key: cord-317501-yblzopc3
authors: Kuhn, Philipp; Fühner, Viola; Unkauf, Tobias; Moreira, Gustavo Marcal Schmidt Garcia; Frenzel, André; Miethe, Sebastian; Hust, Michael
title: Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display
date: 2016-06-21
journal: Proteomics Clin Appl
DOI: 10.1002/prca.201600002
sha: 
doc_id: 317501
cord_uid: yblzopc3

Antibodies are valuable molecules for the diagnostic and treatment of diseases caused by pathogens and toxins. Traditionally, these antibodies are generated by hybridoma technology. An alternative to hybridoma technology is the use of antibody phage display to generate recombinant antibodies. This in vitro technology circumvents the limitations of the immune system and allows—in theory—the generation of antibodies against all conceivable molecules. Phage display technology enables obtaining human antibodies from naïve antibody gene libraries when either patients are not available or immunization is not ethically feasible. On the other hand, if patients or immunized/infected animals are available, it is common to construct immune phage display libraries to select in vivo affinity‐matured antibodies. Because the phage packaged DNA sequence encoding the antibodies is directly available, the antibodies can be smoothly engineered according to the requirements of the final application. In this review, an overview of phage display derived recombinant antibodies against bacterial, viral, and eukaryotic pathogens as well as toxins for diagnostics and therapy is given.

Antibodies are essential molecules as tools for basic research [1], diagnostics [2], and therapy [3] . In the past-and still today-polyclonal antibodies were produced as serum in animals like horses [4] . A milestone in antibody generation was the development of hybridoma technology that allows the production of monoclonal antibodies [5] . But the hybridoma technology has drawbacks like limited number of candidates, possible instability of the aneuploid cell lines [6], inability to provide antibodies against highly conserved antigens and most of all its limited application to generate human antibodies [7] . The hybridoma technology essentially gives murine antibodies that can be used for diagnostic or research purposes. However, their therapeutic applications are limited because repeated administration of murine antibodies can cause human anti-mouse antibody reaction, reducing antibody half-life and leading to severe side effects such as anaphylactic shock [8] . A strategy to circumvent these problems is antibody humanization or the use of transgenic animals in which the original antibody gene repertoire is replaced with a human gene repertoire [9] [10] [11] [12] . Another strategy is the human hybridoma technology resulting in human antibodies [13, 14] , although this technology has the same problems of the murine hybridoma technology regarding the limitation of the immune system.

A technology that overcomes the limitation of the immune system is antibody phage display. Since this technology involves an in vitro selection process, it is completely independent of any immune system. The display method most commonly used today is based on the work of Georg P. Smith on filamentous phage, which infect E. coli [15] . The selection process is called "panning," referring to the gold digger's tool [16] .

M13 phage display technology was further developed 1990/91 for antibodies in three places in parallel: Heidelberg (Germany), Cambridge (UK), and La Jolla (USA) [17] [18] [19] [20] . Two different genetic systems have been developed for the expression of the antibody::pIII (phage protein III) fusion proteins for phage display. First, the antibody genes can be directly inserted into the phage genome fused upstream of the wild-type pIII gene [20] . Second, most successful systems, uncouple antibody expression from phage propagation by providing the genes encoding the antibody::pIII fusion proteins on a separate plasmid (called "phagemid") that contains a phage morphogenetic signal for packaging the vector into the phage particles [18] . No matter the used phage display system, antibody fragments are displayed on the surface of M13 phage and the corresponding antibody gene is packaged in the phage particle. Other phage display technologies using phage lambda [21] or T7 [22] are less suitable for the display of antibody fragments. In addition, these phages are lytic phage that aggravates the practical work. The most common antibody formats used for antibody phage display are the single chain fragment variable (scFv) [23] [24] [25] or fragment antigen binding (Fabs) [26, 27] . Other antibody formats used for phage display are single chain Fabs (scFab), human VH domains (dAbs), the variable domains of camel heavy chains (VHHs) and immunoglobulins of sharks (IgNARs) [28] [29] [30] [31] [32] [33] . Figure 1 shows an antibody phage and different antibody fragments. For veterinary research, chicken libraries are often used [34, 35] . In contrast to what occurs in humans, the diversity of chicken antibody genes is the result of gene conversion, and the N-and C-terminal parts of chicken's VH and VL are always identical, facilitating antibody gene amplification, and library cloning [36, 37] . Also rabbit phage display was used to generate antibodies against pathogens [38] .

In the selection process (panning), the antigen is immobilized to a solid surface, like column matrixes [18] , magnetic beads [39] , or plastic surfaces with high protein-binding capacity such as polystyrene tubes or respectively microtitre wells, which is the most common method [40] . An other option is the panning in solution using biotinylated antigens followed by a "pull-down" with streptavidin beads [41] . The vast excess of nonbinding antibody phage will be removed by stringent washing. Subsequently, the bound antibody phage will be eluted, e.g. by pH shift or trypsin, and reamplified by infection of E. coli. After infection of the phagemid bearing E. coli with a helperphage, new antibody phage will be produced. The selection cycle will be repeated and the number of antigen-specific antibody phage clones should increase with every panning round. Usually 2-3 panning rounds are performed. Finally, monoclonal antibody phage or monoclonal soluble antibodies can be identified by, e.g. ELISA [42] , immunoblot [40] , or flow cytometry [43] . The antibody fragment genes can be subcloned into any other antibody format, e.g. scFv-Fc or IgG [23, 27, 42, 44, 45] . A schema of the selection process is given in Fig. 2 .

Regarding the source of genes, antibodies can be selected from two types of libraries: immune libraries and universal libraries. Immune libraries are constructed from immunized/infected donors and typically used in medical research to obtain an antibody against a particular target antigen, e.g. an infectious pathogen like Ebola virus [46] . An advantage of this kind of library is that the V-genes contain hypermutations and are affinity matured, although its development can be restricted to ethical constraints. The alternative are universal or "single pot" libraries, which includes naïve, semisynthetic and synthetic libraries that are designed to isolate antibody fragments binding to every possible antigen, at least in theory [44, 47] . Naïve libraries are constructed from rearranged V genes from B cells (IgM) of nonimmunized donors. Examples for this library type are the naïve human Fab library constructed by de Haard and colleagues [26] and the HAL scFv libraries [23, 48] . Semisynthetic libraries are constructed from unrearranged V genes from pre-B cells (germline cells) [49] or from one antibody framework [50] in which one or several CDRs, but always the CDR H3, are randomized. Often used semisynthetic libraries are the Tomlinson I and J libraries using one defined framework VH3-23 and Kappa IKV1-39 with randomized CDR2 and CDR3 [51] .

A combination of naïve and synthetic repertoire was used for the FAB310 antibody gene library. In this library, light chains from autoimmune patients were combined with a Fd fragment (VH+CH1) containing synthetic CDR1 and CDR2 in the human VH3-23 framework and naïve CDR3 regions, originated from autoimmune patients [27] . Fully synthetic libraries are made of human frameworks with randomized CDR cassettes [52] [53] [54] . The theoretical size of these universal libraries is usually higher than 10 10 independent clones [24, 48, [54] [55] [56] .

