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Supplementary Data from Architectonics of Phage-Liposome Nanowebs as Optimized Photosensitizer Vehicles for Photodynamic Cancer Therapy

Filamentous M13 phage can be engineered to display cancer cell–targeting or tumor-homing peptides through phage display. It would be highly desirable if the tumor-targeting phage can also carry anticancer drugs to deliver them to the cancer cells. We studied the evolution of structures of the complexes between anionic filamentous M13 phage and cationic serum-stable liposomes that encapsulate the monomeric photosensitizer zinc naphthalocyanine. At specific phage-liposome ratios, multiple phage nanofibers and liposomes are interwoven into a “nanoweb.” The chemical and biological properties of the phage-liposome nanoweb were evaluated for possible application in drug delivery. This study highlights the ability of phage-liposome nanowebs to serve as efficient carriers in the transport of photosensitizers to cancer cells. Mol Cancer Ther; 9(9); 2524–35. ©2010 AACR.

Fusions of single-domain antibodies (sdAbs, nanobodies) to enzymatic reporters make convenient molecular probes to detect the presence of an antigen of interest. We have previously fused the monomeric hyperactive ascorbate peroxidase derivative APEX2 to anti-Ebolavirus and anti-Marburgvirus sdAbs to generate immunoreagents useful in detecting nucleoprotein (NP) on western blots, ELISA, and within cells following transfection of NP expression plasmids or following virus infection. Here we present the methods used to overexpress and purify these sdAb-APEX2 fusion proteins, and to employ them as probes in various scenarios with colorimetric and fluorometric signal development. We also introduce a dimeric hyperactive ascorbate peroxidase derivative dEAPX that enables bivalent sdAb probes to be produced while avoiding the need to generate more complex tandem sdAbs, leveraging avidity for improved signal strength. The APEX2 and dEAPX reagents appear interchangeable with any existing detection platform and the methods described here should enable a user to study their antigen of interest by simply swapping out the sdAb for their recombinant affinity reagent of choice.

Generating and characterizing immunoreagents to enable studies of novel emerging viruses is an area where ensembles of synthetic genes, recombinant antibody pipelines, and modular antibody-reporter fusion proteins can respond rapidly. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread through the global population causing widespread morbidity, mortality, and socioeconomic chaos. Using SARS-CoV-2 as our model and starting with a gBlocks encoded nucleocapsid (N) gene, we purified recombinant protein from E. coli, to serve as bait for selecting semisynthetic nanobodies from our Nomad single-pot library. Clones were isolated in days and first fused to Gaussia luciferase to determine EC50 in the tens of nM range, and second fused to the ascorbate peroxidase derivative APEX2 for sensitive detection of SARS-CoV-2 infected cells. To generate inherently fluorescent immunoreagents, we introduce novel periplasmic sdAb fusions made with mNeonGreen and mScarlet-I, which were produced at milligram amounts. The fluorescent fusion proteins enabled concise visualization of SARS-CoV-2 N in the cytoplasm but not in the nucleus 24 h post infection, akin to the distribution of SARS-CoV N, thereby validating these useful imaging tools. SdAb reactivity appeared specific to SARS-CoV-2 with very much weaker binding to SARS-CoV, and no noticeable cross-reactivity to a panel of overexpressed human codon optimized N proteins from other CoV. High periplasmic expression levels and in silico immortalization of the nanobody constructs guarantees a cost-effective and reliable source of SARS-CoV-2 immunoreagents. Our proof-of-principle study should be applicable to known and newly emerging CoV to broaden the tools available for their analysis and help safeguard human health in a more proactive than reactive manner.

