Sequential DNA immunization of chickens with bivalent heterologous vaccines induce highly reactive and cross-specific antibodies against influenza hemagglutinin.

Vaccines against avian influenza are mostly based on hemagglutinin (HA), which is the main antigen of this virus and a target for neutralizing antibodies. Traditional vaccines are known to be poorly efficient against newly emerging strains, which is an increasing worldwide problem for human health and for the poultry industry. As demonstrated by research and clinical data, sequential exposure to divergent influenza HAs can boost induction of universal antibodies which recognize conserved epitopes. In this work, we have performed sequential immunization of laying hens using monovalent or bivalent compositions of DNA vaccines encoding HAs from distant groups 1 and 2 (H5, H1, and H3 subtypes, respectively). This strategy gave promising results, as it led to induction of polyclonal antibodies against HAs from both groups. These polyclonal antibodies showed cross-reactivity between different HA strains in ELISA, especially when bivalent formulations were used for immunization of birds. However, cross-reactivity of antibodies induced against H3 and H5 HA subtypes was rather limited against each other after homologous immunization. Using a cocktail of HA sequences and/or sequential DNA vaccination with different strains presents a good strategy to overcome the limited effectiveness of vaccines and induce broader immunity against avian influenza. Such a strategy could be adapted for vaccinating laying hens or parental flocks of different groups of poultry.

Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody.

Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display peptide libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitate epitope mapping. Based on the sequences of the affinity- selected polypeptides and the structural model of HA the epitope was located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibiting activity and its antigenic specificity. Additionally, total RNA isolated from the hybridoma cell line secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed differences in specificity were discussed.

3DFlu: database of sequence and structural variability of the influenza hemagglutinin at population scale.

The influenza virus type A (IVA) is an important pathogen which is able to cause annual epidemics and even pandemics. This fact is the consequence of the antigenic shifts and drifts capabilities of IVA, caused by the high mutation rate and the reassortment capabilities of the virus. The hemagglutinin (HA) protein constitutes the main IVA antigen and has a crucial role in the infection mechanism, being responsible for the recognition of host-specific sialic acid derivatives. Despite the relative abundance of HA sequence and serological studies, comparative structure-based analysis of HA are less investigated. The 3DFlu database contains well annotated HA representatives: 1192 models and 263 crystallographic structures. The relations between these proteins are defined using different metrics and are visualized as a network in the provided web interface. Moreover structural and sequence comparison of the proteins can be explored. Metadata information (e.g. protein identifier, IVA strain, year and location of infection) can enhance the exploration of the presented data. With our database researchers gain a useful tool for the exploration of high quality HA models, viewing and comparing changes in the HA viral subtypes at several information levels (sequence, structure, ESP). The complete and integrated view of those relations might be useful to determine the efficiency of transmission, pathogenicity and for the investigation of evolutionary tendencies of the influenza virus.Database URL: http://nucleus3d.cent.uw.edu.pl/influenza.

Role of the host genetic variability in the influenza A virus susceptibility.

The aftermath of influenza infection is determined by a complex set of host-pathogen interactions, where genomic variability on both viral and host sides influences the final outcome. Although there exists large body of literature describing influenza virus variability, only a very small fraction covers the issue of host variance. The goal of this review is to explore the variability of host genes responsible for host-pathogen interactions, paying particular attention to genes responsible for the presence of sialylated glycans in the host endothelial membrane, mucus, genes used by viral immune escape mechanisms, and genes particularly expressed after vaccination, since they are more likely to have a direct influence on the infection outcome.

Mapping of the influenza A hemagglutinin serotypes evolution by the ISSCOR method.

Analyses and visualizations by the ISSCOR method of influenza virus hemagglutinin genes of different A-subtypes revealed some rather striking temporal relationships between groups of individual gene subsets. Based on these findings we consider application of the ISSCOR-PCA method for analyses of large sets of homologous genes to be a worthwhile addition to a toolbox of genomics--allowing for a rapid diagnostics of trends, and ultimately even aiding an early warning of newly emerging epidemiological threats.

The hemagglutinin mutation E391K of pandemic 2009 influenza revisited.

Phylogenetic analyses based on small to moderately sized sets of sequential data lead to overestimating mutation rates in influenza hemagglutinin (HA) by at least an order of magnitude. Two major underlying reasons are: the incomplete lineage sorting, and a possible absence in the analyzed sequences set some of key missing ancestors. Additionally, during neighbor joining tree reconstruction each mutation is considered equally important, regardless of its nature. Here we have implemented a heuristic method optimizing site dependent factors weighting differently 1st, 2nd, and 3rd codon position mutations, allowing to extricate incorrectly attributed sub-clades. The least squares regression analysis of distribution of frequencies for all mutations observed on a partially disentangled tree for a large set of unique 3243 HA sequences, along all nucleotide positions, was performed for all mutations as well as for non-equivalent amino acid mutations - in both cases demonstrating almost flat gradients, with a very slight downward slope towards the 3'-end positions. The mean mutation rates per sequence per year were 3.83×10(-4) for the all mutations, and 9.64×10(-5) for the non-equivalent ones.