To date, 53 antibodies and antibody conjugates were approved by EMA and/or FDA (status January 2016) (http://www.imgt.org/mAb-DB/query.action, Development status: Phase M in search field) and about 350 antibodies were under development in 2013 [57] . Most approved therapeutic antibodies are for cancer and autoimmune diseases and the annual sales of therapeutic antibodies exceeded 50 billion US$ in 2013 [58] . The mechanisms of therapeutic antibodies are manifold and include neutralization of substances, e.g. toxins [59] or cytokines like tumor necrosis factor alpha [60] , blocking of receptors like epidermal growth factor receptor [61] , binding to cells and modulating the host immune system [62] , or combinations of these effects [63] . Currently, two recombinant antibodies are approved for the treatment of pathogens or toxins. Raxibacumab is a human antibody for anthrax treatment derived from a phage display library from Cambridge Antibody Technology (now Medimmune, part of AstraZeneca) in cooperation with Human Genome Science (now GlaxoSmithKline) [64] . The antibody palivizumab for the treatment of Respiratory syncytial virus bronchiolitis is a classical humanized antibody [65] . A further antibody, but also not derived from phage display, the Clostridium difficile antibody bezlotoxumab is in clinical phase 3 [66] .

An overview of recombinant antibodies derived from phage display against bacterial and viral pathogens, eukaryotic pathogens (parasites, fungi), and toxins as well as detailed examples for diagnostic and therapy are given in the next sections.

The most therapeutic antibodies against bacterial targets are generated against toxins. These antibodies are described in the section "recombinant antibodies against toxins." The majority of antibodies against bacteria are developed in order to facilitate diagnostics in patients [67, 68] and environmental samples [69, 70] . In general, cultural and microbial detection of bacteria is regarded as standard in diagnostics for many pathogens, e.g. Mycobacterium tuberculosis [71] or Salmonella Typhimurium [72] . Since these methods are often time-consuming and require experienced lab personal, high throughput analysis is often limited. Real-time PCR measurements have been developed for the diagnostics of many bacteria [73] , offering high sensitivity and specificity of detection. But sample treatment is still needed in many cases, including expensive and complex laboratory devices [74] . An other approach is MS for diagnostic, but this technology needs expensive devices [75] . Antibody-based diagnostic like ELISA would be more simple and easy to use, also in developing countries.

In order to generate antibodies with the desired-binding properties, phage-display has been used to select antibodies on proteins or polysaccharides of Chlamydophila psittaci [76] , Chlamydia trachomatis [77] , Haemophilus influenzae [78] , Listeria monocytogenes [79] , Mycobaterium bovis [35] , Mycobacterium tuberculosis [68, 80, 81] , Porphyromonas gingivalis [67] , Ralstonia solanacearum [69] , Salmonella Typhimurium [2, 82] , and Yersinia pestis [83] . But even selections on cell lysate or whole cells were performed on Mycobacterium avium [84] , Bacillus anthracis [70, 85] , Moraxella catarrhalis [86] , Lawsonia intracellularis [87] , Lactobacillus acidophilus [88] , Helicobacter pylori [89] , Brucella melitensis [90] , and Bordetella pertussis [91] . In the following paragraphs, we give detailed examples for antibody generation using phage display and antibody engineering against different bacterial pathogens.

Tuberculosis (TB) is a bacterial infection caused by Mycobacterium tuberculosis (Mtb), a pathogen with particular cell wall and membrane characteristics that is able to infect and multiply within alveolar macrophages leading to cough, weakness, and fever [92] . In 2013, 9 million people were infected and 1.5 million died worldwide [93] . In order to improve treatment and control disease spread, TB must be diagnosed as early as possible. One prominent target for this purpose is the bacterial Antigen 85 (Ag85) that is the most abundant protein secreted by Mtb [94] . It is a complex of three highly homolog proteins (Ag85A, Ag85B, and Ag85C) with a molecular size of 30-32 kDa each [95] . In a novel approach by Ferrara et al. [68] monoclonal antibodies were selected against the Ag85 complex by combining phage-and yeast-display. First, a naïve scFv-library was preselected against Ag85 by phage display, reducing the diversity from ß10 11 unique scFv clones to ß10 5 colony forming units. Second, the enriched sublibrary was cloned into a yeast-display system. Each clone of the selection output was screened individually for antigen binding by FACS. Those with the highest antigenbinding signals were sorted and further analyzed. In total 192 clones were sequenced revealing 111 genetically unique scFv clones. Further screening assays identified seven antibody pairs, which can detect Ag85 down to a concentration of 6.1 nM in the absence of serum and 22.7 nM in its presence. Interestingly, none of the antibodies were absolutely specific for one of the Ag85 subunits of the complex. All three subunits A, B, and C were bound although not equally. In contrast, Fuchs et al. selected five human antibodies against the recombinant Ag85B protein from the naïve HAL7/8 libraries [81] . Three of them bound specifically to the 85B protein in ELISA (LOD: 5 ng/mL). Even sandwich detection of recombinant 85B in ELISA (LOD: 10 ng/mL) and integration into lateral flow immuno assay (LOD: <5 ng/mL) was shown. But antigen detection in Mtb cell extracts or culture filtrates was restricted to direct ELISA and immunoblot assay. The authors of both papers argued that the selected antibodies must be affinity matured in order to reach required detection limits. Successful implementation of this concept was demonstrated by Sixholo et al. [80] . Chicken antibodies were selected from the semisynthetic Nkuku library against a 16 kDa recombinant antigen of M. tuberculosis, also known as heat shock protein X (HspX). After four rounds of panning, three scFvs were identified in ELISA. The clone with the lowest ELISA signal was selected for in vitro engineering using two different approaches. First, a mutant library was generated from the parental scFv by error-prone PCR with a diversity of ß3 × 10 7 unique scFv. This library was selected again on the HspX antigen under more stringent conditions, leading to identification of three mutant scFvs. These contained one or three individual amino acid exchanges, occuring both in CDR and framework regions. All mutants showed increased ELISA-binding signals, reaching a signal ß11 times higher when compared to the parental scFv. Furthermore, the mutants showed improved association and dissociation kinetics in ELISA and SPR analysis. In the other approach, the length of the scFv linker was reduced from 15 amino acids to a single glycine residue, forcing tetramerization of antibodies. Due to cooperative binding, the apparent functional affinity was improved. In ELISA, tetrameric scFv generated a ß14 times higher ELISA signal, and SPR proved increased association and decreased dissociation rates.

Porphyromonas gingivalis is one of the major pathogens involved in periodontitis [96] . Periodontitis is an inflammatory disease, which causes the loosening and loss of teeth. Secreted cysteine proteases like RgpB contribute to disease pathology and represent potential biomarkers for disease detection and progression [97] . Skottrup et al. [67] generated a naïve antibody library from camels with a diversity of ß5 × 10 7 clones. A special feature is that the antibodies consist of a single monomeric VHH domain, also called nanobodies. Advantages of this format are the small size (ß15 kDa), the ease production in E. coli, and the convex paratope that is formed which enables the targeting of cryptic epitopes. The library was selected on immobilized RgpB. One clone was isolated that binds specifically to cell surface displayed and soluble RgpB with an affinity of 362 pM. A detection limit of ß8 × 10 6 cells/mL of saliva was reached when tested by subtractive inhibition ELISA. But catalytic activity of RgpB was not inhibited by antibody binding.

Salmonella Typhimurium, is one of the most important pathogens of foodborne gastrointestinal infections [98] . Meyer et al. [82] first identified novel immunogenic proteins of S. Typhimurium from a genomic library using oligopeptide phage display. In a second step human antibodies were selected against these targets from the naïve HAL gene libraries. For DIVA (differentiating infected from vaccinated animals) vaccine development, S. Typhimurium strains lacking a marker protein, e.g. OmpD (outer membrane protein D) were developed (DIVA vaccines) [99] . To allow the discrimination between vaccinated animals and infected animals a diagnostic of this specific marker is necessary. Here, Meyer et al.

[2] generated scFvs against OmpD for diagnostics purposes. Only some of the generated antibodies were suitable for a competitive ELISA using swine serum showing also the difficulties when developing diagnostic assays that are often hampered by the complexity of serum samples.