We had previously shown that three anti–Marburg virus nanobodies (VHH or single-domain antibody [sdAb]) targeted a cryptotope within an alpha-helical assembly at the nucleoprotein (NP) C-terminus that was conserved through half a century of viral evolution. Here, we wished to determine whether an anti–Ebola virus sdAb, that was cross-reactive within the Ebolavirus genus, recognized a similar structural feature upstream of the ebolavirus NP C-terminus. In addition, we sought to determine whether the specificities of a less cross-reactive anti–Zaire ebolavirus sdAb and a totally specific anti–Sudan ebolavirus sdAb were the result of exclusion from this region. Binding and X-ray crystallographic studies revealed that the primary determinant of cross-reactivity did indeed appear to be a preference for the helical feature. Specificity, in the case of the Zaire ebolavirus–specific sdAb, arose from the footprint shifting away from the helices to engage more variable residues. While both sdAbs used CDRs, they also had atypical side-on approaches, with framework 2 helping to accommodate parts of the epitope in sizeable paratope gullies. The Sudan ebolavirus–specific sdAb was more remarkable and appeared to bind two C-terminal domains simultaneously via nonoverlapping epitopes—“paratope duality.” One mode involved paratope gullying, whereas the other involved only CDRs, with CDR3 restructuring to wedge in between opposing walls of an interdomain crevice. The varied routes used by sdAbs to engage antigens discovered here deepen our appreciation of the small scaffold's architectural versatility and also reveal lucrative opportunities within the ebolavirus NP C-termini that might be leveraged for diagnostics and novel therapeutic targeting.

Peroxisome proliferator-activated receptor (PPAR)γ is a global transcriptional regulator associated with anti-inflammatory actions. It is highly expressed in alveolar macrophages (AMs), which are unable to clear the intracellular pathogen Mycobacterium tuberculosis (M.tb). Although M.tb infection induces PPARγ in human macrophages, which contributes to M.tb growth, the mechanisms underlying this are largely unknown. We undertook NanoString gene expression analysis to identify novel PPARγ effectors that condition macrophages to be more susceptible to M.tb infection. This revealed several genes that are differentially regulated in response to PPARγ silencing during M.tb infection, including the Bcl-2 family members Bax (pro-apoptotic) and Mcl-1 (pro-survival). Apoptosis is an important defense mechanism that prevents the growth of intracellular microbes, including M.tb, but is limited by virulent M.tb. This suggested that M.tb differentially regulates Mcl-1 and Bax expression through PPARγ to limit apoptosis. In support of this, gene and protein expression analysis revealed that Mcl-1 expression is driven by PPARγ during M.tb infection in human macrophages. Further, 15-lipoxygenase (15-LOX) is critical for PPARγ activity and Mcl-1 expression. We also determined that PPARγ and 15-LOX regulate macrophage apoptosis during M.tb infection, and that pre-clinical therapeutics that inhibit Mcl-1 activity significantly limit M.tb intracellular growth in both human macrophages and an in vitro TB granuloma model. In conclusion, identification of the novel PPARγ effector Mcl-1 has determined PPARγ and 15-LOX are critical regulators of apoptosis during M.tb infection and new potential targets for host-directed therapy for M.tb.

Marburg virus (MARV) is a highly lethal hemorrhagic fever virus that is increasingly re-emerging in Africa, has been imported to both Europe and the US, and is also a Tier 1 bioterror threat. As a negative sense RNA virus, MARV has error prone replication which can yield progeny capable of evading countermeasures. To evaluate this vulnerability, we sought to determine the epitopes of 4 llama single-domain antibodies (sdAbs or VHH) specific for nucleoprotein (NP), each capable of forming MARV monoclonal affinity reagent sandwich assays. Here, we show that all sdAb bound the C-terminal region of NP, which was produced recombinantly to derive X-ray crystal structures of the three best performing antibody-antigen complexes. The common epitope is a trio of alpha helices that form a novel asymmetric basin-like depression that accommodates each sdAb paratope via substantial complementarity-determining region (CDR) restructuring. Shared core contacts were complemented by unique accessory contacts on the sides and overlooks of the basin yielding very different approach routes for each sdAb to bind the antigen. The C-terminal region of MARV NP was unable to be crystallized alone and required engagement with sdAb to form crystals suggesting the antibodies acted as crystallization chaperones. While gross structural homology is apparent between the two most conserved helices of MARV and Ebolavirus, the positions and morphologies of the resulting basins were markedly different. Naturally occurring amino acid variations occurring in bat and human Marburgvirus strains all mapped to surfaces distant from the predicted sdAb contacts suggesting a vital role for the NP interface in virus replication. As an essential internal structural component potentially interfacing with a partner protein it is likely the C-terminal epitope remains hidden or “cryptic” until virion disruption occurs. Conservation of this epitope over 50 years of Marburgvirus evolution should make these sdAb useful foundations for diagnostics and therapeutics resistant to drift.