Alignment free characterization of the influenza-A hemagglutinin genes by the ISSCOR method.

Analyses and visualizations by the ISSCOR method of the influenza virus hemagglutinin genes of three different A-subtypes revealed some rather striking temporal (for A/H3N3), and spatial relationships (for A/H5N1) between groups of individual gene subsets. The application to the A/H1N1 set revealed also relationships between the seasonal H1, and the swine-like novel 2009 H1v variants in a quick and unambiguous manner. Based on these examples we consider the application of the ISSCOR method for analysis of large sets of homologous genes as a worthwhile addition to a toolbox of genomics-it allows a rapid diagnostics of trends, and possibly can even aid an early warning of newly emerging epidemiological threats.

Quick path finding--quick algorithmic solution for unambiguous labeling of phylogenetic tree nodes.

Ever increasing amounts of genetic information stored in sequential databases require efficient methods to automatically reveal their phylogenetic relationships. A framework for in silico unambiguous analysis of phylogenetic trees, based on information contained in tree's topology, together with its branches length, is proposed. The resulting, translated tree has all nodes labeled, with no constraints on nodes' degree, and the subsequent finding of evolutionary pathways from the QPF-translated tree is robust and straightforward. Main features of the method are: small demands on computational time, and the ability to analyze phylogenies obtained prior to the proposed QPF analysis by any traditional tree-building technique.

Probabilistic model of influenza virus transmissibility at various temperature and humidity conditions.

The spread efficiency of influenza virus is significantly affected by several environmental parameters. However, neither the underlying reasons, nor the exact character and magnitude of the phenomena involved are sufficiently well understood. Here we present a probabilistic approach to the virus transmission events. For a sample ensemble, we construct a model of the infectivity as a function of the ambient conditions, and we determine its parameter values on the basis of the available experimental data.

A model of influenza virus spread as a function of temperature and humidity.

The influence that atmospheric conditions might have on the efficiency of the spread of influenza virus is important for epidemiological and evolutionary research. However, it has not been satisfactorily recognized and quantified so far. Here we provide a statistical model of influenza transmission between individuals. It has been derived from the results of recent experiments, which involved infecting guinea pigs with influenza at various temperatures and relative air humidity levels. The wide range of transmission rates in those experiments reflects the ensemble-independent phenomena. The correlation between most of our simulations and the experimental results is satisfactory. For several different conditions, we obtained transmissibility values which seem to be sufficiently accurate to provide partial input for an intended large-scale epidemiological study in the near future.

ISSCOR: Intragenic, Stochastic Synonymous Codon Occurrence Replacement--a new method for an alignment-free genome sequence analysis.

Synonymous codons do not occur at equal frequencies. Codon usage and codon bias have been extensively studied. However, the sequential order in which synonymous codons appear within a gene has not been studied until now. Here we describe an in silico method, which is the first attempt to tackle this problem: to what extent this sequential order is unique, and to what extent the succession of synonymous codons is important. This method, which we called Intragenic, Stochastic Synonymous Codon Occurrence Replacement (ISSCOR), generates, by a Monte Carlo approach, a set of genes which code for the same amino acid sequence, and display the same codon usage, but have random permutations of the synonymous codons, and therefore different sequential codon orders from the original gene. We analyze the complete genome of the bacterium Helicobacter pylori (containing 1574 protein coding genes), and show by various, alignment-free computational methods (e.g., frequency distribution of codon-pairs, as well as that of nucleotide bigrams in codon-pairs), that: (i) not only the succession of adjacent synonymous codons is far from random, but also, which is totally unexpected, the occurrences of non-adjacent synonymous codon-pairs are highly constrained, at strikingly long distances of dozens of nucleotides; (ii) the statistical deviations from the random synonymous codon order are overwhelming; and (iii) the pattern of nucleotide bigrams in codon-pairs can be used in a novel way for characterizing and comparing genes and genomes. Our results demonstrate that the sequential order of synonymous codons within a gene must be under a strong selective pressure, which is superimposed on the classical codon usage. This new dimension can be measured by the ISSCOR method, which is simple, robust, and should be useful for comparative and functional genomics.

Primary sequences of proteins from complete genomes display a singular periodicity: Alignment-free N-gram analysis.

A method is proposed to represent and to analyze complete genome sequences (52 species from procaryotes and eukaryotes), based upon n-gram sequence's frequencies of amino acid pairs (bigrams), separated by a given number of other residues. For each of the species analyzed, it allows us to construct over-abundant and over-deficient occurrence profiles, summarizing amino acid bigram frequencies over the entire genome. The method deals efficiently with a sparseness of statistical representations of individual sequences, and describes every gene sequence in the same way, independently of its length and of the genome sizes. The frequency of over-abundant and over-deficient occurrences of bigrams presents a singular periodicity around 3.5 peptide bonds, suggesting a relation with the alpha helical secondary structure.