An overview of recombinant antibodies generated by phage display against bacterial pathogens is given in Table 1 .

Up to now, a large panel of antibodies against various viruses has been generated from either naïve or immune libraries using phage display technology. Panning against peptides, recombinant viral proteins, or complete virus particles has led to the identification of antibodies directed against human pathogenic viruses such as Sin nombre virus [100] , Dengue virus [101, 102] , Influenza virus [103, 104] , VEEV [105] , Norovirus [106] , SARS coronavirus [107] , or Hepatitis C [108] from naïve antibody gene libraries. Other antibodies were selected from immune antibody gene libraries targeting, e.g. Western equine encephalitis virus (WEEV) [109] , HIV [110, 111] , SARS [112] , Yellow fever virus [113] , or Influenza virus [114, 115] . Semisynthetic libraries were also used to generate antibodies specific for Influenza virus [116] . Beside human and animal viruses, antibodies were also generated against plant viruses [40, 117, 118] . Libraries originated from different species have been successfully employed to isolate virus-specific antibodies in the past such as those from macaque [119] , mouse [120] , chimpanzee [121] , llama [122] , chicken [123] , and human origin [124] .

Most of the virus-specific antibodies have been isolated from libraries in scFv format [125, 126] , although Fab [127, 128] and VHH libraries [122] were also successfully used. An interesting approach was used by Xiao et al. using the antibody CH2 domain as scaffold to generate binders against gp120 of HIV [129] .

Different antibody characteristics have an influence on virus binding and neutralization. Neutralizing antibodies prevent cell binding of the virus. The anti-gp41 antibody HK20 has a higher neutralizaton rate as scFv or Fab compared to IgG showing, that the epitope is less accessable for the IgG format [130] . Another interesting example is the anti-gp41 VHH 2H10. Here, a tryptophan in the CDR3 is not relevant for epitope binding, but essential for virus neutralization [131] .

In the following paragraphs, we give detailed examples for antibody generation using phage display and antibody engineering against different virus groups.

Vaccinia virus is the prototype virus in the genus of Orthopoxvirus. It is a relatively large DNA virus with a genome of about 200 kbp [132] . The genus Orthopoxvirus includes various species such as monkeypox virus, cowpox virus, and especially variola virus that is the causative agent of smallpox in humans. Naturally occurring smallpox has been eradicated in 1977 because of a massive WHO vaccination program that began in 1967. However, no vaccination of the civilian population is conducted nowadays. The potential threat of intentional release has renewed the search for safe and effective smallpox vaccines as case fatality rates of 30% or more among unvaccinated subjects are reported [133] . Because orthopoxviruses are highly related, it is assumed, that immunity against one poxvirus goes along with immunity against most members of the entire virus family [132, 134] . Using an immune scFv phage display library constructed from vaccinia virus immunized patients, a panel of human vaccinia-specific antibodies was selected. Plaque-reduction neutralization tests revealed that seven of these antibodies neutralized vaccinia as well as cowpox virus in vitro. Five of those antibodies additionally neutralized monkeypox virus [124] . Other antibodies were generated from a Fab immune library derived from vaccinia virus immunized chimpanzee. Converted into a chimeric chimpanzee/human IgG format, two antibodies displayed high affinities to vaccinia protein B5 (K d of 0.2 and 0.7 nM). Antibody 8AH8AL was neutralizing in vitro for vaccinia and smallpox virus and proofed to be protective in mice challenged with vaccinia virus even when administered 2 days after challenge. In this model 8AH8AL proved to provide significantly greater protection than that of the previously isolated rat anti-B5 antibody 19C2 [135] . Vaccinia had to be used as model, because the final confirmation of protection against smallpox is not possible.

Ebola virus and Marburg virus, two filoviruses, cause severe hemorrhagic fever and possess high mortality of up to 90% in humans. In addition to public health concerns associated with natural outbreaks, Ebola virus might be a potential agent of biological warfare and bio-terrorism [136] . Human antibodies directed against Ebola virus were selected from a library originated from patients that recovered from infection in the 1995 Ebola virus outbreak in Kikwit, Democratic Republic of Congo [137] . Several antibodies against various viral proteins such as nucleoprotein, envelope glycoprotein, and secreted envelope glycoprotein have been isolated in this study. One antibody (KZ52), specific for envelope glycoprotein, was able to neutralize in vitro as Fab (50% neutralization at 0.4 g/mL) and as full IgG (90% neutralization at 2.6 g/mL) [46] . Follow-up studies were showing effective protection in vivo in a Guinea pig Ebola challenge model when the antibody was administered up to one hour postviral challenge [138] . Interestingly, KZ52 was not protective in macaques challenged with Ebola even if the antibody was given as a two-dose treatment with the first dose one day prior viral challenge and the second dose 4 days postchallenge [139] . A murine scFv and two shark IgNAR V immune libraries were generated against inactivated Zaire Ebola virus to yield various antibodies specific for the viral matrix protein VP40 and the viral nucleoprotein [136] . Interestingly, this work represents the first example of a successful targeted IgNAR V isolation from a shark immune response library. Dengue virus (DENV), a member of the Flaviviridae family, is responsible for at least 100 million symptomatic infections each year and became a major health and economic burden in over 50 countries worldwide [140] [141] [142] . It is a positive strand RNA virus with a ß11 kb genome, that compromise a single open reading frame. The four circulating serotypes of dengue virus show approximately 70% sequence homology [128, 141] . Fab monoclonal antibodies to dengue type 4 virus were isolated from a chimpanzee immune library. Two Fab, namely 5H2 and 5D9 neutralized DENV-4 efficiently with a titer of 0.24-0.58 g/mL by plague reduction neutralization test [143] . Another study selected human scFv antibodies specific to dengue virus envelope protein by panning against recombinant full-length envelope protein and its domain III [102] . Because DENV envelope protein is an essential molecule for virion assembly and virus entry, scFvs selected in this study were shown to exhibit inhibitory effects on DENV infection in vitro [102] . Dengue nonstructural protein 5 (NS5) is essential for viral replication and host immune response modulation, making it an excellent target for dengue-inhibiting antibodies. A naïve human Fab-phage library was screened for NS5-specific antibody fragments using various NS5 variants from Dengue Virus serotypes 1-4 as antigens for panning and characterization [128] . Using NS5 from alternating dengue serotypes for each round of panning, this strategy resulted in the identification of two clones that are cross-reactive against all four dengue serotypes. Another study selected antibodies using phage display by panning with Dengue virus particles directly captured from supernatant of infected Vero cells. Here, highly serotype-specific antibodies were generated. From a total of nine antibodies, seven were shown to be specific to only one serotype. One Dengue-3 selected clone cross-reacted with Dengue 1, whereas another clone showed cross-reactivity with all serotypes despite being selected solely on Dengue 2 particles. Interestingly, all of the obtained antibodies recognized several strains of distinct genotypes within the corresponding serotype [101] . Panning against dengue envelope protein resulted in the identification of an antibody (C9) from a mouse/human chimeric Fab library that cross-reacts with DENV1-3 and neutralizes DENV2 in cell-based assays after conversion into full-length IgG [141] . Besides scFv and Fab, variable domain heavy-chain antibodies (VHH antibodies) were also selected using phage display technology. After four rounds of panning on recombinant DENV 2 NS1 protein, 20 positive clones were selected. Affinity measurements with NS1 revealed a K D value of 2.79 × 10 −8 M for the best VHH antibody P2 [122] .

