Andrew Hayhurst

Viral assembly of oriented quantum dot nanowires

The highly organized structure of M13 bacteriophage was used as an evolved biological template for the nucleation and orientation of semiconductor nanowires. To create this organized template, peptides were selected by using a pIII phage display library for their ability to nucleate ZnS or CdS nanocrystals. The successful peptides were expressed as pVIII fusion proteins into the crystalline capsid of the virus. The engineered viruses were exposed to semiconductor precursor solutions, and the resultant nanocrystals that were templated along the viruses to form nanowires were extensively characterized by using high-resolution analytical electron microscopy and photoluminescence. ZnS nanocrystals were well crystallized on the viral capsid in a hexagonal wurtzite or a cubic zinc blende structure, depending on the peptide expressed on the viral capsid. Electron diffraction patterns showed single-crystal type behavior from a polynanocrystalline area of the nanowire formed, suggesting that the nanocrystals on the virus were preferentially oriented with their [001] perpendicular to the viral surface. Peptides that specifically directed CdS nanocrystal growth were also engineered into the viral capsid to create wurtzite CdS virus-based nanowires. Lastly, heterostructured nucleation was achieved with a dual-peptide virus engineered to express two distinct peptides within the same viral capsid. This work represents a genetically controlled biological synthesis route to a semiconductor nanoscale heterostructure.

Selective proton permeability and pH regulation of the influenza virus M2 channel expressed in mouse erythroleukaemia cells.

1. The M2 protein of influenza A virus is implicated in transmembrane pH regulation during infection. Whole‐cell patch clamp of mouse erythroleukaemia cells expressing the M2 protein in the surface membrane showed a conductance due to M2 which was specifically blocked by the anti‐influenza drug rimantadine. 2. The ion selectivity of the rimantadine‐sensitive current through M2 was determined. Reversal potentials were close to equilibrium potentials for transmembrane pH gradients and not to those for Na+, K+ or Cl‐ concentration gradients. M2 permeability to Na+ relative to H+ was estimated to be less than 6 x 10(‐7). 3. The M2 conductance increased as external pH decreased below 8.5 and approached saturation at an external pH of 4, effects attributable to increased permeability due to increased driving potential and to activation by low external pH. Both activation and permeation could be described by interaction of protons with sites on M2, with apparent dissociation constants of approximately 0.1 microM and 1 microM, respectively, under physiological conditions. 4. The M2 protein can transfer protons selectively across membranes with the H+ electrochemical gradient, properties consistent with its role in modifying virion and trans‐Golgi pH during virus infection.

Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS)

Periplasmic expression with cytometric screening (PECS) is a powerful and rapid “display-less” technology for isolating ligand-binding proteins from diverse libraries. Escherichia coli expressing a library of proteins secreted into the periplasmic space are incubated with a fluorescent conjugate of the target ligand. Under the proper conditions, ligands as large as about 10 kDa can equilibrate within the periplasmic space without compromising the cells integrity or viability. The bacterial cell envelope effectively serves as a dialysis bag to selectively retain receptor–fluorescent probe complexes but not free ligand. Cells displaying increased fluorescence are then isolated by flow cytometry. We demonstrate that scFv antibodies with both very high and low affinity to digoxigenin can be isolated from libraries screened by PECS using a benchtop flow cytometer. We also show that preexisting libraries constructed for display on filamentous bacteriophage can be screened by PECS without the need for subcloning. In fact, PECS was found to select for proteins that could be missed by conventional phage panning and screening methods.

Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis.

Brucella melitensis is a highly infectious animal pathogen able to cause a recurring debilitating disease in humans and is therefore high on the list of biological warfare agents. Immunoglobulin genes from mice immunized with gamma-irradiated B. melitensis strain 16M were used to construct a library that was screened by phage display against similarly prepared bacteria. The selected phage particles afforded a strong enzyme-linked immunosorbent assay (ELISA) signal against gamma-irradiated B. melitensis cells. However, extensive efforts to express the respective single chain antibody variable region fragment (scFv) in soluble form failed due to: (i) poor solubility and (ii) in vivo degradation of the c-myc tag used for the detection of the recombinant antibodies. Both problems could be addressed by: (i) fusing a human kappa light chain constant domain (Ck) chain to the scFv to generate single chain antibody fragment (scAb) antibody fragments and (ii) by co-expression of the periplasmic chaperone Skp. While soluble, functional antibodies could be produced in this manner, phage-displaying scFvs or scAbs were still found to be superior ELISA reagents for immunoassays, due to the large signal amplification afforded by anti-phage antibodies. The isolated phage antibodies were shown to be highly specific to B. melitensis and did not recognize Yersinia pseudotuberculosis in contrast to the existing diagnostic monoclonal YST 9.2.1.

