Toward the discovery of vaccine adjuvants:coupling in silico screening and in vitro analysis of antagonist binding to human and mouse CCR4 receptors

Background Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. Methodology Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cell migration, including that of CCR4+ Tregs. Significance Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Integrating in silico and in vitro analysis of peptide binding affinity to HLA-Cw*0102:a bioinformatic approach to the prediction of new epitopes

Predictive models of peptide-Major Histocompatibility Complex (MHC) binding affinity are important components of modern computational immunovaccinology. Here, we describe the development and deployment of a reliable peptide-binding prediction method for a previously poorly-characterized human MHC class I allele, HLA-Cw*0102.

Coupling in silico and in vitro analysis of peptide-MHC binding:a bioinformatic approach enabling prediction of superbinding peptides and anchorless epitopes

The ability to define and manipulate the interaction of peptides with MHC molecules has immense immunological utility, with applications in epitope identification, vaccine design, and immunomodulation. However, the methods currently available for prediction of peptide-MHC binding are far from ideal. We recently described the application of a bioinformatic prediction method based on quantitative structure-affinity relationship methods to peptide-MHC binding. In this study we demonstrate the predictivity and utility of this approach. We determined the binding affinities of a set of 90 nonamer peptides for the MHC class I allele HLA-A*0201 using an in-house, FACS-based, MHC stabilization assay, and from these data we derived an additive quantitative structure-affinity relationship model for peptide interaction with the HLA-A*0201 molecule. Using this model we then designed a series of high affinity HLA-A2-binding peptides. Experimental analysis revealed that all these peptides showed high binding affinities to the HLA-A*0201 molecule, significantly higher than the highest previously recorded. In addition, by the use of systematic substitution at principal anchor positions 2 and 9, we showed that high binding peptides are tolerant to a wide range of nonpreferred amino acids. Our results support a model in which the affinity of peptide binding to MHC is determined by the interactions of amino acids at multiple positions with the MHC molecule and may be enhanced by enthalpic cooperativity between these component interactions.

AllerTOP - a server for in silico prediction of allergens

Background: Allergy is a form of hypersensitivity to normally innocuous substances, such as dust, pollen, foods or drugs. Allergens are small antigens that commonly provoke an IgE antibody response. There are two types of bioinformatics-based allergen prediction. The first approach follows FAO/WHO Codex alimentarius guidelines and searches for sequence similarity. The second approach is based on identifying conserved allergenicity-related linear motifs. Both approaches assume that allergenicity is a linearly coded property. In the present study, we applied ACC pre-processing to sets of known allergens, developing alignment-independent models for allergen recognition based on the main chemical properties of amino acid sequences.Results: A set of 684 food, 1,156 inhalant and 555 toxin allergens was collected from several databases. A set of non-allergens from the same species were selected to mirror the allergen set. The amino acids in the protein sequences were described by three z-descriptors (z1, z2 and z3) and by auto- and cross-covariance (ACC) transformation were converted into uniform vectors. Each protein was presented as a vector of 45 variables. Five machine learning methods for classification were applied in the study to derive models for allergen prediction. The methods were: discriminant analysis by partial least squares (DA-PLS), logistic regression (LR), decision tree (DT), naïve Bayes (NB) and k nearest neighbours (kNN). The best performing model was derived by kNN at k = 3. It was optimized, cross-validated and implemented in a server named AllerTOP, freely accessible at http://www.pharmfac.net/allertop. AllerTOP also predicts the most probable route of exposure. In comparison to other servers for allergen prediction, AllerTOP outperforms them with 94% sensitivity.Conclusions: AllerTOP is the first alignment-free server for in silico prediction of allergens based on the main physicochemical properties of proteins. Significantly, as well allergenicity AllerTOP is able to predict the route of allergen exposure: food, inhalant or toxin. © 2013 Dimitrov et al.; licensee BioMed Central Ltd.

