UMSBP2 is chromatin remodeler that functions in regulation of gene expression and suppression of antigenic variation in trypanosomes.

Universal Minicircle Sequence binding proteins (UMSBPs) are CCHC-type zinc-finger proteins that bind the single-stranded G-rich UMS sequence, conserved at the replication origins of minicircles in the kinetoplast DNA, the mitochondrial genome of kinetoplastids. Trypanosoma brucei UMSBP2 has been recently shown to colocalize with telomeres and to play an essential role in chromosome end protection. Here we report that TbUMSBP2 decondenses in vitro DNA molecules, which were condensed by core histones H2B, H4 or linker histone H1. DNA decondensation is mediated via protein-protein interactions between TbUMSBP2 and these histones, independently of its previously described DNA binding activity. Silencing of the TbUMSBP2 gene resulted in a significant decrease in the disassembly of nucleosomes in T. brucei chromatin, a phenotype that could be reverted, by supplementing the knockdown cells with TbUMSBP2. Transcriptome analysis revealed that silencing of TbUMSBP2 affects the expression of multiple genes in T. brucei, with a most significant effect on the upregulation of the subtelomeric variant surface glycoproteins (VSG) genes, which mediate the antigenic variation in African trypanosomes. These observations suggest that UMSBP2 is a chromatin remodeling protein that functions in the regulation of gene expression and plays a role in the control of antigenic variation in T. brucei.

Selection and antigenic characterization of immune-escape mutants of H7N2 low pathogenic avian influenza virus using homologous polyclonal sera.

Understanding the dynamics of the selection of influenza A immune escape variants by serum antibody is critical for designing effective vaccination programs for animals, especially poultry where large populations have a short generation time and may be vaccinated with high frequency. In this report, immune-escape mutants of A/turkey/New York/4450/1994 H7N2 low pathogenic avian influenza virus, were selected by serially passaging the virus in the presence of continuously increasing concentrations of homologous chicken polyclonal sera. Amino acid mutations were identified by sequencing the parental hemagglutinin (HA) gene and every 10 passages by both Sanger and deep sequencing, and the antigenic distance of the mutants to the parent strain was determined. Progressively, a total of five amino acid mutations were observed over the course of 30 passages. Based on their absence from the parental virus with deep sequencing, the mutations appear to have developed de novo. The antigenic distance between the selected mutants and the parent strain increased as the number of amino acid mutations accumulated and the concentration of antibodies had to be periodically increased to maintain the same reduction in virus titer during selection. This selection system demonstrates how H7 avian influenza viruses behave under selection with homologous sera, and provides a glimpse of their evolutionary dynamics, which can be applied to developing vaccination programs that maximize the effectiveness of a vaccine over time.

Directed selection of amino acid changes in the influenza hemagglutinin and neuraminidase affecting protein antigenicity.

Influenza virus hemagglutinin (HA) and neuraminidase (NA) proteins elicit protective antibody responses and therefore, are used as targets for vaccination, especially the HA protein. However, these proteins are subject to antigenic drift, decreasing vaccine efficacy, and few to no studies have analyzed antigenic variability of these proteins by growing the viruses under immune pressure provided by human sera. In this work, we show that after growing different influenza virus strains under immune pressure, the selection of amino acid changes in the NA protein is much more limited than the selection in the HA protein, suggesting that the NA protein could remain more conserved under immune pressure. Interestingly, all the mutations in the HA and NA proteins affected protein antigenicity, and many of the selected amino acid changes were located at the same positions found in viruses circulating. These studies could help to inform HA and NA protein residues targeted by antibody responses after virus infection in humans and are very relevant to update the strains used for influenza virus vaccination each year and to improve the currently available vaccines.

Development of an Alternative Modified Live Influenza B Virus Vaccine.