Viruses assemble large macromolecular repeat structures that become part of the infectious particles or virions. Ribonucleocapsids (RNCs) of negative strand RNA viruses are a prime example where repetition of nucleoprotein (NP) along the genome creates a core polymeric helical scaffold that accommodates other nucleocapsid proteins including viral polymerase. The RNCs are transported through the cytosol for packaging into virions through association with viral matrix proteins at cell membranes. We hypothesized that RNC would be ideal targets for crosslinkers engineered to promote aberrant protein–protein interactions, thereby blocking their orderly transport and packaging. Previously, we had generated single-domain antibodies (sdAbs) against Filoviruses that have all targeted highly conserved C-terminal regions of NP known to be repetitively exposed along the length of the RNCs of Marburgvirus (MARV) and Ebolavirus (EBOV). Our crosslinker design consisted of dimeric sdAb expressed intracellularly, which we call Xintrabodies (X- for crosslinking). Electron microscopy of purified NP polymers incubated with purified sdAb constructs showed NP aggregation occurred in a genus-specific manner with dimeric and not monomeric sdAb. A virus-like particle (VLP) assay was used for initial evaluation where we found that dimeric sdAb inhibited NP incorporation into VP40-based VLPs whereas monomeric sdAb did not. Inhibition of NP packaging was genus specific. Confocal microscopy revealed dimeric sdAb was diffuse when expressed alone but focused on pools of NP when the two were coexpressed, while monomeric sdAb showed ambivalent partition. Infection of stable Vero cell lines expressing dimeric sdAb specific for either MARV or EBOV NP resulted in smaller plaques and reduced progeny of cognate virus relative to wild-type Vero cells. Though the impact was marginal at later time-points, the collective data suggest that viral replication can be reduced by crosslinking intracellular NP using relatively small amounts of dimeric sdAb to restrict NP packaging. The stoichiometry and ease of application of the approach would likely benefit from transitioning away from intracellular expression of crosslinking sdAb to exogenous delivery of antibody. By retuning sdAb specificity, the approach of crosslinking highly conserved regions of assembly critical proteins may well be applicable to inhibiting replication processes of a broad spectrum of viruses.

The use of Xenopus laevis as a model for vertebrate developmental biology is limited by a lack of antibodies specific for embryonic antigens. This study evaluated the use of immune and non-immune phage display libraries for the isolation of single domain antibodies, or nanobodies, with specificities for Xenopus embryonic antigens. The immune nanobody library was derived from peripheral blood lymphocyte RNA obtained from a llama immunized with Xenopus gastrula homogenates. Screening this library by immunostaining of embryonic tissues with pooled periplasmic material and sib-selection led to the isolation of several monoclonal phages reactive with the cytoplasm and nuclei of gastrula cells. One antigen recognized by a group of nanobodies was identified using a reverse proteomics approach as nucleoplasmin, an abundant histone chaperone. As an alternative strategy, a semi-synthetic non-immune llama nanobody phage display library was panned on highly purified Xenopus proteins. This proof-of-principle approach isolated monoclonal nanobodies that specifically bind Nuclear distribution element-like 1 (Ndel1) in multiple immunoassays. Our results suggest that immune and non-immune phage display screens on crude and purified embryonic antigens can efficiently identify nanobodies useful to the Xenopus developmental biology community.

We explore evolved soybean ascorbate peroxidase (APEX2) as a reporter when fused to the C-termini of llama nanobodies (single-domain antibodies, sdAb, variable domains of heavy chain-only antibodies, VHH) targeted to the E. coli periplasm. Periplasmic expression preserves authentic antibody N-termini, intra-domain disulphide bond(s), and capitalizes on efficient haem loading through the porous E. coli outer membrane. Using monomeric and dimeric anti-nucleoprotein (NP) sdAb cross-reactive within the Marburgvirus genus and cross-reactive within the Ebolavirus genus, we show that periplasmic sdAb&ndash, APEX2 fusion proteins are easily purified at multi-mg amounts. The fusions were used in Western blotting, ELISA, and microscopy to visualize NPs using colorimetric and fluorescent imaging. Dimeric sdAb&ndash, APEX2 fusions were superior at binding NPs from viruses that were evolutionarily distant to that originally used to select the sdAb. Partial conservation of the anti-Marburgvirus sdAb epitope enabled the recognition of a novel NP encoded by the recently discovered Mĕngl&agrave, virus genome. Antibody&ndash, antigen interactions were rationalized using monovalent nanoluciferase titrations and contact mapping analysis of existing crystal structures, while molecular modelling was used to reveal the potential landscape of the Mĕngl&agrave, NP C-terminal domain. The sdAb&ndash, APEX2 fusions also enabled live Marburgvirus and Ebolavirus detection 24 h post-infection of Vero E6 cells within a BSL-4 laboratory setting. The simple and inexpensive mining of large amounts of periplasmic sdAb&ndash, APEX2 fusion proteins should help advance studies of past, contemporary, and perhaps Filovirus species yet to be discovered.