Influence of iron deprivation and sub-inhibitory concentrations of antifungal antibiotics on surface antigens of candida albicans yeast cells

This study examined the effect of iron deprivation and sub-inhibitory concentrations of antifungal agents on yeast cell surface antigen recognition by antibodies from patients with Candida infections. Separation of cell wall surface proteins by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological detection by immunoblotting, revealed that antigenic profiles of yeasts were profoundly influenced by the growth environment. Cells grown under iron-depleted conditions expressed several iron-regulated proteins that were recognized by antibodies from patient sera. An attempt to characterize these proteins by lectin blotting with concanavalin A revealed that some could be glycoprotein in nature. Furthermore, these proteins which were located within cell walls and on yeast surfaces, were barely or not expressed in yeasts cultivated under iron-sufficient conditions. The magnitude and heterogeneity of human antibody responses to these iron-regulated proteins were dependent on the type of Candida infection, serum antibody class and yeast strain. Hydroxamate-type siderophores were also detected in supernatants of iron depleted yeast cultures. This evidence suggests that Candida albicans expresses iron-regulated proteins/glycoproteins in vitro which may play a role in siderophore-mediated iron uptake in Candida albicans. Sequential monitoring of IgG antibodies directed against yeast surface antigens during immunization of rabbits revealed that different antigens were recognized particularly during early and later stages of immunization in iron-depleted cells compared to iron-sufficient cells. In vitro and in vivo adherence studies demonstrated that growth phase, yeast strain and growth conditions affect adhesion mechanisms. In particular, growth under iron-depletion in the presence of sub-inhibitory concentrations of polyene and azole antifungals enhanced the hydrophobicity of C.albicans. Growth conditions also influenced MICs of antifungals, notably that of ketoconazole. Sub-inhibitory concentrations of amphotericin B and fluconazole had little effect on surface antigens, whereas nystatin induced profound changes in surface antigens of yeast cells. The effects of such drug concentrations on yeast cells coupled with host defence mechanisms may have a significant affect on the course of Candida infections.

New horizons in mouse immunoinformatics:reliable in silico prediction of mouse class I histocompatibility major complex peptide binding affinity

Quantitative structure–activity relationship (QSAR) analysis is a main cornerstone of modern informatic disciplines. Predictive computational models, based on QSAR technology, of peptide-major histocompatibility complex (MHC) binding affinity have now become a vital component of modern day computational immunovaccinology. Historically, such approaches have been built around semi-qualitative, classification methods, but these are now giving way to quantitative regression methods. The additive method, an established immunoinformatics technique for the quantitative prediction of peptide–protein affinity, was used here to identify the sequence dependence of peptide binding specificity for three mouse class I MHC alleles: H2–Db, H2–Kb and H2–Kk. As we show, in terms of reliability the resulting models represent a significant advance on existing methods. They can be used for the accurate prediction of T-cell epitopes and are freely available online (http://www.jenner.ac.uk/MHCPred).

In vivo analysis of ciliary muscle morphologic changes with accommodation and axial ametropia

Purpose. To use anterior segment optical coherence tomography (AS-OCT) to analyze ciliary muscle morphology and changes with accommodation and axial ametropia. Methods. Fifty prepresbyopic volunteers, aged 19 to 34 years were recruited. High-resolution images were acquired of nasal and temporal ciliary muscles in the relaxed state and at stimulus vergence levels of -4 and -8 D. Objective accommodative responses and axial lengths were also recorded. Two-way, mixed-factor analyses of variance (ANOVAs) were used to assess the changes in ciliary muscle parameters with accommodation and determine whether these changes are dependent on the nasal–temporal aspect or axial length, whereas linear regression analysis was used to analyze the relationship between axial length and ciliary muscle length. Results. The ciliary muscle was longer (r = 0.34, P = 0.02), but not significantly thicker (F = 2.84, P = 0.06), in eyes with greater axial length. With accommodation, the ciliary muscle showed a contractile shortening (F = 42.9. P < 0.001), particularly anteriorly (F = 177.2, P < 0.001), and a thickening of the anterior portion (F= 46.2, P < 0.001). The ciliary muscle was thicker (F = 17.8, P < 0.001) and showed a greater contractile response on the temporal side. Conclusions. The accommodative changes observed support an anterior, as well as centripetal, contractile shift of ciliary muscle mass.