High-throughput antibody isolation

To define the proteome of an organism, there is a need for robust and reproducible methods for the quantitative detection of all the polypeptides in a cell. High-density arrays of receptors specific for each of the polypeptides in a complex sample hold great promise for the analysis of complex protein mixtures. Because of their high affinity, specificity and their ability to bind to virtually any protein, antibodies appear particularly promising as the receptor element in protein-detection arrays. For proteomic-scale analyses, the ability to isolate and produce antibodies en masse to large numbers of target molecules is critical. A variety of systems for the high-throughput isolation of antibodies from combinatorial libraries are being developed and are outlined in this review. However, there are several other important considerations to be borne in mind before such systems can realistically be applied on a large scale.

Differences in conductance of M2 proton channels of two influenza viruses at low and high pH

The M2 protein of influenza A viruses forms a proton channel involved in modifying virion and trans Golgi pH during infection. Previous studies of the proton current using whole‐cell patch clamp of mouse erythroleukaemia (MEL) cells expressing the M2 protein of the ‘Weybridge’ strain provided evidence for two protonation sites, one involved in permeation, the other in activation by acid pH. The present report compares the M2 channels of two different strains of influenza virus, ‘Weybridge’ (WM2) and ‘Rostock’ (RM2). Whereas with external acid pH the current‐voltage relations showed similar small degrees of inward rectification, a similar apparent Kd of approximately 10 μm for proton permeation and a high selectivity for protons over Na+, the two M2 proteins differed in whole‐cell conductance at low and high pH. The proton conductance of unit membrane area was on average 7‐fold greater in RM2‐ than WM2‐expressing MEL cells. At high external pH WM2 was shown previously to have small conductance for outward current at positive driving potential. In contrast, RM2 shows high conductance for outward current with high external pH, but shows small conductance for inward current with high internal pH, conditions in which WM2 shows high conductance for inward current. The different properties of the conductances due to the two channels at high pH were determined by three amino acids in their transmembrane domains. All intermediate mutants possessed one or other property and transformation of the WM2 phenotype into that of RM2 required substitution in all three residues V27I, F38L and D44N; single substitutions in RM2 effected the opposite phenotypic change. The significance of this difference for virus replication is not clear and it may be that the higher proton flux associated with RM2 is the main factor determining its increased ability to dissipate pH gradients. It is apparent, however, from the specific differences in the sidedness of the pH‐induced changes in voltage dependence of the whole‐cell current that this is an intrinsic property of the virus proton channel which may have parallels with regulation of other proton channels.

Llama Single Domain Antibodies Specific for the 7 Botulinum Neurotoxin Serotypes as Heptaplex Immunoreagents

Background There are currently 7 known serotypes of botulinum neurotoxin (BoNT) classified upon non-cross reactivity of neutralizing immunoglobulins. Non-neutralizing immunoglobulins, however, can exhibit cross-reactivities between 2 or more serotypes, particularly mosaic forms, which can hamper the development of highly specific immunoassays, especially if based on polyclonal antisera. Here we employ facile recombinant antibody technology to subtractively select ligands to each of the 7 BoNT serotypes, resulting in populations with very high specificity for their intended serotype. Methods and Findings A single llama was immunized with a cocktail of 7 BoNT toxoids to generate a phage display library of single domain antibodies (sdAb, VHH or nanobodies) which were selected on live toxins. Resulting sdAb were capable of detecting both toxin and toxin complex with the best combinations able to detect 100s-10s of pg per 50 µL sample in a liquid bead array. The most sensitive sdAb were combined in a heptaplex assay to identify each of the BoNT serotypes in buffer and milk and to a lesser extent in carrot juice, orange juice and cola. Several anti-A(1) sdAb recognized A2 complex, showing that subtype cross-reactivity within a serotype was evident. Many of our sdAb could act as both captor and tracer for several toxin and toxin complexes suggesting sdAb can be used as architectural probes to indicate BoNT oligomerisation. Six of 14 anti-A clones exhibited inhibition of SNAP-25 cleavage in the neuro-2A assay indicating some sdAb had toxin neutralizing capabilities. Many sdAb were also shown to be refoldable after exposure to high temperatures in contrast to polyclonal antisera, as monitored by circular dichroism. Conclusions Our panel of molecularly flexible antibodies should not only serve as a good starting point for ruggedizing assays and inhibitors, but enable the intricate architectures of BoNT toxins and complexes to be probed more extensively.

