Generation and Characterisation of recombinant antibody fragments specific for Venezuelan and Western Equine Encephalitis Virus

Das Venzuelanische und das Westliche Pferdeenzephalitis-Virus (VEEV, WEEV) gehören zur Gattung der Alphaviren. Einige Subtypen dieser Viren sind humanpathogen und zählen als enzephalizide Viren zu den bioterroristischen Agenzien der Kategorie B. Bisher existieren nur wenige monoklonale Antikörper, die nur im Bereich der Diagnostik Verwendung finden. Die Behandlung bei Infektionen erfolgt nur unterstützend, eine antivirale Therapie steht bisher nicht zur Verfügung. Ziel dieser Arbeit war die Generierung von Antikörpergenbibliotheken aus dem Knochenmark immunisierter Makaken (Macaca fascicularis) und die Isolierung virusspezifischer Antikörper mittels Phagen-Display-Technologie. Es konnten spezifisch bindende scFv-Fragmente aus den Immunbibliotheken isoliert werden, welche in das scFv-Fc Antikörperformat überführt und charakterisiert wurden. Die Antikörper wurden erfolgreich als Fänger- und Detektionsantikörper in ELISA- und immunhistochemischen Experimenten eingesetzt. Der einzige gegen das VEE-Virus isolierte Antikörper, ToR67-3B4, wurde im Folgenden auf neutralisierende Eigenschaften untersucht. Dabei wurde in vitro eine Verhinderung der Infektion für einige Virustypen nachgewiesen. Zusätzlich dazu konnten in vivo Mäuse vor der Infektion mit den VEE-Subtypen TrD, Mena II, Fe37c und BeAn8 geschützt werden, wenn eine Behandlung mit dem scFv-Fc-Antikörper ToR67-3B4 6 Stunden nach Infektion durchgeführt wurde. Damit wurde eine Protektion durch den Antikörper nachgewiesen, welcher die Mäuse somit effektiv vor einer Infektion schützt. In dieser Arbeit wurden erstmals virusspezifische Antikörper aus einer Immunantikörpergenbibliothek nach Selektion auf aktiven Viruspartikeln isoliert. Diese Antikörper könnten zur schnellen Identifikation und Detektion von VEEV und WEEV eingesetzt werden. Für den VEEV-spezifischen Antikörper ToR67-3B4 besteht außerdem die Möglichkeit zum Einsatz in der Therapie, da eine Protektion vor Infektionen in vivo nachgewiesen werden konnte.Venezuelan and Western Equine Encephalitis virus (VEEV and WEEV) belong to the alphavirus genus. Some subtypes of these viruses are pathogenic to humans and are classified as potential agents of biological warfare and terrorism. Today, there are only few monoclonal antibodies available, which are mainly used in diagnostics. Treatment of infection is only symptomatic and supportive, an antiviral therapy is not available. Aim of this study was the generation of immune antibody gene libraries from the bone marrow of immunised macaques (Macaca fascicularis) and the isolation of virus binding antibodies via phage display technology. By using this technique, specifically binding scFv-fragments were isolated from the immune libraries. The scFv fragments were transformed to scFv-Fc antibody format and their binding properties were characterised. The VEEV and WEEV specific antibodies were successfully used as capture and detector antibodies in ELISAs and were used to identify infected cells in immunohistochemistry studies. The neutralising properties from scFv-Fc antibody ToR67-3B4, the only antibody isolated from the immune library derived from the VEEV immunised macaque, were examined in vitro and in vivo. The antibody was able to prevent infection of cells with several virus subtypes. Additionally it was able to prevent mice from being infected by the VEEV subtypes TrD, Mena II, Fe37c and BeAn8, when administered 6 hours after infection. This indicates that scFv-Fc ToR67-3B4 is able to protect mice effectively from infection. This study describes an isolation of virus specific antibodies from an immune antibody gene library via selection on complete and active virus particles which was never shown before. The antibodies show great potential to be used for fast identification and detection of VEEV and WEEV. Especially the VEEV specific antibody ToR67-3B4 could probably be used in therapy because of its ability to protect mice in in vivo challenge studies

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

Isolation and Characterisation of a Human-Like Antibody Fragment (scFv) That Inactivates VEEV In Vitro and In Vivo

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required

<i>In vitro</i> neutralisation activity of scFv-Fc fusion ToR67-3B4 to different VEEV strains and Everglades virus.

<p>VEEV strains of subtype IA/B (Panel A and B) and Everglades virus (strain Fe37c, Panel C) with a TCID₅₀/ml of 5×10⁴ were incubated with serial dilutions of scFv-Fc ToR67-3B4 and other mAbs at 37°C for 2 hours. Starting concentrations were between 0.5 to 1.0 mg/ml. Subsequently the mixtures were subjected to cell culture infection. Residual infectious activity of virus was estimated by specific immunostaining one day post infection with VEEV-specific antibodies. Absorbance (OD450 nm) values obtained with virus samples not preincubated with any antibody served as positive control and were set as 100% infectivity. Non-infected Vero cells were used as negative control. The mean values of two independent experiments are shown.</p

scFv-Fc ToR67-3B4 protects mice against VEEV disease.