Venezuelan equine encephalitis virus (VEEV), an alphavirus of the Togoviridae family, causes equine epidemics but can also cause encephalitis in humans [144, 145] . Because this virus is classified as Category B agent by the Centers for Disease Control and Prevention (CDC), much research has been done to generate neutralizing antibodies against it. Antibodies were generated from an immune library from human donors targeting both VEEV envelope glycoproteins E1 and E2 [146] . The isolated Fabs L1A7 and F5 were neutralizing in vitro, with F5 being 300 times more effective than L1A7. Subsequently, F5 was converted into full IgG format and was employed to generate neutralization-escape variants of VEEV for epitope mapping. Within another study, an immune macaque library was used to generate human-like antibodies [119] . One of these antibodies, scFv-Fc ToR67-3B4, was protective in mice when administered 6 h postviral challenge with VEEV Trinidiad strains, showing 80-100% survival after a challenge with 100-fold LD 50 . However, scFv-Fc ToR67-3B4 was not able to neutralize Trinidad strain in vitro, but other VEEV strains instead, showing that neutralization is not mandatory for an in vivo protective antibody [119] . Another study describes the selection of antibodies from a human naïve scFv gene library using complete, active VEEV particles as antigen. In this case, specific detection of the VEEV strains TC83, H12/93, and 230 by the selected antibodies was proven. Remarkably, none of the selected scFv phage clones showed cross-reactivity with Alphavirus species of the Eastern equine encephalitis virus and WEEV antigenic complex or with Chikungunya virus, making them ideal tools for the immunological detection and diagnostic of Alphavirus species [105] . Two different scFv antibody libraries were constructed from WEEV immunized macaques. Subcloned as scFv-Fc, three antibodies from these libraries specifically bound WEEV in ELISA with little or no cross-reactivity with other alphaviruses and were found to be neutralizing in vitro. In this study, the first antibodies against WEEV, that were shown to be neutralizing in vitro, were developed. About 1 ng/mL of the best antibody (ToR69-3A2) neutralized 50% of 5 × 10 4 TCID50/mL WEEV [109] .

Flu is a disease caused by influenza viruses. In the last years, the bird flu (H5N1) and the pandemic swine flu (a variant of H1N1) moved in the research focus. Due to many genetic events, such as antigenic drift and shift, new influenza variants will occur in the future and will challenge vaccine and diagnostic development [147, 148] . Different groups developed antibodies by phage display against influenza viruses. Sui et al. [104] selected antibodies from a nonimmune scFv library against the H5 hemagglutinin ectodomain. Hemagglutinin is a trimer and the extracelluar part consists of a stalk domain and a globular head domain [149] . They identified ten antibodies binding to the trimeric H5, but not monomeric H5. Interestingly, nine of these antibodies shared the same germline framework (VH1-69). These antibodies were converted into IgG1 and were protective in mice with 10 or 15 mg/kg in a prophylactic or therapeutic challenge model, respectively. Very remarkably, some antibodies crossneutralized H1, H2, H5, H6, H8, and H9 influenza strains. These are phage display derived antibodies that are candidates for broad-spectrum influenza immunotherapy [104] .

Rabies is caused by the rabies virus that infects the central nervous system. Before Louis Pasteur developed the rabies vaccination, the disease was always fatal. The current postexposure therapy is based on vaccination-as performed by Pasteur in 1885-and polyclonal anti-rabbies immunoglobulins [150] [151] [152] . One hundred forty-seven unique recombinant antibodies against the rabies glycoprotein were selected from two immune scFv libraries [153] . The neutralization of 27 street rabies viruses was tested in vitro and the best neutralizing antibodies were tested in vivo in a hamster rabies infection model. Here, the antibody CR4098 showed 100% postexposure protection with 40 IU/kg [154] . This antibody was further analyzed in combination with an other human antibody CR57, derived by somatic cell hybridization technique [155] , in in vitro and in vivo models [152] . The safety of this mAb cocktail named CL184 was tested in a clinical phase 1 study [156] and subsequently in phase 2 studies. The antibodies were named Foravirumab (CR4098) and Rafivirumab (CR57).

An overview of recombinant antibodies generated by phage display against viruses is given in Table 2 .

Using phage display technology, a large panel of antibodies against a broad range of eukaryotic pathogens has been generated. These antibodies are directed against pathogenic animals, e.g. Taenia solium [157] , protozoa, e.g. Cryptosporidium parvum [158, 159] , Plasmodium falciparum [160, 161] , or Toxoplasma gondii [162] and fungi such as Aspergillus fumigatus [41] . Not only human pathogens have been in the focus of antibody generation, but also veterinary pathogens like Myxobolus rotundus [163] (a fish pathogen) or Babesia gibsoni (a dog pathogen) [164] and plant pathogens like Aspergillus niger [165] , Fusarium verticilloides [166] , or Sclerotinia sclerotiorum [167] .

Most antibodies generated in these studies derive from human antibody gene libraries, but libraries from mouse [168] , chicken [166] , camel [169] , or macaque [41] origin have been also successfully applied in phage display to generate monoclonal antibodies against eukaryotic pathogens.

In the following paragraphs detailed examples for the use of phage display for the generation of antibodies against different eukaryotic pathogens are given.

Aspergillus fumigates is the causative pathogen of allergic bronchopulmonary aspergillosis, chronic necrotizing aspergillosis, saprophytic aspergilloma, and highly lethal invasive aspergillosis, the most important Aspergillus-related disease [170, 171] . Invasive aspergillosis occurs in immunocompromised individuals for example after hematopoietic stem cell transplantation or solid organ transplantation [172] . Due to its severity, an early diagnosis is crucial for a successful treatment. In 2009, Schütte et al. [41] reported the generation of several antibodies binding specifically to Crf2, an A. fumigates antigen of the glycosyl hydrolase family that is located in the cell wall of the growing hyphae. These antibodies could serve as tools for new diagnostic assays such as serum ELISA or histopathological immunofluorescence microscopy. The antibodies described in this study were isolated from two different phage-displayed scFv libraries: One was a macaque immune library with a theoretical diversity of 1.5 × 10 7 independent clones; while the other was human naïve antibody library HAL4/7 [23] . Both libraries were used in two different panning strategies: the first was performed on recombinant antigen immobilized on immunostrips; the second was carried out in solution with biotinylated antigen. Finally, 16 individual scFv clones were selected, with six clones deriving from the naïve library and ten from the immune library. Interestingly, all antibodies generated on immobilized antigen bind to linear epitopes, whereas all antibodies generated by panning in solution bind to conformational epitopes. Seven of these antibodies bound to their native antigen on growing hyphae of Aspergillus fumigatus and did not show crossreactions to other Aspergillus species or Candida albicans.

