the results are influenced by a strong inactivation of the bacteriophages due to the experimental conditions.

Effects of ribosomes on the kinetics of Qß replication.

Bacteriophage Qß utilizes some host cell translation factors during replication. Previously, we constructed a kinetic model that explains replication of long RNA molecules by Qß replicase. Here, we expanded the previous kinetic model to include the effects of ribosome concentration on RNA replication. The expanded model quantitatively explained single- and double-strand formation kinetics during replication with various ribosome concentrations for two artificial long RNAs. This expanded model and the knowledge obtained in this study provide useful frameworks to understand the precise replication mechanism of Qß replicase with ribosomes and to design amplifiable RNA genomes in translation-coupling systems.

Involvement of type I and type II mechanisms on the photoinactivation of non-enveloped DNA and RNA bacteriophages.

Microbial photodynamic inactivation (PDI), involving the use of a photosensitizer (PS), light and molecular oxygen, with the subsequent production of reactive oxygen species (ROS), has been considered a promising and effective technology for viral inactivation. Although singlet oxygen is generally accepted as the main damaging species in PDI, ROS like free radicals may also be involved in the process, inducing damages to proteins, lipids, nucleic acids and other molecular structures. In this study, the relative importance of each mechanism (type I and type II) on the photoinactivation of non-enveloped DNA (T4-like phage) and RNA (Qß phage) viruses was evaluated. For this purpose, two cationic porphyrins (Tri-Py(+)-Me-PF and Tetra-Py(+)-Me) and four different ROS scavengers were used. The scavenging effect of sodium azide and L-histidine (singlet oxygen quenchers) and of D-mannitol and L-cysteine (free radical scavengers) was assessed by exposure of both phages (T4-like and Qß) to each cationic porphyrin (5.0µM for T4-like phage and 0.5µM for Qß phage) and white light (40Wm(-2)) in the presence of different concentrations of the scavengers (5, 10, 50 and 100mM). Sodium azide and L-histidine gave the best protection, reducing the phototoxic effect of Tri-Py(+)-Me-PF on T4-like phage respectively by 80% and 72% and in the presence of Tetra-Py(+)-Me by 90% and 78%. Free radical scavengers D-mannitol and L-cysteine did not significantly reduce the rate of T4-like phage photoinactivation (around 20% protection, for both PS). The sodium azide protection on Qß phage photoinactivation, in the presence of Tri-Py(+)-Me-PF, was lower (39%) when compared with T4-like phage. D-mannitol did not exert on Qß phage any protective effect after 90min of irradiation. The effect of the simultaneous presence of singlet oxygen and free radicals scavengers at 100mM confirmed that singlet oxygen (type II mechanism) is clearly the main ROS involved in T4-like and Qß phages photoinactivation by these two cationic PS. As RNA-type phages are more easily photoinactivated when compared with DNA-type ones, the protection conferred by the scavengers during the PDI process is lower and this should be taken into account when the main mechanism involved in PDI of different viruses is to be studied.

Glycan-targeted virus-like nanoparticles for photodynamic therapy.

Virus-like particles (VLPs) have proven to be versatile platforms for chemical and genetic functionalization for a variety of purposes in biomedicine, catalysis, and materials science. We describe here the simultaneous modification of the bacteriophage Qß VLP with a metalloporphyrin derivative for photodynamic therapy and a glycan ligand for specific targeting of cells bearing the CD22 receptor. This application benefits from the presence of the targeting function and the delivery of a high local concentration of singlet oxygen-generating payload.

Cell targeting with hybrid Qß virus-like particles displaying epidermal growth factor.