Immunological and biochemical techniques in the analysis of tear proteins

This study is concerned with the analysis of tear proteins, paying particular attention to the state of the tears (e.g. non-stimulated, reflex, closed), created during sampling, and to assess their interactions with hydrogel contact lenses. The work has involved the use of a variety of biochemical and immunological analytical techniques for the measurement of proteins, (a), in tears, (b), on the contact lens, and (c), in the eluate of extracted lenses. Although a diverse range of tear components may contribute to contact lens spoilation, proteins were of particular interest in this study because of their theoretical potential for producing immunological reactions. Although normal host proteins in their natural state are generally not treated as dangerous or non-self, those which undergo denaturation or suffer a conformational change may provoke an excessive and unnecessary immune response. A novel on-lens cell based assay has been developed and exploited in order to study the role of the ubiquitous cell adhesion glycoprotein, vitronectin, in tears and contact lens wear under various parameters. Vitronectin, whose levels are known to increase in the closed eye environment and shown here to increase during contact lens wear, is an important immunoregulatory protein and may be a prominent marker of inflammatory activity. Immunodiffusion assays were developed and optimised for use in tear analysis, and in a series of subsequent studies used for example in the measurement of albumin, lactoferrin, IgA and IgG. The immunodiffusion assays were then applied in the estimation of the closed eye environment; an environment which has been described as sustaining a state of sub-clinical inflammation. The role and presence of a lesser understood and investigated protein, kininogen, was also estimated, in particular, in relation to contact lens wear. Difficulties arise when attempting to extract proteins from the contact lens in order to examine the individual nature of the proteins involved. These problems were partly alleviated with the use of the on-lens cell assay and a UV spectrophotometry assay, which can analyse the lens surface and bulk respectively, the latter yielding only total protein values. Various lens extraction methods were investigated to remove protein from the lens and the most efficient was employed in the analysis of lens extracts. Counter immunoelectrophoresis, an immunodiffusion assay, was then applied to the analysis of albumin, lactoferrin, IgA and IgG in the resultant eluates.

IN VIVO analysis of ocular morphological changes during phakic accommodation

The principal theme of this thesis is the in vivo examination of ocular morphological changes during phakic accommodation, with particular attention paid to the ciliary muscle and crystalline lens. The investigations detailed involved the application of high-resolution imaging techniques to facilitate the acquisition of new data to assist in the clarification of aspects of the accommodative system that were poorly understood. A clinical evaluation of the newly available Grand Seiko Auto Ref/ Keratometer WAM-5500 optometer was undertaken to assess its value in the field of accommodation research. The device was found to be accurate and repeatable compared to subjective refraction, and has the added advantage of allowing dynamic data collection at a frequency of around 5 Hz. All of the subsequent investigations applied the WAM-5500 for determination of refractive error and objective accommodative responses. Anterior segment optical coherence tomography (AS-OCT) based studies examined the morphology and contractile response of youthful and ageing ciliary muscle. Nasal versus temporal asymmetry was identified, with the temporal aspect being both thicker and demonstrating a greater contractile response. The ciliary muscle was longer in terms of both its anterior (r = 0.49, P <0.001) and overall length (r = 0.45, P = 0.02) characteristics, in myopes. The myopic ciliary muscle does not appear to be merely stretched during axial elongation, as no significant relationship between thickness and refractive error was identified. The main contractile responses observed were a thickening of the anterior region and a shortening of the muscle, particularly anteriorly. Similar patterns of response were observed in subjects aged up to 70 years, supporting a lensocentric theory of presbyopia development. Following the discovery of nasal/ temporal asymmetry in ciliary muscle morphology and response, an investigation was conducted to explore whether the regional variations in muscle contractility impacted on lens stability during accommodation. A bespoke programme was developed to analyse AS-OCT images and determine whether lens tilt and decentration varied between the relaxed and accommodated states. No significant accommodative difference in these parameters was identified, implying that any changes in lens stability with accommodation are very slight, as a possible consequence of vitreous support. Novel three-dimensional magnetic resonance imaging (MRI) and analysis techniques were used to investigate changes in lens morphology and ocular conformation during accommodation. An accommodative reduction in lens equatorial diameter provides further evidence to support the Helmholtzian mechanism of accommodation, whilst the observed increase in lens volume challenges the widespread assertion that this structure is incompressible due to its high water content. Wholeeye MRI indicated that the volume of the vitreous chamber remains constant during accommodation. No significant changes in ocular conformation were detected using MRI. The investigations detailed provide further insight into the mechanisms of accommodation and presbyopia, and represent a platform for future work in this field.

