Dataset Information

Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

ABSTRACT: In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prN???) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prN???. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prN?85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prN??? in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus infections.

SUBMITTER: Pereira SS

PROVIDER: S-EPMC4171512 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

Pereira Soraya S SS Moreira-Dill Leandro S LS Morais Michelle S S MS Prado Nidiane D R ND Barros Marcos L ML Koishi Andrea C AC Mazarrotto Giovanny A C A GA Gonçalves Giselle M GM Zuliani Juliana P JP Calderon Leonardo A LA Soares Andreimar M AM Pereira da Silva Luiz H LH Duarte dos Santos Claudia N CN Fernandes Carla F C CF Stabeli Rodrigo G RG

PloS one 20140922 9

In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the ...[more]

PMID: 25243411

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Project description:Despite innovations in cancer therapeutics, cancer remains associated with high mortality and is one of biggest health challenges worldwide. Therefore, developing precise cancer imaging and effective treatments is an unmet clinical need. A relatively novel type of therapeutics are heavy chain variable domain antibody fragments (VHHs) derived from llamas. Here, we explored the suitability of VHHs for cancer imaging and therapy through reviewing the existing literature. We searched the MEDLINE, EMBASE and Cochrane databases and identified 32 papers on molecular imaging and 41 papers on therapy that were suitable for comprehensive reviewing. We found that VHHs harbor a higher specificity and affinity compared to mAbs, which contributes to high-quality imaging and less side-effects on healthy cells. The employment of VHHs in cancer imaging showed remarkably shorter times between administration and imaging. Studies showed that 18F and 99mTc are two optimal radionuclides for imaging with VHHs and that site-specific labelling is the optimal conjugation modality for VHHs with radionuclide or fluorescent molecules. We found different solutions for reducing kidney retention and immunogenicity of VHHs. VHHs as anticancer therapeutics have been tested in photodynamic therapy, targeted radionuclide therapy, immunotherapy and molecular targeted therapy. These studies showed that VHHs target unique antigen epitopes, which are distinct from the ones recognized by mAbs. This advantage means that VHHs may be more effective for targeted anticancer therapy and can be combined with mAbs. We found that high cellular internalization and specificity of VHHs contributes to the effectiveness and safety of VHHs as anticancer therapeutics. Two clinical trials have confirmed that VHHs are effective and safe for cancer imaging and therapy. Together, VHHs seem to harbor several advantages compared to mAbs and show potential for application in personalized treatment for cancer patients. VHH-based imaging and therapy are promising options for improving outcomes of cancer patients.

Project description:Solubility of recombinant proteins (i.e., the extent of soluble versus insoluble expression in heterogeneous hosts) is the first checkpoint criterion for determining recombinant protein quality. However, even soluble proteins often fail to represent functional activity because of the involvement of non-functional, misfolded, soluble aggregates, which compromise recombinant protein quality. Therefore, screening of solubility and folding competence is crucial for improving the quality of recombinant proteins, especially for therapeutic applications. The issue is often highlighted especially in bacterial recombinant hosts, since bacterial cytoplasm does not provide an optimal environment for the folding of target proteins of mammalian origin. Antibody fragments, such as single-chain variable fragment (scFv), single-chain antibody (scAb), and fragment antigen binding (Fab), have been utilized for numerous applications such as diagnostics, research reagents, or therapeutics. Antibody fragments can be efficiently expressed in microorganisms so that they offer several advantages for diagnostic applications such as low cost and high yield. However, scFv and scAb fragments have generally lower stability to thermal stress than full-length antibodies, necessitating a judicious combination of designer antibodies, and bacterial hosts harnessed with robust chaperone function. In this review, we discuss efforts on not only the production of antibodies or antibody fragments in microorganisms but also scFv stabilization via (i) directed evolution of variants with increased stability using display systems, (ii) stabilization of the interface between variable regions of heavy (V H ) and light (V L ) chains through the introduction of a non-native covalent bond between the two chains, (iii) rational engineering of V H -V L pair, based on the structure, and (iv) computational approaches. We also review recent advances in stability design, increase in avidity by multimerization, and maintaining the functional competence of chimeric proteins prompted by various types of chaperones.

Dataset Information

Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

Publications

Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

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