Filamentous M13 phage can be engineered to display cancer cell–targeting or tumor-homing peptides through phage display. It would be highly desirable if the tumor-targeting phage can also carry anticancer drugs to deliver them to the cancer cells. We studied the evolution of structures of the complexes between anionic filamentous M13 phage and cationic serum-stable liposomes that encapsulate the monomeric photosensitizer zinc naphthalocyanine. At specific phage-liposome ratios, multiple phage nanofibers and liposomes are interwoven into a “nanoweb.” The chemical and biological properties of the phage-liposome nanoweb were evaluated for possible application in drug delivery. This study highlights the ability of phage-liposome nanowebs to serve as efficient carriers in the transport of photosensitizers to cancer cells. Mol Cancer Ther; 9(9); 2524–35. ©2010 AACR.

Single-domain antibodies (sdAb) specific for botulinum neurotoxin serotype A (BoNT A) were selected from an immune llama phage display library derived from a llama that was immunized with BoNT A toxoid. The constructed phage library was panned using two methods: panning on plates coated with BoNT A toxoid (BoNT A Td) and BoNT A complex toxoid (BoNT Ac Td) and panning on microspheres coupled to BoNT A Td and BoNT A toxin (BoNT A Tx). Both panning methods selected for binders that had identical sequences, suggesting that panning on toxoided material may be as effective as panning on bead-immobilized toxin for isolating specific binders. All of the isolated binders tested were observed to recognize bead-immobilized BoNT A Tx in direct binding assays, and showed very little cross-reactivity towards other BoNT serotypes and unrelated protein. Sandwich assays that incorporated selected sdAb as capture and tracer elements demonstrated that all of the sdAb were able to recognize soluble ("live") BoNT A Tx and BoNT Ac Tx with virtually no cross-reactivity with other BoNT serotypes. The isolated sdAb did not exhibit the high degree of thermal stability often associated with these reagents; after the first heating cycle most of the binding activity was lost, but the portion of the protein that did refold and recover antigen-binding activity showed only minimal loss on subsequent heating and cooling cycles. The binding kinetics of selected binders, assessed by both an equilibrium fluid array assay as well as surface plasmon resonance (SPR) using toxoided material, gave dissociation constants (K(D)) in the range 2.2 x 10(-11) to 1.6 x 10(-10) M. These high-affinity binders may prove beneficial to the development of recombinant reagents for the rapid detection of BoNT A, particularly in field screening and monitoring applications.

Phage display is a well-known technique that facilitates the selection of peptides or proteins that bind to a desired target. Using this tool, binding elements contained in the natural immune repertoires of the source animal or from a synthetically generated collection may be selected. The unpaired variable domain of the llama's heavy-chain-only classes of immunoglobulins represents an ideal source of genetic material to create phage display libraries. Initial panning of a semi-synthetic llama library yielded only one binder to the toxin ricin. In an effort to increase the number of monoclonal phage binders selected, the Luminex xMAP technology (Luminex, Austin, TX, USA) was used in addition to the enzyme-linked immunosorbent assay (ELISA) to screen clonal populations of phage after three rounds of selection. The xMAP technology detected phage displayed single domain antibody (sdAb) bound to ricin immobilized on the surface of microspheres under equilibrium conditions. This enhanced capability led directly to the identification of additional single domain antibodies of interest. The selected sdAbs were expressed, purified, and then evaluated for their specificity as well as enhanced thermal stability in comparison to conventional immunoglobulin G (IgG). We determined equilibrium dissociation constants and demonstrated their use as effective capture molecules in sandwich immunoassays.

Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (naive) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naive display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.

Llamas possess a class of unconventional immunoglobulins that have only heavy chains; unpaired heavy variable domains are responsible for antigen binding. These domains have previously been cloned and expressed as single domain antibodies (sdAbs); they comprise the smallest known antigen binding fragments. SdAbs have been shown to bind antigens at90 degrees C and to refold after being denatured. To take advantage of the remarkable properties of sdAbs, we constructed a large, semisynthetic llama sdAb library. This library facilitated the rapid selection of binders to an array of biothreat targets. We selected sdAb specific for live vaccinia virus (a smallpox virus surrogate), hen egg lysozyme, cholera toxin, ricin, and staphylococcal enterotoxin B. The selected sdAb possessed high specificity as well as enhanced thermal stability in comparison to conventional IgG and scFv antibodies. We also determined equilibrium dissociation constants as well as demonstrated the use of several antitoxin sdAbs as effective capture and reporter molecules in sandwich assays on the Luminex instrument. The ability to rapidly select such rugged antibodies will enhance the reliability of immunoassays by extending shelf life and the capacity to function in hostile environments.

We report a simple and versatile approach for the conjugation of luminescent CdSe–ZnS core-shell quantum dots (QDs) to proteins through coordination of engineered C-terminal oligohistidine sequences. Several histidine tail containing proteins were self-assembled onto the QD surface using this method. A recombinant antibody specific for the high explosive 2,4,6-trinitrotoluene (TNT) was conjugated to QDs through a carboxy terminal histidine tail and the bioconjugate used to detect TNT by competitive immunoassay. TNT was detected over the range of 10 μg/ml down to 41 ng/ml using the scFv conjugated to QDs. These results open up the possibility to conjugate luminescent QDs to a whole range of proteins to form QD bioconjugates that can be effectively used in bio-oriented applications, such as sensing, imaging, immunoassay and other diagnostics.

Anchored periplasmic expression (APEx) is a technology for the isolation of ligand-binding proteins from combinatorial libraries anchored on the periplasmic face of the inner membrane of Escherichia coli . After disruption of the outer membrane by Tris-EDTA-lysozyme, the inner-membrane-anchored proteins readily bind fluorescently labeled ligands as large as 240 kDa. Fluorescently labeled cells are isolated by flow cytometry, and the DNA of isolated clones is rescued by PCR. By using two rounds of APEx, the affinity of a neutralizing antibody to the Bacillus anthracis protective antigen was improved >200-fold, exhibiting a final K D of 21 pM. This approach has several technical advantages compared with previous library screening technologies, including the unique ability to screen for ligand-binding proteins that bind endogenously expressed ligands fused to a short-lived GFP. Further, APEx is able to display proteins either as an N - terminal fusion to a six-residue sequence derived from the native E. coli lipoprotein NlpA, or as a C-terminal fusion to the phage gene three minor coat protein of M13. The latter fusions allow hybrid phage display/APEx strategies without the need for further subcloning.

We report a virus-based scaffold for the synthesis of single-crystal ZnS, CdS, and freestanding chemically ordered CoPt and FePt nanowires, with the means of modifying substrate specificity through standard biological methods. Peptides (selected through an evolutionary screening process) that exhibit control of composition, size, and phase during nanoparticle nucleation have been expressed on the highly ordered filamentous capsid of the M13 bacteriophage. The incorporation of specific, nucleating peptides into the generic scaffold of the M13 coat structure provides a viable template for the directed synthesis of semiconducting and magnetic materials. Removal of the viral template by means of annealing promoted oriented aggregation-based crystal growth, forming individual crystalline nanowires. The unique ability to interchange substrate-specific peptides into the linear self-assembled filamentous construct of the M13 virus introduces a material tunability that has not been seen in previous synthetic routes. Therefore, this system provides a genetic toolkit for growing and organizing nanowires from semiconducting and magnetic materials.

