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

Spontaneous charging affects the motion of sliding drops

Water drops moving on surfaces are not only an everyday phenomenon seen on windows but also form an essential part of many industrial processes. Previous understanding is that drop motion is dictated by viscous dissipation and activated dynamics at the contact line. Here we demonstrate that these two effects cannot fully explain the complex paths of sliding or impacting drops. To accurately determine the forces experienced by moving drops, we imaged their trajectory when sliding down a tilted surface, and applied the relevant equations of motion. We found that drop motion on low-permittivity substrates is substantially influenced by electrostatic forces. Our findings confirm that electrostatics must be taken into consideration for the description of the motion of water, aqueous electrolytes and ethylene glycol on hydrophobic surfaces. Our results are relevant for improving the control of drop motion in many applications, including printing, microfluidics, water management and triboelectric nanogenerators

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

Kinetic drop friction

Abstract Liquid drops sliding on tilted surfaces is an everyday phenomenon and is important for many industrial applications. Still, it is impossible to predict the drop’s sliding velocity. To make a step forward in quantitative understanding, we measured the velocity  $$(U)$$ ( U ) , contact width  $$(w)$$ ( w ) , contact length $$(L)$$ ( L ) , advancing  $$({\theta }_{{{{{{\rm{a}}}}}}})$$ ( θ a ) , and receding contact angle $$({\theta }_{{{{{{\rm{r}}}}}}})$$ ( θ r ) of liquid drops sliding down inclined flat surfaces made of different materials. We find the friction force acting on sliding drops of polar and non-polar liquids with viscosities ( $${\eta }$$ η ) ranging from 10−3 to 1 $${{{{{\rm{Pa}}}}}}\cdot {{{{{\rm{s}}}}}}$$ Pa ⋅ s can empirically be described by $${F}_{{{{{{\rm{f}}}}}}}(U)={F}_{0}+\beta w\eta U$$ F f ( U ) = F 0 + β w η U for a velocity range up to 0.7 ms−1. The dimensionless friction coefficient $$(\beta )$$ ( β ) defined here varies from 20 to 200. It is a material parameter, specific for a liquid/surface combination. While static wetting is fully described by $${\theta }_{{{{{{\rm{a}}}}}}}$$ θ a and $${\theta }_{{{{{{\rm{r}}}}}}}$$ θ r , for dynamic wetting the friction coefficient is additionally necessary

Exploring Wedge and Bulk Viscous Dissipation in Droplets Moving on Inclined Surfaces by Means of Direct Numerical Simulations

The motion of droplets on inclined surfaces is a ubiquitous phenomenon, yet the underlying dissipative mechanisms remain poorly understood. Employing direct numerical simulations, we investigate water and water-glycerol (85% wt.) droplets (∼25 μL) moving on smooth surfaces, with contact angles of around 90º, at varying inclinations. Our focus is on elucidating the role of wedge and bulk viscous dissipation in the droplets. We observe that, for fast-moving droplets, both mechanisms contribute comparably, while the wedge dissipation dominates in slow-moving cases. Comparisons with existing estimates reveal the inadequacy of previous predictions in capturing the contributions of wedge and bulk dissipation forces in fast-moving droplets. Furthermore, we demonstrate that droplets with identical sliding velocities can exhibit disparate viscous dissipation forces due to variations in internal fluid dynamics

<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