In silico identification of novel G protein coupled receptors

G-protein coupled receptors (GPCRs) are a superfamily of membrane integral proteins responsible for a large number of physiological functions. Approximately 50% of marketed drugs are targeted toward a GPCR. Despite showing a high degree of structural homology, there is a large variance in sequence within the GPCR superfamily which has lead to difficulties in identifying and classifying potential new GPCR proteins. Here the various computational techniques that can be used to characterize a novel GPCR protein are discussed, including both alignment-based and alignment-free approaches. In addition, the application of homology modeling to building the three-dimensional structures of GPCRs is described.

Time-resolved Q-factor measurement and its application in performance analysis of 42.6 Gbit/s packets generated by SGDBR lasers

We demonstrate a novel time-resolved Q-factor measurement technique and demonstrate its application in the analysis of optical packet switching systems with high information spectral density. For the first time, we report the time-resolved Q-factor measurement of 42.6 Gbit/s AM-PSK and DQPSK modulated packets, which were generated by a SGDBR laser under wavelength switching. The time dependent degradation of Q-factor performance during the switching transient was analyzed and was found to be correlated with different laser switching characteristics in each case.

Applicability of contact angle techniques used in the analysis of contact lenses, part 1:comparative methodologies

Objectives and Methods: Contact angle, as a representative measure of surface wettability, is often employed to interpret contact lens surface properties. The literature is often contradictory and can lead to confusion. This literature review is part of a series regarding the analysis of hydrogel contact lenses using contact angle techniques. Here we present an overview of contact angle terminology, methodology, and analysis. Having discussed this background material, subsequent parts of the series will discuss the analysis of contact lens contact angles and evaluate differences in published laboratory results. Results: The concepts of contact angle, wettability and wetting are presented as an introduction. Contact angle hysteresis is outlined and highlights the advantages in using dynamic analytical techniques over static methods. The surface free energy of a material illustrates how contact angle analysis is capable of providing supplementary surface characterization. Although single values are able to distinguish individual material differences, surface free energy and dynamic methods provide an improved understanding of material behavior. The frequently used sessile drop, captive bubble, and Wilhelmy plate techniques are discussed. Their use as both dynamic and static methods, along with the advantages and disadvantages of each technique, is explained. Conclusions: No single contact angle technique fully characterizes the wettability of a material surface, and the application of complimenting methods allows increased characterization. At present, there is not an ISO standard method designed for soft materials. It is important that each contact angle technique has a standard protocol, as small protocol differences between laboratories often contribute to a variety of published data that are not easily comparable. © 2013 Contact Lens Association of Ophthalmologists.

Immunoinformatics and the in silico prediction of immunogenicity:an introduction

Immunoinformatics is the application of informatics techniques to molecules of the immune system. One of its principal goals is the effective prediction of immunogenicity, be that at the level of epitope, subunit vaccine, or attenuated pathogen. Immunogenicity is the ability of a pathogen or component thereof to induce a specific immune response when first exposed to surveillance by the immune system, whereas antigenicity is the capacity for recognition by the extant machinery of the adaptive immune response in a recall response. In thisbook, we introduce these subjects and explore the current state of play in immunoinformatics and the in silico prediction of immunogenicity.