<p>BALB/c mice were challenged by the aerosol route with approximately 100 LD₅₀ VEEV strain TrD (Panel A and B), Mena II, Fe37c and Mucambo virus (BeAn8, Panel C and D). Six, twenty-four, forty-eight and seventy-two hours later they were injected with 100 µg scFv-Fc ToR67-3B4 intraperitoneally (Panel A, B and C, n = 6 or 10). As negative control mice remained either untreated or were injected with a human IgG1 antibody (Panel B) or a nonspecific antibody (anti-WEEV, Panel D, n = 10 or 5). Animals were observed twice daily for clinical signs of infection and were culled when appropriate using humane endpoints.</p

Titration of monkey sera after the final boost.

<p>Either VRS-purified culture supernatants from VEEV TC83 infected Vero cell or uninfected cells (negative control) were immobilised on 96well-microtiter plates. For the detection of a VEEV specific antibody titer, serial dilutions of pre-immune serum (PI 543) and immune serum (S543) after the sixth boost were applied. Bound antibodies were detected by rabbit anti-monkey IgG conjugated to horseradish peroxidase and staining with TMB.</p

Immunological analysis of VEEV glycoproteins.

<p>Panel A and B show the immunoblot analysis of VEEV glycoproteins with scFv-Fc ToR67-3B4 and monoclonal antibodies. Proteins from VEEV strain TC-83 were either resolved on an 8–15% gradient (panel A) or a linear 12% polyacrylamide gel (panel B) under denaturing and non-reducing conditions at 56°C (panel A) or at 56°C and 95°C (panel B) under denaturing conditions with or without 100 mM DTT. After Western blotting the membrane was cut in stripes and was probed with either scFv-Fc ToR67-3B4 (Panel A lane 2 and 6; panel B all lanes) or E1- and E2-specific mAbs (Panel A lane 1 and 5: SFV 8/6 (E1-specific); lane 3: WIS-VEE1 (E1-specific); lane 4: 1A3B7 (E2-specific)). Scale indicates protein size in kDa. Panel C depicts the immunohistochemistry of Vero cells infected with VEEV TrD, 1 day post infection. Photomicrographs were obtained from infected (I–IV) and non–infected (V) cells with a 10fold magnification lens. Cells in panel I and II were stained for VEEV antigen with biotinylated scFv-Fc ToR67-3B4, infected cells in panel III and IV were detected with biotinylated mAb SFV 8/6 as positive control. Non-infected Vero cells in panel V were stained with scFv-Fc ToR67-3B4 as negative control.</p

Reactivity of scFv-Fc ToR 67- 3B4 to Alphavirus subspecies and species.

<p>Panel <b>2A</b> shows the antigen binding efficiency of ToR67-3B4 on different immobilised VEEV subtypes at a concentration of 1 µg/ml, n = 1 for all data points. Panel <b>2B</b> illustrates the cross-reactivity of scFv-Fc ToR67-3B4 to a range of Alphavirus species analysed by a sandwich ELISA. Antigens were captured by an anti-Alphavirus mAb mix, specifically bound virus was detected by biotinylated scFv-Fc ToR67-3B4 (1∶5000). The insert in the upper right corner of the bar chart shows the positive control and demonstrates that all Alphaviruses except SINV are specifically captured by the antibody mixture. Detection of virus was performed with the same biotinylated anti- Alphavirus mAb mix. All viral antigens were applied with a TCID₅₀/ml of 5×10⁷ to 10⁸. Culture supernatant of non-infected Vero cells was used as negative control. The mean values of two ELISAs from two independent experiments are shown. In panel <b>2C</b> scFv-Fc ToR67-3B4 was applied as capture antibody in combination with the cognate Alphavirus-specific antibody mixture for group-specific detection. Viral antigens were applied with a TCID₅₀/ml of 5×10⁷ to 10⁸. Culture supernatant of non-infected Vero was used as negative control. The mean values of two ELISAs from three independent experiments are shown. Panel <b>2D</b> shows the detection limit for vaccine strain TC83 (subtype IA/B) using capture antibody mAb VEE WIS1 paired with biotinylated scFv-Fc ToR67-3B4 detector antibody (1∶5000). Virus was titrated in 2-fold dilutions. The data represents 2 separate experiments with 3 replicates of each concentration. Abbreviations used in this legend are BHK: Baby hamster kidney cells, VEEV: Venezuelean equine encephalitis virus, WEEV: Western equine encephalitis virus, EEEV: Eastern equine encephalitis virus, CHIKV: Chikungunya virus, SINV: Sindbis virus and SFV: Semliki Forest virus.</p