Structurally uniform protein nanoparticles derived from the self-assembly of viral capsid proteins are attractive platforms for the multivalent display of cell-targeting motifs for use in nanomedicine. Virus-based nanoparticles are of particular interest because the scaffold can be manipulated both genetically and chemically to simultaneously display targeting groups and carry a functional payload. Here, we displayed the human epidermal growth factor (EGF) on the exterior surface of bacteriophage Qß as a C-terminal genetic fusion to the Qß capsid protein. The co-assembly of wild-type Qß and EGF-modified subunits resulted in structurally homogeneous nanoparticles displaying between 5 and 12 copies of EGF on their exterior surface. The particles were found to be amenable to bioconjugation by standard methods as well as the high-fidelity copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC). Such chemical derivatization did not impair the ability of the particles to specifically interact with the EGF receptor. Additionally, the particle-displayed EGF remained biologically active promoting autophosphorylation of the EGF receptor and apoptosis of A431 cells. These results suggest that hybrid Qß-EGF nanoparticles could be useful vehicles for targeted delivery of imaging and/or therapeutic agents.

Evolution and protein packaging of small-molecule RNA aptamers.

A high-affinity RNA aptamer (K(d) = 50 nM) was efficiently identified by SELEX against a heteroaryldihydropyrimidine structure, chosen as a representative drug-like molecule with no cross reactivity with mammalian or bacterial cells. This aptamer, its weaker-binding variants, and a known aptamer against theophylline were each embedded in a longer RNA sequence that was encapsidated inside a virus-like particle by a convenient expression technique. These nucleoprotein particles were shown by backscattering interferometry to bind to the small-molecule ligands with affinities similar to those of the free (nonencapsidated) aptamers. The system therefore comprises a general approach to the production and sequestration of functional RNA molecules, characterized by a convenient label-free analytical technique.

Engineered virus-like nanoparticles reverse heparin anticoagulation more consistently than protamine in plasma from heparin-treated patients.

Heparin is widely used for anticoagulation, often requiring the subsequent administration of a reversal agent. The only approved reversal agent for heparin is protamine sulfate, which induces well described adverse reactions in patients. Previously we reported a novel class of heparin antagonists based on the bacteriophage Qß platform, displaying polyvalent cationic motifs which bind with high affinity to heparin. Here we report heparin reversal by the most effective of these virus-like particles (VLP) in samples from patients who were administered heparin during cardiac procedures or therapeutically for treatment of various thrombotic conditions. The VLP consistently reversed heparin in these samples, including those from patients that received high doses of heparin, with greater efficiency than a negative control VLP and with significantly less variability than protamine sulfate. These results provide the first step towards validation of heparin antagonist VLPs as viable alternatives to protamine.

Multivalent display and receptor-mediated endocytosis of transferrin on virus-like particles.

The structurally regular and stable self-assembled capsids derived from viruses can be used as scaffolds for the display of multiple copies of cell- and tissue-targeting molecules and therapeutic agents in a convenient and well-defined manner. The human iron-transfer protein transferrin, a high affinity ligand for receptors upregulated in a variety of cancers, has been arrayed on the exterior surface of the protein capsid of bacteriophage Qbeta. Selective oxidation of the sialic acid residues on the glycan chains of transferrin was followed by introduction of a terminal alkyne functionality through an oxime linkage. Attachment of the protein to azide-functionalized Qbeta capsid particles in an orientation allowing access to the receptor binding site was accomplished by the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. Transferrin conjugation to Qbeta particles allowed specific recognition by transferrin receptors and cellular internalization through clathrin-mediated endocytosis, as determined by fluorescence microscopy on cells expressing GFP-labeled clathrin light chains. By testing Qbeta particles bearing different numbers of transferrin molecules, it was demonstrated that cellular uptake was proportional to ligand density, but that internalization was inhibited by equivalent concentrations of free transferrin. These results suggest that cell targeting with transferrin can be improved by local concentration (avidity) effects.

Assembly of hybrid bacteriophage Qbeta virus-like particles.

