Identification of antigenic epitopes in the haemagglutinin protein of H7 avian influenza virus

ABSTRACT

The H7 subtype avian influenza virus (AIV) has been reported to infect not only poultry but also humans. The haemagglutinin (HA) protein is the major surface antigen of AIV and plays an important role in viral infection. In this study, five monoclonal antibodies (mAbs, 2F8, 3F6, 5C11, 5E2 and 5C12) against the HA protein of H7 virus were produced and characterized. Epitope mapping indicated that ¹⁰³RESGSS¹⁰⁷ was the minimal linear epitope recognized by the mAbs 2F8/3F6/5C11, and mAbs 5E2/5C12 recognized the epitope 103-145aa. The protein sequence alignment of HA indicated that the two epitopes were not found in other subtypes of AIV, and none of the five mAbs cross-reacted with other subtypes, suggesting these mAbs are specific to H7 virus. The epitope ¹⁰³RESGSS¹⁰⁷ was highly conserved among Eurasian lineage strains of H7 AIV, whereas three amino acid substitutions (E104R, E104K and E104G) in the epitope occurred in 98.44% of North-American lineage strains. Any of these single mutations prevented the mutated epitope from being recognized by mAbs 2F8/3F6/5C11; thus, these mAbs can distinguish between Eurasian and North-American lineages of H7 strains. Furthermore, the mAbs 2F8, 3F6 and 5C11 could be highly blocked with H7-positive serum in blocking assays, revealing that ¹⁰³RESGSS¹⁰⁷ may be a dominant epitope stimulating the production of antibodies during viral infection. These results may facilitate future investigations into the structure and function of HA protein, as well as surveillance and detection of H7 virus.

RESEARCH HIGHLIGHTS

• Five mAbs against HA protein of H7 AIV were generated and characterized.

• Two novel epitopes ¹⁰³RESGSS¹⁰⁷ and 103-145aa were identified.

• The epitope ¹⁰³RESGSS¹⁰⁷ differs between Eurasian and North-American lineages.

• The mAbs 2F8, 3F6 and 5C11 could distinguish two lineages of H7 strains.

     

Determination of rate-constants as a method to describe passive expiration

To describe the relaxed expiration by a two-compartment model, we introduced a gas/energy transfer between the lung compartment (V₁) and a second one (V₂). If V₂ were a real volume, the rate-constants (i.e. the flow/volume ratios) of the compartments would describe a real gas-exchange. Alternatively, if a viscoelastic behaviour of the lung or an energy-exchange between compartments was simulated, V₂ would become a "pseudo-volume". We studied nine mechanically ventilated subjects. Changes in volume were transduced by respiratory inductive plethysmography. The rate-constants were assumed (together with the initial volumes of the compartments) as parameters to fit the total volume [V₁(t)+V₂(t)]. Once the best fitting was performed using these "physiological" parameters, the system was directly identified and the compartments were independently analysed. The time profile of the second compartment showed a maximum that depended on the value of the rate-constants. Appropriate tests confirmed the reliability of our procedure. In conclusion, our analysis demonstrated that the energy/volume of the second compartment may increase at the beginning of expiration and then decrease, showing a maximum, even though the total curve can only be a decreasing one. In other words, the slowing down of the curve representing expiratory volume is due not only to the longer emptying of the second compartment, but also to the interaction between the two compartments. As presently proposed, this interaction can be represented by either a gas exchange between two actual volumes, or a mechanical energy transfer between the lung and the tissue compartment.     

“Smart” Materials for Biosensing Devices: Cell-Mimicking Supramolecular Assemblies and Colorimetric Detection of Pathogenic Agents

Mimicking cell membrane and the biomolecular recognition associated with membranes represents a great technical challenge, yet it has opened doors to innovative diagnostic and therapeutic methods. Our work has focused on design and synthesis of a class of smart materials exploiting biological principals for use in biosensors: these materials are functional polymeric assemblies that mimic the cell membrane and conveniently report the presence of pathogens with a color change. Biologically active cell membrane components are incorporated into conjugated polymers with desirable optical properties and the binding of the target molecules onto the material triggers conformational and electronic shifts that are reflected in a chromatic change (a so-called biochromic shift) that is conveniently observed and recorded. Langmuir–Blodgett thin films and vesicle bilayers provide ideal configurations for precise delivery of the biological binding entity to the sensing interface, and for control of molecular orientation for effective biomolecular interaction. Polydiacetylenic membrane-mimicking materials containing cell surface receptor gangliosides and sialic acid residues, respectively were formulated into these architectures and used for colorimetric detection of bacterial toxins and influenza virus. One advantage of these biochromic conjugated polymer (BCP) sensors is that their molecular recognition and signal transduction functionalities are resident in a single functional unit, making them amenable to convenient microfabrication and use.     