Malaria is a life-threatening protozoal infection of the red blood cells and one of the most common mosquito-borne diseases. Currently, an estimated 3.2 billion people in tropical and subtropical countries live at risk of malaria [173, 174] . In humans malaria is elicited by at least five different species of Plasmodium, P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi [175] . P. falciparum is responsible for most malaria-related deaths globally, while P. vivax is the most widespread parasite [176] . Three different approaches to generate antibodies against P. falciparum using phage display are described. In these studies various structures of different developmental stages of the parasite served as antigens. In a first study Roeffen et al. [160] used two scFv immune libraries, constructed from B-lymphocytes from P. falciparum patients with transmission-blocking immunity, to generate antibodies against Pfs48/45, a surface protein of P. falciparum that is expressed during zygote-and macrogamete stages. Pfs48/45 is a potential vaccine candidate since it is a target of transmissionblocking antibodies that are taken up by the mosquito together with the blood meal and block within the mosquito's intestinal tract the oocyte development [177] [178] [179] . The panning was performed on an extract of gametocytes immobilized in immunotubes and the elution was performed by competition with a mixture of four rat monoclonal mAbs, which recognize distinct epitopes on Pfs48/45 (epitopes I, IIb, III, and V). Interestingly, all selected 14 antibody clones bound to epitope III of Pfs48/45. In 2001, Sowa et al. [180] reported the isolation of a human monoclonal antibody against the Block 2 region of Plasmodium falciparum merozoite surface protein-1 (PfMSP-1) by phage display from a malaria patient derived scFv library. Lundquist et al. [161] generated a Fab-immune library from leukocytes of 13 adults with acquired immunity to malaria. Three individual Fabs designated RAM1, RAM2, and RAM3 were isolated from this immune library by panning on a recombinant fragment of the merozite surface protein 3 (MSP-3 ). MSP-3 is involved in heme binding, and antibodies against this protein promote eradication of the parasite by monocytes [181, 182] . A synthetic peptide of the N-terminal fragment of MSP-3 is even used in human clinical trials as vaccine [183] . The antibodies were subcloned into IgG1 and IgG3 format for further analysis. Binding of the antibodies to native parasite protein was demonstrated for all three antibodies in immuno blot and immunofluorescence microscopy. RAM1 and RAM2 also bound to their antigen in fixed and permeabilized cells in flow cytometry. The IgG3 format of RAM1 showed in a functional assay (antibody-dependent cellular inhibition assay, ACDI) an inhibition rate that is comparable to affinity-purified polyclonal anti-MSP-3 211-237 antibodies derived from immune donors. The IgG1 format also showed inhibition in the ACDI, but lower compared to IgG3 [161] . An overview of recombinant antibodies generated by phage display against eukaryotic pathogens is given in Table 3 .

Several toxins are classified by the CDC as category A or B agents (for definition see: www.niaid.nih.gov/topics/ biodefenserelated/biodefense/pages/cata.aspx) that are relevant for diagnostics and therapeutics. They can easily be disseminated and result in high or moderate mortality rates [184] . In this context, the antibody phage display offers a powerful tool for antibody selection and allows the isolation of neutralizing antibodies against complete active toxins or special domains by using different human naïve antibody libraries with high diversity [185] [186] [187] . For toxins, the aim is to find antibodies binding to the cell binding domain of the toxin and neutralize the interaction of this domain with the corresponding cellular target, mainly cell surface proteins. But also antibodies directed against the translocation domain or the enzymatic domain can neutralize the toxicitiy. For the isolation of high-affinity antibodies against specific targets, animals are often immunized with toxoids, nontoxic subunits or selected toxin domains. Well suited are macaques for the construction of immune libraries, because macaque V-genes are very similar to their human counterparts [59, 119, [188] [189] [190] [191] .

In the following paragraphs, we give detailed examples for antibody generation using phage display and antibody engineering against different toxins.

So far, antibody phage display was successfully used for antibody selection against a panel of toxins classified as category A agents, such as from Clostridium botulinum (botulism) [190, [192] [193] [194] and Bacillus anthracis (anthrax) [59] and also against different category B agents, such as staphylococcal enterotoxin B [195, 196] and ricin toxin from Ricinus communis [191, 197] . An example for a high-risk microorganism that [198, 199] . Especially, serotype A is recognized as the most toxic substance known, showing LD 50 of about 1 ng/kg by intravenous route, about 10 ng/kg by the pulmonary route and about 1 g/kg for the oral route [200] . BoNTs are composed of a disulfide bond-linked 50 kDa light chain and a 100 kDa heavy chain. The heavy chain contains two functional domains (Hc and Hn) that are responsible for toxin uptake into nerve cells by receptor-mediated endocytosis and for the translocation of the light chain across the membrane into the neuronal cytosol. Whereas the catalytic domain of the light chain is responsible for the BoNT toxicity. The current approach for treatment of botulism includes the application of human anti-botulism immunoglobulins, such as BabyBIG, or equine anti-toxin serum. But the human serum stock of BabyBIG is limited and the equine anti-toxin may cause hypersensitivity and serum sickness. In these situations, antibody phage display provides a technology to generate toxinneutralizing antibodies against each serotype. For instance, a macaque immune library was used to isolate neutralizing scFv with nM affinities against the light chain of BoNT/A [190, 201, 202] , but also antibodies against the heavy chain or other relevant serotypes of BoNT are of therapeutic interest. Phage display technology was also used for isolation of single domain antibodies (VHH) after immunization of a llama with a cocktail of 7 BoNT toxoids (A-F) [193] . Another approach was the generation of a human antibody gene library after inducing a BoNT/A-specific immune response by in vitro immunization [194] . Furthermore, antibody phage display was used to generate antibodies against other clostridial toxins such as those from Clostridium tetani or Clostridium difficile [188, 203] . Anthrax, another serious infectious disease is caused by Bacillus anthracis, an aerobic, Gram-positive, spore-forming bacterium that is found in soils around the world. Bacillus anthracis secrets two toxins: the lethal toxin (LT) and the edema toxin (ET) [204] . Both are composed of two subunits: the LT consists of the lethal factor (LF), and the protective antigen (PA); while the ET is formed by the edema factor, and PA. It was demonstrated that only LT has an essential role in the pathogenesis of anthrax [205] . The subunit PA is the basis of current vaccines and induces the generation of neutralizing antibodies. In combination with antibiotics, commercial monoclonal antibodies against PA, such as raxibacumab, are commonly used for treatment [206] . In 2012, the FDA approved raxibacumab to treat inhalational anthrax. Due to security issues the use of anti-PA antibodies alone is questionable, since PA could be modified and lose the recognized epitopes while retaining biological activity. An alternative to anti-PA antibodies are antibodies targeting the LF, such as 2LF, which was isolated from an immune library via antibody phage display technology [59] . A combination of an anti-PA antibody with an anti-LF antibody could lead to a synergistic effect and improve the efficacy of the therapy.

An example for bacterial toxins classified as category B agent is staphylococcal enterotoxin B (SEB) from Staphylococcus aureus. This bacterium is a potential causative agent for food-borne illness and produces twenty-one types of staphylococcal enterotoxins that cause symptoms of food poisoning such as abdominal cramps, vomiting, and diarrhea [207, 208] . SEB is a single polypeptide of 28 kDa and is the most potent toxin secreted by S. aureus. As a superantigen, it stimulates T cells and leads to an overproduction of cytokines, causing clinical symptoms such as fever, hypertension, and in some cases death. Phage display was used to generate recombinant antibodies from a murine immune library [196] and to identify the epitope of a SEB-specific monoclonal antibody using a peptide phage library [209] . Furthermore, a human monoclonal antibody against SEB was isolated from a synthetic human antibody gene library that inhibited SEB binding to MHC-II [195] .

The phage display technology was also used to isolate antibodies against ricin, which is also classified as category B agent by CDC. Ricin is a 61-kDa glycoprotein from the castor bean plant (Ricinus communis), which consists of two distinct subunits (RTA and RTB). RTB is a galactose-and N-acetylgalactosamine-specific lectin that binds to specific sugar residues on the cell surface, allowing internalization of the toxic RTA by endocytosis [210] . RTA is an RNA N-glycosidase that irreversibly inactivates eukaryotic ribosomes, leading to the inhibition of protein synthesis [211] . Human-like antibodies were selected by phage display from a macaque immunized with RTA. One antibody, 43RCA, had a picomolar affinity and neutralized the biological activity of ricin in vitro [191] . Furthermore, neutralizing VHH with high affinity was selected from a llama immune library. The best antibody C8 was able to neutralize 100% ricin activity in an in vitro assay using 40 g/mL VHH [197] .