Biological selection using the M13 bacteriophage was used to isolate peptide–phage fusions capable of specific recognition and nucleation of zinc sulfide (ZnS) and cadmium sulfide (CdS) nanocrystals. Advanced analytical electron microscopy data revealed that the phage constructs were found to control both crystal size and structure of semiconductor nanocrystals synthesized in aqueous solution at room temperature. Most significantly, different engineered peptide–phage constructs were found that directed the specific nucleation of either the zinc-blende or wurtzite structure for both ZnS and CdS nanocrystals. The ZnS specific peptides were found to function the same in either of two different fusion constructs, a pIII and pVIII fusion. As such, this method provides a new route to the synthesis of nanocrystals with specified physical characteristics such as size and crystal phase and has the potential to assemble biological II–VI hybrid materials into spatially complex functional mesoscopic electronic and photonic structures.

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 K(d) of approximately 10 microM 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.

Expression of single-chain antibody fragments (scAb)in the periplasm of Escherichia coli often results in low soluble product yield and cell lysis. We have increased scAb solubility and prevented cell culture lysis by coexpressing the E. coli Skp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scAb gene within an isopropyl beta-d-thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scAb solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scAb and should prove useful in expanding library sizes.

A bottle-neck in recombinant antibody sandwich immunoassay development is pairing, demanding protein purification and modification to distinguish captor from tracer. We developed a simple pairing scheme using microliter amounts of E. coli osmotic shockates bearing site-specific biotinylated antibodies and demonstrated proof of principle with a single domain antibody (sdAb) that is both captor and tracer for polyvalent Marburgvirus nucleoprotein. The system could also host pairs of different sdAb specific for the 7 botulinum neurotoxin (BoNT) serotypes, enabling recognition of the cognate serotype. Inducible supE co-expression enabled sdAb populations to be propagated as either phage for more panning from repertoires or expressed as soluble sdAb for screening within a single host strain. When combined with streptavidin-g3p fusions, a novel transdisplay system was formulated to retrofit a semi-synthetic sdAb library which was mined for an anti-Ebolavirus sdAb which was immediately immunoassay ready, thereby speeding up the recombinant antibody discovery and utilization processes.

Conventional antibodies, widely used in detection schemes, such point detectors for biological threats, are large 150 kDa proteins that aggregate irreversibly under harsh conditions such as excessive heating. There is a need for the development of high affinity, highly specific robust recognition elements to overcome the limitations of conventional antibodies. One option is single domain antibodies (sdAb), the molecularly engineered binding elements derived from camelids and shark. SdAb are small 12-16 kDa robust recognition elements able to function after exposure to chemical denaturants and elevated temperatures. These antibody fragments contain only 3 hypervariable loops but can still bind with affinity equivalent to conventional antibody fragments having twice the number of complementarity-determining regions (CDRs) available to interact with the target. We developed and characterized sdAb towards toxins (ricin, abrin and SEB) and viral (filovirus) targets. We employed a combination of strategies, including sandwich immunoassays, surface plasmon resonance, circular dichroism, mutagenesis, and crystallography to help us to understand the mechanism of this bio-molecular recognition and provide critical insights to the basis of the sdAb-target interaction and sdAb properties.

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.

Publisher Summary Camelids and sharks possess a class of unconventional immunoglobulins consisting of heavy-chain homodimers in which antigen binding is mediated through a single variable domain. When expressed recombinantly, these variable domains, referred to as “single-domain antibodies” (sdAb), comprise the smallest known antigen-binding fragments. Both camelid- and shark-derived sdAb have been incorporated into multivalent and/or bivalent constructs. These reagents have the advantage that through avidity, they can have a higher apparent affinity than a monomeric sdAb. Strategies including DNA shuffling, random PCR, and mutational hotspot randomization have been used to generate and select sdAb variants with improved protein expression, stability, and affinity. The ability to design or tailor sdAb to meet an application's particular needs is a strong advantage that favors their future adoption in detection schemes. Furthermore, the biophysical properties of sdAb make them attractive recognition elements in biosensors. However, there are several limitations to the use of sdAb in biosensors. Although sdAb can be rapidly developed from naive libraries, the highest affinity reagents have been derived from immunized animals. Using animals adds both time and expense to reagent development. Finally, protein engineering offers the potential to create fusions of sdAb with protein domains to impart specific functionalities, such as the construction of monomolecular protein lattices that can be used to pattern surfaces, this cannot currently be accomplished with conventional IgG. Directed evolution enables the selection of sdAb variants with desirable traits such as improved thermodynamic stability and proteolytic stability. These features combined make sdAb ideal reagents for use in the development of tomorrow's rugged biosensors.

