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

Pelat, Thibaut; Hust, Michael; Hale, Martha L.; Lefranc, Marie‐Paule; Dübel, Stefan; Thullier, Philippe

doi:10.1186/1472-6750-9-60

Cited by 88 publications

(84 citation statements)

References 45 publications

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“…Mechaly and coworkers (31) reported ScFv antibodies against Bacillus anthracis spores with an affinity in the lownanomolar range (30 nM). On the other hand, even a picomolar level of K D (affinity constant) (41 pM) has also been reported for ricin toxin (36). Further, anti-SEB ScFv antibody did not cross-react with SEs (SEA, SEC1, SEC2, SEC3, and SED) commonly implicated in SFP outbreaks, making it useful for SEB detection.…”

Section: Discussionmentioning

confidence: 95%

“…Further, anti-SEB ScFv antibody did not cross-react with SEs (SEA, SEC1, SEC2, SEC3, and SED) commonly implicated in SFP outbreaks, making it useful for SEB detection. Reports on the construction of recombinant antibodies for the detection of food-borne pathogens (29,34), toxins (9,10), and biological warfare agents (9,10,14,31,36) are scanty. Recombinant Fab antibodies, isolated from a phage display library, have been used in detection of botulinum toxin (9,10).…”

Section: Discussionmentioning

confidence: 99%

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Construction of a Single-Chain Variable-Fragment Antibody against the Superantigen Staphylococcal Enterotoxin B

Singh

Agrawal

Kamboj

et al. 2010

Appl Environ Microbiol

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Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detection of SEB is of the utmost importance. High-affinity and specific monoclonal antibodies are being used for detection of SEB, but hybridoma clones tend to lose their antibody-secreting ability over time. This problem can be overcome by the use of recombinant antibodies produced in a bacterial system. In the present investigation, genes from a hybridoma clone encoding monoclonal antibody against SEB were immortalized using antibody phage display technology. A murine phage display library containing single-chain variable-fragment (ScFv) antibody genes was constructed in a pCANTAB 5E phagemid vector. Phage particles displaying ScFv were rescued by reinfection of helper phage followed by four rounds of biopanning for selection of SEB binding ScFv antibody fragments by using phage enzyme-linked immunosorbent assay (ELISA). Soluble SEB-ScFv antibodies were characterized from one of the clones showing high affinity for SEB. The anti-SEB ScFv antibody was highly specific, and its affinity constant was 3.16 nM as determined by surface plasmon resonance (SPR). These results demonstrate that the recombinant antibody constructed by immortalizing the antibody genes from a hybridoma clone is useful for immunodetection of SEB.Staphylococcus aureus is one of the most prevalent causative agents of food-borne illness throughout the world. The illness occurs following ingestion of staphylococcal enterotoxins (SEs) produced by S. aureus in the contaminated food. The symptoms of food poisoning include nausea, vomiting, abdominal cramps, and diarrhea. Twenty-one different types of SEs, i.e., staphylococcal enterotoxin A (SEA) to staphylococcal enterotoxin E (SEE) and staphylococcal enterotoxin G (SEG) to staphylococcal enterotoxin V (SEV), have already been discovered (33, 40). The immunomodulatory effects of SEs, such as immunosuppression, enhancement of endotoxic shock, and induction of cytokine release, can be attributed to the fact that SEs are potent T-cell mitogens. The ability of SEs to induce proliferation of T cells is somewhat similar to conventional antigen presentation by major histocompatibility complex (MHC) class II molecules to T-cell receptors (TCRs). However, unlike conventional antigens, the T-cell mitogenicity of SEs does not require antigen processing and lacks the normal specificity to the TCR for specific epitopes in response to conventional antigens. This bypass of normal TCR specificity for conventional T-cell epitopes results in the stimulation of a substantial proportion of the total T-cell population, and therefore, staphylococcal enterotoxins are referred to as superantigens. The stimulation of T cells leads to over...

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Construction of a Single-Chain Variable-Fragment Antibody against the Superantigen Staphylococcal Enterotoxin B

Singh

Agrawal

Kamboj

et al. 2010

Appl Environ Microbiol

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“…With these mice, human antibodies can be generated by hybridoma technology but these transgenic animals are available in a very limited number of laboratories only (Fishwild et al, 1996;Jakobovits, 1995;Lonberg and Huszar, 1995;Nelson et al, 2010). For the isolation of antibodies directed against toxins, animals are immunised by non-toxic subunits or even selected domains from these subunits (Pelat et al, 2009a;Pelat et al, 2007;Scotcher et al, 2010;Winterroth et al, 2010). A technology which circumvents the limitations of the immune system is antibody phage display.…”

Section: Antibody Phage Displaymentioning

confidence: 99%

Development of Human and Macaque Antibodies Using Antibody Phage Display for the Detection of Equine Encephalitis Viruses

Thullier¹,

Hülseweh²,

Pelat³

et al. 2011

Pathogenesis of Encephalitis

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“…Combinations of forward and reverse primers were used to amplify the regions coding for the variable regions VLK and VH as previously described. 27 PCR products were cloned in the pGemT vector (Promega, Madison, Wisconsin) according to the manufacturer's instructions, yielding two sub-libraries encoding the heavy chains (Fd fragment) or the kappa light chains.…”

Section: Construction and Screening Of The Anti-marv Antibody Gene LImentioning

confidence: 99%

Generation and characterization of protective antibodies to Marburg virus

Froude

Pelat²,

Miethe

et al. 2017

mAbs

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Marburg virus (MARV) and Ebola virus (EBOV) have been a source of epidemics and outbreaks for several decades. We present here the generation and characterization of the first protective antibodies specific for wild-type MARV. Non-human primates (NHP), cynomolgus macaques, were immunized with viral-replicon particles expressing the glycoproteins (GP) of MARV (Ci67 isolate). An antibody fragment (single-chain variable fragment, scFv) phage display library was built after four immunogen injections, and screened against the GP 1-649 of MARV. Sequencing of 192 selected clones identified 18 clones with distinct V H and V L sequences. Four of these recombinant antibodies (R4A1, R4B11, R4G2, and R3F6) were produced in the scFv-Fc format for in vivo studies. Mice that were challenged with wild-type Marburg virus (Ci67 isolate) receiving 100 mg of scFv-Fc on days ¡1, 1 and 3 demonstrated protective efficacies ranging from 75-100%. The amino-acid sequences of the scFv-Fcs are similar to those of their human germline counterparts, sharing an identity ranging between 68 and 100% to human germline immunoglobulin. These results demonstrate for the first time that recombinant antibodies offer protection against wild-type MARV, and suggest they may be promising candidates for further therapeutic development especially due to their human homology.

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

Abstract: Background: Ricin is a lethal toxin that inhibits protein synthesis. It is easily extracted from a ubiquitously grown plant, Ricinus communis, and thus readily available for use as a bioweapon (BW). Anti-ricin antibodies provide the only known therapeutic against ricin intoxication.

Cited by 88 publications

References 45 publications

Construction of a Single-Chain Variable-Fragment Antibody against the Superantigen Staphylococcal Enterotoxin B

Construction of a Single-Chain Variable-Fragment Antibody against the Superantigen Staphylococcal Enterotoxin B

Development of Human and Macaque Antibodies Using Antibody Phage Display for the Detection of Equine Encephalitis Viruses

Generation and characterization of protective antibodies to Marburg virus

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