Bacteriophage Qbeta coat protein forms uniform virus-like particles when expressed recombinantly in a variety of organisms. We have inserted the IgG-binding Z domain at the carboxy terminus of the coat protein and coexpressed this chimeric subunit with native coat protein to create hybrid, IgG-binding virus-like particles. Extracellular osmolytes were found to have an effect on the efficiency of incorporation of fusion proteins into VLPs in Escherichia coli when a carbenicillin, but not a kanamycin, selection marker was used. The addition of sucrose to the growth medium decreased the incorporation efficiency; the osmoprotectant glycine betaine eliminated this effect. The decrease in efficiency was not observed when carbenicillin was omitted from the final expression culture. The addition of sodium chloride instead of sucrose gave rise to particles with a larger number of fusion proteins than the standard conditions. These results illustrate that cellular conditions should be taken into account even in apparently simple systems when natural or engineered protein nanoparticles are made.

Immobilization of bacteriophage Qbeta on metal-derivatized surfaces via polyvalent display of hexahistidine tags.

Metal-binding peptide motifs are widely used for protein purification, catalysis, and metal-mediated self assembly in the construction of novel materials and multivalent light harvesting complexes. Herein we describe hexahistidine sequences incorporated into the virus-like particle derived from bacteriophage Qbeta via co-expression of the wild-type (WT) and hexahistidine-modified coat proteins in Escherichia coli. The resulting polyvalent display of approximately 37 hexahistidine moieties per virion gave rise to altered properties of Zeta potential and hydrodynamic radius, but no observed change in stability compared to WT. While the resulting display density did not permit hexahistidine chains to cooperate in the coordination of heme, the multiple tags did impart a strong affinity for immobilized metal ions. A dissociation constant for binding to Ni-NTA of approximately 10nM was measured by SPR under non-competitive, physiological conditions. Affinity chromatography over immobilized metal columns was used to purify the particles from both crude cell lysates and after chemical derivatization. These results illustrate the potential of metal-NTA surfaces for the self-assembled presentation of multi-functionalized particles to interrogate systems ranging from small molecule binding to whole cell interactions.

Unnatural amino acid incorporation into virus-like particles.

Virus-like particles composed of hepatitis B virus (HBV) or bacteriophage Qbeta capsid proteins have been labeled with azide- or alkyne-containing unnatural amino acids by expression in a methionine auxotrophic strain of E. coli. The substitution does not affect the ability of the particles to self-assemble into icosahedral structures indistinguishable from native forms. The azide and alkyne groups were addressed by Cu(I)-catalyzed [3 + 2] cycloaddition: HBV particles were decomposed by the formation of more than 120 triazole linkages per capsid in a location-dependent manner, whereas Qbeta suffered no such instability. The marriage of these well-known techniques of sense-codon reassignment and bioorthogonal chemical coupling provides the capability to construct polyvalent particles displaying a wide variety of functional groups with near-perfect control of spacing.

Assembly of bacteriophage Qbeta virus-like particles in yeast Saccharomyces cerevisiae and Pichia pastoris.

Recombinant bacteriophage Qbeta coat protein (CP), which has been proposed as a promising carrier of foreign epitopes via their incorporation either by gene engineering techniques or by chemical coupling, efficiently self-assembles into virus-like particles (VLPs) when expressed in Escherichia coli. Here, we demonstrate expression and self-assembly of Qbeta CP in yeast Saccharomyces cerevisiae and Pichia pastoris. Production reached 3-4 mg/1g of wet cells for S. cerevisiae and 4-6 mg for P. pastoris, which was about 15-20% and 20-30% of the E. coli expression level, respectively. Qbeta VLPs were easily purified by size-exclusion chromatography in both cases and contained nucleic acid, shown by native agarose gel electrophoresis. The obtained particles were highly immunogenic in mice and the resulting sera recognized both E. coli- and yeast-derived Qbeta VLPs equally well.

The influence of ionic strength on the interaction of viruses with charged surfaces under environmental conditions.