We have developed several conjugation strategies based on noncovalent self-assembly for the attachment of proteins and other biomolecules to water-soluble luminescent colloidal semiconductor nanocrystals (quantum dots [QDs]). The resulting QD-protein conjugates were employed in designing a variety of bioinspired applications, including single and multiplexed immunosorbent assays to detect toxins and small molecule explosives. In these studies we showed that QD fluorophores offer several important advantages. In particular, their tunable broad excitation spectra combined with narrow fluorescence emission peaks permit single-line excitation of multiple color nanocrystals, with facile signal deconvolution to extract individual contributions from each population (e.g., size) of QDs in multiplexed assays. Furthermore, the QDs strong resistance to photobleaching under continuous illumination relative to many organic dyes makes them ideal fluorophores for long-term cellular imaging studies. This chapter details the materials and methods for the synthesis of surface-functionalized CdSe-ZnS core-shell QDs, the construction and preparation of recombinant proteins, the conjugation of antibodies (and antibody fragments) to QDs, and the use of antibody-conjugated QDs in fluoroimmunoassays.

We have developed several conjugation strategies based on noncovalent self-assembly for the attachment of proteins and other biomolecules to water-soluble luminescent colloidal semiconductor nanocrystals (quantum dots [QDs]). The resulting QD-protein conjugates were employed in designing a variety of bioinspired applications, including single and multiplexed immunosorbent assays to detect toxins and small molecule explosives. In these studies we showed that QD fluorophores offer several important advantages. In particular, their tunable broad excitation spectra combined with narrow fluorescence emission peaks permit single-line excitation of multiple color nanocrystals, with facile signal deconvolution to extract individual contributions from each population (e.g., size) of QDs in multiplexed assays. Furthermore, the QDs strong resistance to photobleaching under continuous illumination relative to many organic dyes makes them ideal fluorophores for long-term cellular imaging studies. This chapter details the materials and methods for the synthesis of surface-functionalized CdSe-ZnS core-shell QDs, the construction and preparation of recombinant proteins, the conjugation of antibodies (and antibody fragments) to QDs, and the use of antibody-conjugated QDs in fluoroimmunoassays.

Genetically modified Salmonella typhimurium VNP20009 (VNP) is a useful vehicle for cancer therapy and vaccine development but exhibits limited tumor targeting in vivo. We engineered a novel VNP derivative that expressed carcinoembryonic antigen (CEA)-specific single chain antibody fragments (scFv) on the cell surface to increase tumor-specific targeting. There was significant scFv cell surface display visualized by flow cytometry and confocal microscopy when cells were probed with fluorescently labeled CEA. Atomic force microscopy (AFM) measurements on whole bacteria confirmed binding of unlabeled CEA to the displayed scFv. The modified VNP strain exhibited increased localization in the upper gastrointestinal tract of CEA transgenic mice and accumulated in CEA-expressing tumors. Furthermore, treatment with a single dose of the VNP derivative inhibited growth of MC38CEA tumors and was associated with local accumulation of CD3(+) T cells and CD11b(+) macrophages. The display of antibody fragments on the surface of VNP represents a novel strategy for both targeting CEA-expressing tumors and increasing the immunogenicity of Salmonella-based vaccines for cancer.

We demonstrate the use of luminescent QDs conjugated to antibody fragments to develop solution-phase nanoscale sensing assemblies, based on fluorescence resonance energy transfer (FRET) for the specific detection of the explosive 2,4,6-trinitrotoluene (TNT) in aqueous environments. The hybrid sensor consists of anti-TNT specific antibody fragments attached to a hydrophilic QD via metal-affinity coordination. A dye-labeled TNT analogue prebound in the antibody binding site quenches the QD photoluminescence via proximity-induced FRET. Analysis of the data collected at increasing dye-labeled analogue to QD ratios provided an insight into understanding how the antibody fragments self-assemble on the QD. Addition of soluble TNT displaces the dye-labeled analogue, eliminating FRET and resulting in a concentration-dependent recovery of QD photoluminescence. Sensor performance and specificity were evaluated.