DC-SIGN mediates the binding of Aspergillus fumigatus and keratinophylic fungi by human dendritic cells

Human contact with fungi does not usually lead to pathological consequences, as the immune system manages to defeat the invader pathogens. Nevertheless, under immune suppression, fungi overcome immune defenses and cause diseases that range from nonserious colonizations of keratinizated tissue (Dermatophytosis) to life threatening disseminated infections (Aspergillosis). Host defenses against fungi rely on innate and adaptative responses, with dendritic cell (DC) and macrophage surface receptors having a major role in the recognition of fungal pathogens and in the orchestration of an effective immune response. DC-SIGN is a C-type lectin involved in the recognition of bacterial, viral and parasitic pathogens, as well as in interactions between cells of the immune system. Its expression is restricted to DCs and subsets of macrophages. Here we show that DC-SIGN mediates the binding and capture of Aspergillus fumigatus and keratinophylic fungi, including Chrysosporium tropicum, by human DCs, describe the requirements of these interactions and discuss their potential involvement in the onset and persistence of pulmonary fungal infections.     

Analysis of dynamic cerebral autoregulation using an ARX model based on arterial blood pressure and middle cerebral artery velocity simulation

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Determining appropriate models for joint control using surface electrical stimulation of soleus in spinal cord injury

The mechanical impedance of the ankle joint during electrical stimulation of the soleus is studied by applying constant-velocity 10° angular perturbations to the ankle and measuring the resultant torque. Both neurologically intact subjects and spinal cord injured subjects are tested. Lumped, piecewise linear models are developed to predict the torque from the measured displacement and acceleration signals. The commonly used second-order mass-spring-dashpot model fails to predict the changes in torque that occur following imposed movements. A fiveelement, directionally-dependent piecewise linear model is much better at predicting the measured responses for velocities up to 50° s⁻¹. Numerical least squared error indentification techniques are used to estimate the model parameters for three neurologically intact and three spinal cord injured subjects. The average error between the model’s response and the measured response across all subjects is 10·9%. There is some evidence that a velocity-dependent non-linear model could produce better results than the directionally-dependent piecewise linear model.     

Dynamic relationship between EMG and torque at the human ankle: Variation with contraction level and modulation

Stochastic system identification techniques were used to determine the dynamic relationship between the electromyogram (EMG) and torque in the ankle muscles of normal human subjects. EMG and torque were recorded while subjects modulated ankle torque by tracking a computer-generated stochastic waveform. Nonparametric impulse response functions (IRFs) relating EMG to ankle torque were computed and parameterised by determining the parameters of the second-order system which provided the best least-squares fit. Two sets of experiments were carried out. In the first, the mean level of torque was varied from 5 per cent of the maximum voluntary contraction (MVC) to 30 per cent MVC while the depth of modulation was held constant at ±5 per cent of MVC. In the second series of experiments the mean torque was held constant at 25 per cent MVC while the depth of modulation was varied from ±2.5 per cent to ±25 per cent. The major findings were: (1) A second-order, low-pass filter provided a good quasilinear model of the EMG/force dynamics under all conditions; (2) The model parameters depended only weakly on the mean level of torque; (3) In contrast, the model parameters depended strongly on the amplitude with which the contraction was modulated; the natural frequency increased significantly with the depth of modulation.     

Presence of a linear plasmid-like DNA molecule in the fungal pathogen Ceratocystis fimbriata Ell. & Halst.

Summary A plasmid-like molecule was detected in a strain of the ascomycete Ceratocystis fimbriata Ell. & Halst., a pathogenic fungus of Populus spp. The DNA replicon, designated pFQ501, was found to have a linear structure with a length of 6.0 kb (3.9 × 10⁶ daltons) and a density of 1.685 g/cc. This molecule was found to be associated with the mitochondria and was isolated from the gel; its restriction map was deduced from single and double digestions.