Influenza B virus (IBV) is considered a major human pathogen, responsible for seasonal epidemics of acute respiratory illness. Two antigenically distinct IBV hemagglutinin (HA) lineages cocirculate worldwide with little cross-reactivity. Live attenuated influenza virus (LAIV) vaccines have been shown to provide better cross-protective immune responses than inactivated vaccines by eliciting local mucosal immunity and systemic B cell- and T cell-mediated memory responses. We have shown previously that incorporation of temperature-sensitive (ts) mutations into the PB1 and PB2 subunits along with a modified HA epitope tag in the C terminus of PB1 resulted in influenza A viruses (IAV) that are safe and effective as modified live attenuated (att) virus vaccines (IAV att). We explored whether analogous mutations in the IBV polymerase subunits would result in a stable virus with an att phenotype. The PB1 subunit of the influenza B/Brisbane/60/2008 strain was used to incorporate ts mutations and a C-terminal HA tag. Such modifications resulted in a B/Bris att strain with ts characteristics in vitro and an att phenotype in vivo Vaccination studies in mice showed that a single dose of the B/Bris att candidate stimulated sterilizing immunity against lethal homologous challenge and complete protection against heterologous challenge. These studies show the potential of an alternative LAIV platform for the development of IBV vaccines.IMPORTANCE A number of issues with regard to the effectiveness of the LAIV vaccine licensed in the United States (FluMist) have arisen over the past three seasons (2013-2014, 2014-2015, and 2015-2016). While the reasons for the limited robustness of the vaccine-elicited immune response remain controversial, this problem highlights the critical importance of continued investment in LAIV development and creates an opportunity to improve current strategies so as to develop more efficacious vaccines. Our laboratory has developed an alternative strategy, the incorporation of 2 amino acid mutations and a modified HA tag at the C terminus of PB1, which is sufficient to attenuate the IBV. As a LAIV, this novel vaccine provides complete protection against IBV strains. The availability of attenuated IAV and IBV backbones based on contemporary strains offers alternative platforms for the development of LAIVs that may overcome current limitations.

Characteristics and immunobiology of grass pollen allergens.

Grass pollens are one of the most important airborne allergen sources worldwide. About 20 species from five subfamilies are considered to be the most frequent causes of grass pollen allergy, and the allergenic relationships among them closely follow their phylogenetic relationships. The allergic immune response to pollen of several grass species has been studied extensively over more than three decades. Eleven groups of allergens have been identified and described, in most cases from more than one species. The allergens range from 6 to 60 kD in apparent molecular weight and display a variety of physicochemical properties and structures. The most complete set of allergens has so far been isolated and cloned from Phleum pratense (timothy grass) pollen. Based on the prevalence of IgE antibody recognition among grass pollen-sensitized individuals, several allergens qualify as major, but members of two groups, groups 1 and 5, have been shown to dominate the immune response to grass pollen extract. Isoform variation has been detected in members of several of the allergen groups, which in some cases can be linked to observed genetic differences. N-linked glycosylation occurs in members of at least three groups. Carbohydrate- reactive IgE antibodies have been attributed to grass pollen sensitization and found to cross-react with glycan structures from other allergen sources, particularly vegetable foods. Another cause of extensive cross-reactivity are the group 12 allergens (profilins), which belong to a family of proteins highly conserved throughout the plant kingdom and present in all tissues. Members of eight allergen groups have been cloned and expressed as recombinant proteins capable of specific IgE binding. This development now allows diagnostic dissection of the immune response to grass pollen with potential benefits for specific immunotherapy.

The significance of isoallergenic variations in present and future specific immunotherapy.

The isoallergenic variation of the tree pollen major allergens has been studied by 2D gel electrophoresis, and by analysis of several recombinant clones. The studies have included both antibody-based and T cell stimulation assays. Bet v 1, the major allergen of birch, forms at least 24 spots when conventional extracts are analyzed by 2D gel electrophoresis. Comparison of Bet v 1-encoding DNA sequences reveals a considerable number of amino acid substitutions. This sequence variation can theoretically account for the number of spots observed in 2D gels. Whereas pools of serum from allergic individuals and monospecific antibodies raised in rabbits bind to most if not all spots in 2D gels, analyses of individual serum and/or murine monoclonal antibodies show individual patterns of reactivity with various subsets of spots. These observations point to a model in which amino acid substitutions induce local perturbations of the allergen surface, causing differences in epitope structure. Furthermore, analysis of pollen from individual trees shows that each tree produces individual subsets of Bet v 1 spots. When analyzed in stimulation assays, T cell clones also display differences in reactivity to different isoallergens. In conclusion, we have shown that Bet v 1 is heterogeneous, and that individual trees produce various subsets of isoallergens which display differences in reactivity both towards antibodies and T cells. A careful selection of isoform may therefore be of major importance if recombinant allergens or synthetic peptides are to be used for conventional immunotherapy.