Quantum dots (QDs) are a versatile synthetic photoluminescent nanomaterial whose chemical and photo-physical properties suggest that they may be superior to conventional organic fluorophores for a variety of biosensing applications. We have previously investigated QD-fluorescence resonance energy transfer (FRET) interactions by using the E. coli bacterial periplasmic binding protein - maltose binding protein (MBP) which was site-specifically dye-labeled and self assembled onto the QD surface and allowed us to monitor FRET between the QD donor and the acceptor dye. FRET efficiency increased as a function of the number of dye-acceptor moieties arrayed around the QD donor. We used this system to further demonstrate a prototype FRET based biosensor that functioned in the chemical/nutrient sensing of maltose. There are a number of potential benefits to using this type of QD-FRET based biosensing strategy. The protein attached to the QDs surface functions as a biosensing and biorecognition element in this configuration while the QD acts as both nanoscaffold and FRET energy donor. In this report, we show that the sensor design can be extended to target a completely unrelated analyte, namely the explosive TNT. The sensor consists of anti-TNT antibody fragments self-assembled onto the QD surface with a dye-labeled analog of TNT (TNB coupled to AlexaFluor 555 dye) prebound in the fragment binding site. The close proximity of dye to QD establishes a baseline level of FRET and addition of TNT displaces the TNB-dye analog, recovering QD photoluminescence in a concentration dependent manner. Potential benefits of this QD sensing strategy are discussed.

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.

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.

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.

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 cell's 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.

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.

We would like to comment on the paper recently published by Marks and colleagues (Garcia-Rodriguez et al., 2011) developing human monoclonal antibodies binding multiple serotypes of botulinum neurotoxin. It is stated in the introduction that, ‘Polyclonal sera raised against one serotype does not cross react with other serotypes. Moreover, no mAbs have been reported which bind to more than one BoNT serotype,’ and in the discussion, ‘. . .we can find no examples in the literature of such cross-serotype binding BoNT antibodies’, ‘. . .polyclonal sera uniquely define each “serotype”’. We would like to suggest that the authors qualify these statements to more accurately capture the intricacies of BoNT antibody reactivity discovered by their predecessors. In the course of our own work to generate absolute specificity to the seven BoNT serotypes (Conway et al., 2010) we came across several examples of cross-reactivity among the serotypes in the literature and were made aware of more by an insightful reviewer. It is certainly the case that not all cross-reactivities will be neutralizing, one such example being the mouse monoclonal antibody characterized by Tsuzuki et al. (1988) that recognizes a light chain epitope common to serotypes B, C1, D and E. Yet, some crossreactivities do show neutralization, one example being the hyperimmunization response to the heavy chain receptor binding domain of serotype E offering cross-protection to serotype A in a mouse model (Baldwin et al., 2005). We hope that by clarifying the serological distances and similarities of the seven serotypes, the exciting work of Garcia-Rodriguez et al., may be more accurately placed in context within the vast and often complex scope of BoNT diversity. References

Lama immunoglobulins consist of conventional antibody (IgG1) and unique forms that lack light chains, called heavy chain antibodies (IgG2 and IgG3). These unusual antibodies possess unique properties ideal for diagnostics and therapeutics. To evaluate the IgG from a llama immunised with botulinum complex toxoids A through F each IgG subclass was tested as capture

The ability to detect low levels of 2,4,6-trinitrotoluene (TNT) in aqueous samples is important due to the toxicity of both TNT and its breakdown products. We have been characterizing recombinant anti-TNT antibodies isolated from the Griffin library of phage displayed scFvs by selection for binders to the TNT-surrogate 2,4,6-trinitrobenzene (TNB) coupled to the protein bovine serum albumin. Two candidate antibody fragments, TNB1 and TNB2, were isolated and evaluated by ELISA for their ability to bind to TNB coupled to the protein ovalbumin. Competition ELISA was then used to demonstrate antibody fragment binding to TNT in solution and to examine cross-reactivity towards several TNT-related compounds and other explosives. Both recombinant antibody fragments were incorporated into a continuous flow assay for the detection of TNT. TNB2, the best single chain antibody, showed a limit of detection of 1 ng ml(-1), comparable to a commercially available anti-TNT antibody in the same assay format.