In addition to the different toxins that are classified by the CDC as category A or B agents, the number of toxins is almost endless. Different animals are known to produce high potential toxins containing a complex composition. For example, Tityus serrulatus, known as Brazilian yellow scorpion and the most dangerous scorpion in Brazil, produces a 61-amino acid peptide, called gamma toxin, which is the major toxic component in the venom [212, 213] . Regarding this toxin, a neutralizing antibody was isolated from a human library via phage display and was capable of protecting mice [185] . The same procedure was used for Bothrops jararacussu, a venomous pit viper species endemic in South America. By using a human antibody gene library, different antibodies were selected to inhibit the phospholipase activity of the venom in vitro and reduce the myotoxicity in vivo [214] . Marine organisms can also produce toxins, e.g. the tetrodotoxin (TTX) from pufferfish. ELISA, enzyme-linked immunosorbent assays.

Here, scFv were selected from a human naïve antibody gene library neutralizing 99% of TTX activity in vitro [215] . An Overview of recombinant antibodies generated by phage display against toxins is given in Table 4 .

Antibody phage display allows the generation of recombinant antibodies from different species, including human, llama, camel, macaque, shark, or mice. These antibodies are mainly derived from two types of sources: immune, or naïve libraries. Immune libraries should be preferred when immunized animals or convalescent patients are available, offering the chance to directly isolate affinity matured antibodies. If immunization is not possible or ethically not feasible, naïve antibody gene libraries are an alternative. In such an approach, the antibody generation process is not limited by the immune system. Using antibody phage display a variety of recombinant antibodies was generated for diagnostics and therapy against bacterial, viral pathogens, and eukaryotic pathogens as well as toxins. 

Man-made antibodies

Development of primary and secondary immune responses to mouse monoclonal antibodies used in the diagnosis and therapy of malignant neoplasms

High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice

Production of fully human antibodies by transgenic mice

Human antibodies from transgenic mice

Development trends for human monoclonal antibody therapeutics

Human monoclonal antibodies

Accessing the human repertoire for broadly neutralizing HIV antibodies. mAbs

Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface

Antibody-selectable filamentous fd phage vectors: affinity purification of target genes

Assembly of combinatorial antibody libraries on phage surfaces: the gene III site

A surface expression vector for antibody screening

Making antibody fragments using phage display libraries

Phage antibodies: filamentous phage displaying antibody variable domains

Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library

Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda

A human scFv antibody generation pipeline for proteome research

Application of phage display to high throughput antibody generation and characterization

Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library

A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies

Generation of high-affinity human antibodies by combining donor-derived and synthetic complementaritydetermining-region diversity

Domain antibodies: proteins for therapy

Single chain Fab (scFab) fragment

Single domain camel antibodies: current status

Camelid immunoglobulins and nanobody technology

Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1

Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries

Serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scFvs in inhibition ELISAs

Chicken scFvs and bivalent scFv-C(H) fusions directed against HSP65 of Mycobacterium bovis

Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus

Singlechain antibody fragments from a display library derived from chickens immunized with a mixture of parasite and viral antigens

Recombinant rabbit single-chain antibodies bind to the catalytic and Cterminal domains of HIV-1 integrase protein and strongly inhibit HIV-1 replication

Methodology for selection of human antibodies to membrane proteins from a phage-display library

The production of a genus-specific recombinant antibody (scFv) using a recombinant potyvirus protease

Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus

Construction of human antibody gene libraries and selection of antibodies by phage display

Highthroughput screening of single-chain antibodies using multiplexed flow cytometry

Generating recombinant antibodies to the complete human proteome

High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

Recombinant human monoclonal antibodies to Ebola virus

Making antibodies by phage display technology

Which are the antibodies to watch in 2013? mAbs

The therapeutic monoclonal antibody market

Highaffinity, human antibody-like antibody fragment (singlechain variable fragment) neutralizing the lethal factor (LF) of Bacillus anthracis by inhibiting protective antigen-LF complex formation

Tumor necrosis factor alpha drugs in rheumatoid arthritis: systematic review and metaanalysis of efficacy and safety

Anti-epidermal growth factor receptor monotherapy in the treatment of metastatic colorectal cancer: where are we today? The Oncologist

CD3-specific antibodies: a portal to the treatment of autoimmunity

Monoclonal antibody therapy of cancer

Reduction of respiratory syncytial virus (RSV) in tracheal aspirates in intubated infants by use of humanized monoclonal antibody to RSV F protein

Mechanisms of protection against Clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab

Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis

Using phage and yeast display to select hundreds of monoclonal antibodies: application to antigen 85, a tuberculosis biomarker

Development of specific recombinant monoclonal antibodies against the lipopolysaccharide of Ralstonia solanacearum race 3

Development and implementation of a single-chain Fv antibody for specific detection of Bacillus anthracis spores

Challenges and perspectives in the diagnosis of extrapulmonary tuberculosis

Detecting non-typhoid Salmonella in humans by ELISAs: a literature review

Real-time PCR as a diagnostic tool for bacterial diseases

Sample preparation: the forgotten beginning

A systematic review of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry compared to routine microbiological methods for the time taken to identify microbial organisms from positive blood cultures

Analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from Chlamydophila psittaci

Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human singlechain antibodies to protein antigens

Human Fab fragments specific for the Haemophilus influenzae b polysaccharide isolated from a bacteriophage combinatorial library use variable region gene combinations and express an idiotype that mirrors in vivo expression

High affinity anti-Internalin B VHH antibody fragments isolated from naturally and artificially immunized repertoires

Improving the characteristics of a mycobacterial 16 kDa-specific chicken scFv

Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B

Identification of immunogenic proteins and generation of antibodies against Salmonella typhimurium using phage display

Development of phage-based single chain Fv antibody reagents for detection of Yersinia pestis

Nimotuzumab and cetuximab block ligand-independent EGF receptor signaling efficiently at different concentrations

Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells

Phage antibodies obtained by competitive selection on complement-resistant Moraxella (Branhamella) catarrhalis recognize the high-molecular-weight outer membrane protein

Isolation of Lawsonia intracellularis specific single-chain Fv antibody fragments from phage display library

Using phage display selected antibodies to dissect microbiomes for complete de novo genome sequencing of low abundance microbes

Generation and characterization of human monoclonal scFv antibodies against Helicobacter pylori antigens

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

Construction and characterization of single-chain variable fragment antibodies directed against the Bordetella pertussis surface adhesins filamentous hemagglutinin and pertactin

Is Mycobacterium tuberculosis a closer relative to Gram-positive or Gram-negative bacterial pathogens? Tuberc

The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microbiol

A family of cross-reacting proteins secreted by Mycobacterium tuberculosis

Microbial complexes in subgingival plaque

The role of gingipains in the pathogenesis of periodontal disease

The Salmonella enterica pan-genome

Immunization of pigs to prevent disease in humans: construction and protective efficacy of a Salmonella enterica serovar Typhimurium live negative-marker vaccine

Selection and characterization of scFv antibodies against the Sin Nombre hantavirus nucleocapsid protein

Selection of phage-displayed human antibody fragments on Dengue virus particles captured by a monoclonal antibody: application to the four serotypes

Human monoclonal single-chain antibodies specific to dengue virus envelope protein

Neutralizing human monoclonal antibody against H5N1 influenza HA selected from a Fab-phage display library

Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses

Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV)

Isolation of cross-reactive human monoclonal antibodies that prevent binding of human noroviruses to histoblood group antigens

Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association

Genetically engineered singlechain Fvs of human immunoglobulin against hepatitis C virus nucleocapsid protein derived from universal phage display library. Asian Pac

Human-like antibodies neutralizing Western equine encephalitis virus

Production of recombinant scFv against p24 of human immunodeficiency virus type 1 by phage display technology

Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3-and a trimer-specific gp41 antibody

Human neutralizing Fab molecules against severe acute respiratory syndrome coronavirus generated by phage display

Antibody responses against wild-type yellow fever virus and the 17D vaccine strain: characterization with human monoclonal antibody fragments and neutralization escape variants

Generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza A H5N1 viruses

Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells

Molecular signatures of hemagglutinin stem-directed heterosubtypic human neutralizing antibodies against influenza A viruses

Cucumber mosaic cucumovirus antibodies from a synthetic phage display library

Generation and characterization of a recombinant antibody fragment that binds to the coat protein of grapevine leafroll-associated virus 3

Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in vitro and in vivo

Selection and characterization of single-chain recombinant antibodies against infectious haematopoietic necrosis virus from mouse phage display library

Humanized monoclonal antibodies derived from chimpanzee Fabs protect against Japanese encephalitis virus in vitro and in vivo

Development of VHH antibodies against dengue virus type 2 NS1 and comparison with monoclonal antibodies for use in immunological diagnosis

A large semi-synthetic single-chain Fv phage display library based on chicken immunoglobulin genes

The neutralizing human recombinant antibodies to pathogenic Orthopoxviruses derived from a phage display immune library

Protective and therapeutic capacity of human single-chain Fv-Fc fusion proteins against West Nile virus

Novel phage display-derived H5N1-specific scFvs with potential use in rapid avian flu diagnosis

Alternative recognition of the conserved stem epitope in influenza A virus hemagglutinin by a VH3-30-encoded heterosubtypic antibody

Identification and molecular characterization of human antibody fragments specific for dengue NS5 protein

A large library based on a novel (CH2) scaffold: identification of HIV-1 inhibitors

Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition

Vaccination of BALB/c mice with Escherichia coli-expressed vaccinia virus proteins A27L, B5R, and D8L protects mice from lethal vaccinia virus challenge

Smallpox as a biological weapon: medical and public health management. Working Group on Civilian Biodefense

Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox

Chimpanzee/human mAbs to vaccinia virus B5 protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus

Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries

Ebola virus can be effectively neutralized by antibody produced in natural human infection

Pre-and postexposure prophylaxis of Ebola virus infection in an animal model by passive transfer of a neutralizing human antibody

Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys

Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions

Phage display approaches for the isolation of monoclonal antibodies against dengue virus envelope domain III from human and mouse derived libraries

High affinity human antibody fragments to dengue virus non-structural protein 3

Identification of chimpanzee Fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin G1 antibody that is highly efficient for neutralization of dengue type 4 virus

Venezuelan equine encephalitis

Venezuelan equine encephalitis

The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity

Continuing challenges in influenza

Emerging influenza strains in the last two decades

Influenza virus hemagglutinin stalkbased antibodies and vaccines

The role of vaccination in rabies prevention

The prevention and management of rabies

Comparison of an anti-rabies human monoclonal antibody combination with human polyclonal anti-rabies immune globulin

The human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries

Novel human monoclonal antibody combination effectively neutralizing natural rabies virus variants and individual in vitro escape mutants

Biological characterization of human monoclonal antibodies to rabies virus

First administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity

Development of specific scFv antibodies to detect neurocysticercosis antigens and potential applications in immunodiagnosis

Single-chain variable fragment antibodies selected by phage display against the sporozoite surface antigen P23 Of Cryptosporidium parvum

Single chain variable fragment antibodies selected by phage display against the sporozoite surface antigen S16 of Cryptosporidium parvum

Recombinant human antibodies specific for the Pfs48/45 protein of the malaria parasite Plasmodium falciparum

Human recombinant antibodies against Plasmodium falciparum merozoite surface protein 3 cloned from peripheral blood leukocytes of individuals with immunity to malaria demonstrate antiparasitic properties

Construction and characterization of recombinant single-chain variable fragment antibodies against Toxoplasma gondii MIC2 protein. Parasitology

A combined phage display ScFv library against Myxobolus rotundus infecting crucian carp, Carassius auratus auratus (L.), in China

Detection of Babesia gibsoni by reaction of phage display single chain antibodies with P50 proteins

Isolation and characterization of the human monoclonal antibodies C10 in single-chain fragment variable (scFv) format to glucose oxidase from Aspergillus niger

Generation of a highly reactive chicken-derived single-chain variable fragment against Fusarium verticillioides by phage display

Isolation, expression and characterization of two single-chain variable fragment antibodies against an endopolygalacturonase secreted by Sclerotinia sclerotiorum

Isolation from phage display libraries of single chain variable fragment antibodies that recognize conformational epitopes in the malaria vaccine candidate, apical membrane antigen-1

Parallel selection of multiple anti-infectome nanobodies without access to purified antigens

Pulmonary aspergillosis: a spectrum of disease

Aspergillus fumigatus and aspergillosis

Patients at high risk of invasive fungal infections: when and how to treat

Mosquito-borne diseases

Detection of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae merozoite surface protein 1-p19 antibodies in human malaria patients and experimentally infected nonhuman primates

Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening

Challenges in antimalarial drug treatment for vivax malaria control

Target antigens of transmission-blocking immunity on gametes of Plasmodium falciparum

Biosynthesis of the target antigens of antibodies blocking transmission of Plasmodium falciparum

Sequential expression of antigens on sexual stages of Plasmodium falciparum accessible to transmissionblocking antibodies in the mosquito

Isolation of a monoclonal antibody from a malaria patient-derived phage display library recognising the Block 2 region of Plasmodium falciparum merozoite surface protein-1

Merozoite surface protein-3: a malaria protein inducing antibodies that promote Plasmodium falciparum killing by cooperation with blood monocytes

Plasmodium falciparum merozoite surface protein 3: oligomerization, self-assembly, and heme complex formation

Phase I malaria vaccine trial with a long synthetic peptide derived from the merozoite surface protein 3 antigen. Infect. Immun

Antibodies for biodefense

Isolation and characterization of a human antibody fragment specific for Ts1 toxin from Tityus serrulatus scorpion

Human monoclonal ScFv that inhibits cellular entry and metalloprotease activity of tetanus neurotoxin. Asian Pac

Single chain variable fragment antibodies against Shiga toxins isolated from a human antibody phage display library

A highaffinity macaque antibody Fab with human-like framework regions obtained from a small phage display immune library

Development of human-Like scFv-Fc neutralizing botulinum neurotoxin E

Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library

Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity

Genetic and immunological comparison of anti-botulinum type A antibodies from immune and non-immune human phage libraries

Llama single domain antibodies specific for the 7 botulinum neurotoxin serotypes as heptaplex immunoreagents

Generation of a recombinant full-length human antibody binding to botulinum neurotoxin A

Inhibition of toxic shock by human monoclonal antibodies against staphylococcal enterotoxin B

Construction of a single-chain variable-fragment antibody against the superantigen Staphylococcal enterotoxin B

Development of antiricin single domain antibodies toward detection and therapeutic reagents

Botulinum toxin as a biological weapon: medical and public health management

A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins

Botulism: cause, effects, diagnosis, clinical and laboratory identification, and treatment modalities

Isolation of a nanomolar scFv inhibiting the endopeptidase activity of botulinum toxin A, by single-round panning of an immune phage-displayed library of macaque origin

Isolation of nanomolar scFvs of non-human primate origin, cross-neutralizing botulinum neurotoxins A1 and A2 by targeting their heavy chain

Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain

Anthrax lethal and edema toxins in anthrax pathogenesis

Anthrax as a biological weapon, 2002: updated recommendations for management

Raxibacumab: potential role in the treatment of inhalational anthrax

Identification and characterization of two novel staphylococcal enterotoxins, types S and T

Staphylococcal enterotoxin-like toxins U2 and V, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster

Generation of Fab fragment-like molecular recognition proteins against staphylococcal enterotoxin B by phage display technology

Mechanism of cell entry and toxicity of an affinity-purified lectin from Ricinus communis and its differential effects on normal and virus-transformed fibroblasts

