Generation of recombinant antibodies against the Venezuelan equine encephalitis virus

Das Venezuelanische Pferdeenzephalitis-Virus (VEEV) gehoert zu der Gattung Alphaviren. Bisher sind nur wenige monoklonale, hochaffine Antikoerper gegen diese Viren fuer die schnelle Diagnostik und Therapie verfuegbar. Ziel dieser Arbeit war die Generierung humaner rekombinanter Antikoerperfragmente gegen VEEV aus universellen Antikoerpergenbibliotheken zur verbesserten Detektion. Mittels der Phagen-Display-Technologie wurden erstmals virusspezifische Antikoerperfragmente durch Selektionen mit kompletten VEEV-Partikeln isoliert. Durch Optimierung der Selektionsstrategien konnten zwoelf VEEV-spezifische scFv-Klone aus zwei naiven Antikoerpergenbibliotheken isoliert werden. Zur Charakterisierung wurden scFv-praesentierende, filamentoese M13-Phagen dieser Klone produziert und zur Detektion verschiedener Viruspraeparationen eingesetzt. Die scFv-praesentierenden Phagen erkannten, eingesetzt als Detektionsantikoerper, verschiedene VEEV-Staemme, jedoch keine anderen Alphaviren. Der vielseitige und hochsensitive Einsatz von scFv-praesentierenden Phagen zur Virusdetektion wurde in dieser Arbeit demonstriert. Mittels Immunfaerbungen wurde gezeigt, dass strukturelle Epitope von den scFv-praesentierenden Phagen erkannt werden. Diese Strukturen konnten im Lysat mittels ELISA und auf der Zelloberflaeche VEEV-infizierter Zellen mittels Immunhistochemie spezifisch nachgewiesen werden. Zur Optimierung der Erkennungseigenschaften von VEEV wurde ein Kettenaustausch der variablen Domaene der leichten Antikoerperkette des generierten humanen Antikoerperfragments MK220-IG12 mittels Phagen-Display durchgefuehrt. Die generierten humanen Antikoerperfragmente koennten die schnelle Identifikation und Detektion von VEEV verbessern, sowie potentiell zur Therapie von VEEV-Infektionen eingesetzt werden.Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus. Monoclonal highly affine antibodies against viruses of this group are rare for rapid diagnostic procedures and therapy. The aim of this work was the generation of recombinant antibody fragments against VEEV from universal antibody gene libraries for an improved detection. Virus specific scFv were isolated with selection strategies on whole VEEV particles using phage display for the first time. By optimisation of the selection strategy twelve VEEV specific scFv could be isolated from two naive libraries. Monoclonal scFv presenting filamentous M13 phage of the obtained antibody fragments were produced for characterisation and used for detection of several virus preparations. Used as detection antibodies, the scFv presenting phage were specific for VEEV strains and did not show any cross-reactivity with other alphaviruses. In this study the broad und sensitive approach of scFv presenting phage for virus detection was demonstrated. Immunostains of VEEV proteins indicated the recognition of structural epitopes by the scFv presenting phage. These structures were detected specifically in the lysate by ELISA and on the surface of VEEV infected Vero cells by immunhistochemistry. For an improved recognition of VEEV a chain shuffling of the variable domain of the human antibody fragment MK220-IG12 light chain was performed using phage display. The generated human antibody fragments could improve the fast identification and diagnosis of VEEV and will be potentially used in the therapy of VEEV infections

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

<p>Abstract</p> <p>Background</p> <p>Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV.</p> <p>Results</p> <p>In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) antigenic complex, nor did they react with Chikungunya virus (CHIKV), if they were used as detection reagent.</p> <p>Conclusion</p> <p>For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.</p

Identification of a Putative Crf Splice Variant and Generation of Recombinant Antibodies for the Specific Detection of Aspergillus fumigatus

BACKGROUND: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. RESULTS: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. CONCLUSION: Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus

Classification of the selected anti-Crf2 scFv gene fragments with human gene segments according to VBASE2.

<p>Abbreviations: HV: V (variable) gene segments of the heavy chain; D: D (diversity) gene segment; HJ: J (joining) gene segment of the heavy chain; LV: V gene segment of the light chain; LJ: J gene segment of the light chain; HAL4/7: human, naive antibody gene library; immune library: immune library derived from Crf2 immunized macaque. The germinality index describes the similarity of the anti-Crf2 scFvs to the most similiar human germline genes identified by VBASE2 (<a href="http://www.vbase2.org" target="_blank">www.vbase2.org</a>) indicated by percental identity of the VH or VL framework region.</p

Fluorescence microscopy using anti-Crf2 scFv-Fc fusion proteins.

<p>A Binding of anti-Crf2 scFv-Fc fusion proteins to <i>A. fumigatus</i> (left panel brightfield, right panel FITC). B Binding of MS112-IIB1 to different <i>Aspergillus</i> strains (<i>A. fumigatus</i>, <i>A. nidulans</i>, <i>A. terreus</i>, <i>A. flavus</i>, <i>A. clavatus</i>) and <i>Candida albicans</i>. The staining was performed using 2 µg/mL scFv-Fc fusion protein and goat anti-human IgG (Fc specific) conjugated with Alexa 488 (1∶500).</p

Detection of recombinant biotinylated Crf2 in human serum using anti-Crf2 scFvs.

<p>400 ng anti-Crf2 scFvs were coated for capturing. Human serum was spiked with a dilution series of recombinant Crf2. The bound Crf2 was detected with 100 µL streptavidin HRP conjugate (1 µg/mL).</p

Production and purification of Crf2.

<p>IMAC and IEC purified Crf2 was separated on a reducing 12% SDS-PAGE. A Coomassie staining. B the recombinant Crf was detected with mouse anti-his tag (1∶10,000) and goat anti-mouse IgG (Fc specific) AP conjugate (1∶10,000).</p

Epitope mapping of serum of mice infected with <i>A. fumigatus</i> on Crf1 and Crf2.

<p>A Epitope mapping membrane (15mer oligopeptide, 3 aminoacid overlap) stained with mouse serum (1∶400). The bound mouse antibodies were detected with goat anti-mouse IgG Fc specific AP conjugated (1∶2000). The stained Crf2 specific epitope was marked with a square. B Aligment of Crf1, Crf2 and Asp f9. The recognized epitopes of Crf1 and Crf2 are marked in red (the shorter polypeptide Asp f9 is shown for comparison, Asp f16 is not shown due to a frameshift resulting in a divergent amino acid sequence compared to Crf1, Crf2 and Asp f9).</p