Thermostable Llama Single Domain Antibodies for Detection of Botulinum A Neurotoxin Complex

Immunoglobulins from animals of the Camelidae family boast unique forms that do not incorporate light chains. Antigen binding in these unconventional heavy-chain homodimers is mediated through a single variable domain. When expressed recombinantly these variable domains are termed single domain antibodies (sdAb) and are among the smallest naturally IgG-derived antigen binding units. SdAb possess good solubility, thermostability, and can refold after heat and chemical denaturation making them promising alternative recognition elements. We have constructed a library of phage-displayed sdAb from a llama immunized with a cocktail of botulinum neurotoxin (BoNT) complex toxoids and panned the library for binders for BoNT A complex toxoid. Six unique binders were isolated and found to specifically bind BoNT A complex in toxoid and untoxoided forms and when used in optimal combinations in buffer and milk could detect 100 pg/mL untoxoided complex. All sdAb retained their ability to specifically bind target after heating to 85 degrees C for 1 h, in contrast to conventional polyclonal sera. All of the sdAb were highly specific for subtype A1 rather than A2 and demonstrated binding to the 33 kDa hemagglutinin, potentially to a somewhat overlapping linear epitope. The unique properties of these sdAb may provide advantages for many diagnostic applications where long-term storage and in-line monitoring require very rugged yet highly specific recognition elements.

Rapid assembly of sensitive antigen-capture assays for Marburg virus, using in vitro selection of llama single-domain antibodies, at biosafety level 4.

There is a pressing need for rapid and reliable approaches to the delivery of sensitive yet rugged diagnostic assays specific for emerging viruses, to hasten containment of outbreaks when and wherever they occur. Within 3 weeks, we delivered an antigen-capture assay for Marburg virus (MARV) that was based on llama single-domain antibodies (sdAbs) selected at biosafety level 4. Four unique sdAbs were capable of independently detecting MARV variants Musoke, Ravn, and Angola without cross-reactivity with the 4 Ebola virus species. The unoptimized assays could be performed in <30 min and, at best, provided a visual read of 10-100 pfu in a 100-microL sample when a colorimetric substrate was used and 0.1-1 pfu when a chemiluminescent substrate was used. All the sdAbs were specific for nucleoprotein, with an assay sensitivity that was reliant on detergent-mediated exposure of polyvalent antigen. Our strategy highlights the potential of direct antibody selection on filoviruses as a guide for effective and fast diagnostic development.

An efficient method for the rescue and analysis of functional HIV-1 env genes: Evidence for recombination in the vicinity of the tat/rev splice site

ObjectiveTo establish a robust procedure for the isolation and characterization of full-length expression-competent HIV-1 env genes directly from patient samples. DesignHIV exists as a quasispecies which can be disturbed by in vitro culture, in which numerous members of the population are likely to be defective due to the high error rate of the viral reverse transcriptase. Defective viruses are unlikely to play a dominant role in disease progression. Since env gene translation products play major roles in the initiation and spread of infection we need to study genes with open reading frames. MethodsA nested polymerase chain reaction (PCR) approach has been used to rescue intact (2.6kb) env genes, which are cloned into a T7-promoter-containing vector. Expression of gp160 in CV-1 cells is detected by Western blot. Expression-competent clones are sequenced and resulting sequences used for phylogenetic studies. Translation products are analysed in relation to the known immunogenic structure of gp160. ResultsFrom random patient samples collected in London clinics, only HIV-1 subtype B was found. Two of the samples contained viruses with an additional pair of cysteine residues in their V1 regions. For samples collected in Uganda, HIV-1 subtypes A, D and an A/D recombinant were recovered. ConclusionAn effective procedure is described for the isolation of HIV-1 env genes directly from patient samples, which has worked for A, B and D subtypes to date. The PCR primers can be utilized with other subtypes with the possible exception of subtype O viruses. Phyiogenetic analyses revealed the potential importance of a G/C-rich region near the tat/rev splice site as a site of recombination. The sequences and translation products generated may be more relevant to disease progression in vivo and vaccine formulations than those obtained from viruses selected in long-term culture.