The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins

Modification of Na channel gating by an alpha scorpion toxin from Tityus serrulatus

Scorpion toxins from Centruroides noxius and Tityus serrulatus. Primary structures and sequence comparison by metric analysis

Human antibody fragments specific for Bothrops jararacussu venom reduce the toxicity of other Bothrops sp. venoms

Human ScFv that block sodium ion channel activity of tetrodotoxin

Protein Targeting Compounds

Phage-display antibody detection of Chlamydia trachomatis-associated antigens

Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile

Isolation of high-affinity single-chain antibodies against Mycobacterium avium subsp. paratuberculosis surface proteins from sheep with Johne's disease

Generation and characterization of a novel recombinant antibody against LMP1-TES1 of Epstein-Barr virus isolated by phage display

Production and application of recombinant antibodies to foot-and-mouth disease virus non-structural protein 3ABC

Isolation of recombinant antibodies (scFvs) to grapevine virus B

Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of hantavirus pulmonary syndrome

Neutralising human recombinant antibodies to human cytomegalovirus glycoproteins gB and gH

Potent neutralization of Hendra and Nipah viruses by human monoclonal antibodies

Four chimpanzee monoclonal antibodies isolated by phage display neutralize hepatitis a virus

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen

Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

Human recombinant antibodies specific for hepatitis C virus core and envelope E2 peptides from an immune phage display library

Identification of a human epitope in hepatitis C virus (HCV) core protein using a molecularly cloned antibody repertoire from a non-symptomatic, anti-HCVpositive patient

Nonneutralizing human antibody fragments against hepatitis C virus E2 glycoprotein modulate neutralization of binding activity of human recombinant Fabs

Identification by phage display and characterization of two neutralizing chimpanzee monoclonal antibodies to the hepatitis E virus capsid protein

Recombinant human Fab to glycoprotein D neutralizes infectivity and prevents cell-to-cell transmission of herpes simplex viruses 1 and 2 in vitro

Directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries

Detection and typing of herpes simplex viruses by using recombinant immunoglobulin fragments produced in bacteria

Cross-reactive HIV-1 neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp140) isolated from a patient (R2) with broadly HIV-1 neutralizing antibodies. Virology

Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C

A monoclonal Fab derived from a human nonimmune phage library reveals a new epitope on gp41 and neutralizes diverse human immunodeficiency virus type 1 strains

A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope

Human neutralizing human immunodeficiency virus type 2-specific Fab molecules generated by phage display

A recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo

Human single-chain antibodies that neutralize homologous and heterologous strains and clades of influenza A virus subtype H5N1

Fab MAbs specific to HA of influenza virus with H5N1 neutralizing activity selected from immunized chicken phage library

Isolation of recombinant phage antibodies targeting the hemagglutinin cleavage site of highly pathogenic avian influenza virus

Human monoclonal ScFv specific to NS1 protein inhibits replication of influenza viruses across types and subtypes

Construction of human Fab (gamma1/kappa) library and identification of human monoclonal Fab possessing neutralizing potency against Japanese encephalitis virus

Neutralizing human Fab fragments against measles virus recovered by phage display

Identification of human single-chain antibodies with broad reactivity for noroviruses

Development of Norwalk virus-specific monoclonal antibodies with therapeutic potential for the treatment of Norwalk virus gastroenteritis

Chimpanzee-human monoclonal antibodies for treatment of chronic poliovirus excretors and emergency postexposure prophylaxis

Human recombinant Puumala virus antibodies: crossreaction with other hantaviruses and use in diagnostics

A neutralizing recombinant human antibody Fab fragment against Puumala hantavirus

Selection of single chain variable fragments (scFv) against the glycoprotein antigen of the rabies virus from a human synthetic scFv phage display library and their fusion with the Fc region of human IgG1

The analysis of VH and VL genes repertoires of Fab library built from peripheral B cells of human rabies virus vaccinated donors

Generation and characterization of neutralizing human recombinant antibodies against antigenic site II of rabies virus glycoprotein

Single domain antibody multimers confer protection against rabies infection

Single-chain variable fragment (scFv) antibodies against rotavirus NSP4 enterotoxin generated by phage display

Selection of single-chain antibodies against the VP8* subunit of rotavirus VP4 outer capsid protein and their expression in Lactobacillus casei

SARS patientsderived human recombinant antibodies to S and M proteins efficiently neutralize SARS-coronavirus infectivity

Chicken single-chain variable fragments against the SARS-CoV spike protein

Simian immunodeficiency virus (SIV) envelopespecific Fabs with high-level homologous neutralizing activity: recovery from a long-term-nonprogressor SIVinfected macaque

Isolation of single-chain antibody fragments against Venezuelan equine encephalomyelitis virus from two different immune sources

Human monoclonal Fab antibodies against West Nile virus and its neutralizing activity analyzed in vitro and in vivo

Identification of a WSSV neutralizing scFv antibody by phage display technology and in vitro screening

Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens

Differentiation of Candida albicans and Candida dubliniensis by using recombinant human antibody single-chain variable fragments specific for hyphae

Inhibition of Candida albicans adhesion by recombinant human antibody single-chain variable fragment specific for Als3p

A phagedisplayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

Singlechain antibodies produced by phage display against the C-terminal 19 kDa region of merozoite surface protein-1 of Plasmodium yoelii reduce parasite growth following challenge

Structural and functional characterization of a novel scFv anti-HSP60 of Strongyloides sp

Nanobodies, a promising tool for species-specific diagnosis of Taenia solium cysticercosis

VHH, bivalent domains and chimeric Heavy chainonly antibodies with high neutralizing efficacy for scorpion toxin AahI

Isolation of single chain variable fragment (scFv) specific for Cry1C toxin from human single fold scFv libraries

Production of human antibody fragments binding to melittin and phospholipase A2 in Africanised bee venom: minimising venom toxicity

Monoclonal antibody fragment from combinatorial phage display library neutralizes alpha-latrotoxin activity and abolishes black widow spider venom lethality, in mice

A V H H that neutralizes the zinc metalloproteinase activity of botulinum neurotoxin type A

Molecular characterization of murine humoral immune response to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries

Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B. mAbs

In vivo neutralization of botulinum neurotoxins serotype E with heavy-chain camelid antibodies (VHH)

A novel multivalent, single-domain antibody targeting TcdA and TcdB prevents fulminant Clostridium difficile infection in mice

Selection of nanobodies that block the enzymatic and cytotoxic activities of the binary Clostridium difficile toxin

Phagedisplay derived single-chain fragment variable (scFv) antibodies recognizing conformational epitopes of Escherichia coli heat-labile enterotoxin B-subunit

Production of a single-chain variable fragment antibody against fumonisin B1

A strategy for the generation of specific human antibodies by directed evolution and phage display. An example of a single-chain antibody fragment that neutralizes a major component of scorpion venom

Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library

Single chain antibody fragment with serine protease inhibitory property capable of neutralizing toxicity of Trimeresurus mucrosquamatus venom

Development and characterization of recombinant antibody fragments that recognize and neutralize in vitro Stx2 toxin from Shiga toxin-producing Escherichia coli

A single VHH-based toxin-neutralizing agent and an effector antibody protect mice against challenge with Shiga toxins 1 and 2

Construction of a single chain variable fragment antibody (scFv) against tetrodotoxin (TTX) and its interaction with TTX. Toxicon off

Human monoclonal ScFv neutralize lethal Thai cobra, Naja kaouthia, neurotoxin

Screening for a single-chain variable-fragment antibody that can effectively neutralize the cytotoxicity of the Vibrio parahaemolyticus thermolabile hemolysin

A human single-chain variable fragment targeting to Vibrio vulnificus RtxA toxin