The influence of ionic strength on the electrostatic interaction of viruses with environmentally relevant surfaces was determined for three viruses, MS2, Q beta, and Norwalk. The virus is modeled as a particle comprised of ionizable amino acid residues in a shell surrounding a spherical RNA core of negative charge, these charges being compensated for by a Coulomb screening due to intercalated ions. A second model of the virus involving surface charges only is included for comparison. Surface potential calculations for each of the viruses show excellent agreement with electrophoretic mobility and zeta potential measurements as a function of pH. The environmental surface is modeled as a homogeneous plane held at constant potential with and without a finite region (patch) of opposite potential. The results indicate that the electrostatic interaction between the virus and the oppositely charged patch is significantly influenced by the conditions of ionic strength, pH and size of the patch. Specifically, at pH 7, the Norwalk virus interacts more strongly with the patch than MS2 (approximately 51 vs approximately 9kT) but at pH 5, the Norwalk-surface interaction is negligible while that of MS2 is approximately 5.9kT. The resulting ramifications for the use of MS2 as a surrogate for Norwalk are discussed.

Rapid response of marginal zone B cells to viral particles.

Marginal zone (MZ) B cells are thought to be responsible for the first wave of Abs against bacterial Ags. In this study, we assessed the in vivo response of MZ B cells in mice immunized with viral particles derived from the RNA phage Qbeta. We found that both follicular (FO) and MZ B cells responded to immunization with viral particles. MZ B cells responded with slightly faster kinetics, but numerically, FO B cells dominated the response. B1 B cells responded similarly to MZ B cells. Both MZ and FO B cells underwent isotype switching, with MZ B cells again exhibiting faster kinetics. In fact, almost all Qbeta-specific MZ B cells expressed surface IgG by day 5. Histological analysis demonstrated that a population of activated B cells remain associated with the MZ, probably due to the elevated integrin levels expressed by these cells. Thus, both MZ and FO B cells respond with rapid proliferation to viral infection and both populations undergo isotype switching, but MZ B cells remain in the MZ and may be responsible for local Ab production, opsonizing pathogens entering the spleen.

A protein antibiotic in the phage Qbeta virion: diversity in lysis targets.

A(2), a capsid protein of RNA phage Qbeta, is also responsible for host lysis. A(2) blocked synthesis of murein precursors in vivo by inhibiting MurA, the catalyst of the committed step of murein biosynthesis. An A(2)-resistance mutation mapped to an exposed surface near the substrate-binding cleft of MurA. Moreover, purified Qbeta virions inhibited wild-type MurA, but not the mutant MurA, in vitro. Thus, the two small phages characterized for their lysis strategy, Qbeta and the small DNA phage phiX174, effect host lysis by targeting different enzymes in the multistep, universally conserved pathway of cell wall biosynthesis.

CCA initiation boxes without unique promoter elements support in vitro transcription by three viral RNA-dependent RNA polymerases.

It has previously been observed that the only specific requirement for transcriptional initiation on viral RNA in vitro by the RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus is the CCA at the 3' end of the genome. We now compare the abilities of this RdRp, turnip crinkle virus RdRp, and Qbeta replicase, an enzyme capable of supporting the complete viral replication cycle in vitro, to transcribe RNA templates containing multiple CCA boxes but lacking specific viral sequences. Each enzyme is able to initiate transcription from several CCA boxes within these RNAs, and no special reaction conditions are required for these activities. The transcriptional yields produced from templates comprised of multiple CCA or CCCA repeats relative to templates derived from native viral RNA sequences vary between 2:1 and 0.1:1 for the different RdRps. Control of initiation by such redundant sequences presents a challenge to the specificity of viral transcription and replication. We identify 3'-preferential initiation and sensitivity to structural presentation as two specificity mechanisms that can limit initiation among potential CCA initiation sites. These two specificity mechanisms are used to different degrees by the three RdRps. The finding that three viral RdRps representing two of the three supergroups within the positive-strand RNA viral RdRp phylogeny support substantial transcription in the absence of unique promoters suggests that this phenomenon may be common among positive-strand viruses. A framework is presented arguing that replication of viral RNA in the absence of unique promoter elements is feasible.