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.

HA gene amino acid mutations contribute to antigenic variation and immune escape of H9N2 influenza virus.

Based on differences in the amino acid sequence of the protein haemagglutinin (HA), the H9N2 avian influenza virus (H9N2 virus) has been clustered into multiple lineages, and its rapidly ongoing evolution increases the difficulties faced by prevention and control programs. The HA protein, a major antigenic protein, and the amino acid mutations that alter viral antigenicity in particular have always been of interest. Likewise, it has been well documented that some amino acid mutations in HA alter viral antigenicity in the H9N2 virus, but little has been reported regarding how these antibody escape mutations affect antigenic variation. In this study, we were able to identify 15 HA mutations that were potentially relevant to viral antigenic drift, and we also found that a key amino acid mutation, A180V, at position 180 in HA (the numbering for mature H9 HA), the only site of the receptor binding sites that is not conserved, was directly responsible for viral antigenic variation. Moreover, the recombinant virus with alanine to valine substitution at position 180 in HA in the SH/F/98 backbone (rF/HAA180V virus) showed poor cross-reactivity to immune sera from animals immunized with the SH/F/98 (F/98, A180), SD/SS/94 (A180), JS/Y618/12 (T180), and rF/HAA180V (V180) viruses by microneutralization (MN) assay. The A180V substitution in the parent virus caused a significant decrease in cross-MN titres by enhancing the receptor binding activity, but it did not physically prevent antibody (Ab) binding. The strong receptor binding avidity prevented viral release from cells. Moreover, the A180V substitution promoted H9N2 virus escape from an in vitro pAb-neutralizing reaction, which also slightly affected the cross-protection in vivo. Our results suggest that the A180V mutation with a strong receptor binding avidity contributed to the low reactors in MN/HI assays and slightly affected vaccine efficacy but was not directly responsible for immune escape, which suggested that the A180V mutation might play a key role in the process of the adaptive evolution of H9N2 virus.

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.

Is seasonal vaccination a contributing factor to the selection of influenza epidemic variants?

Influenza A/H3N2 viruses are the most common and virulent subtypes for humans. Antigenic drift, changes in antigenicity through the accumulation of mutations in the hemagglutinin (HA) gene is chiefly responsible for the continuing circulation of A/H3N2 viruses, resulting in frequent updates of vaccine strains based on new variant analyses. In humans, these drift-related mutations are considered to be primarily caused by the immune pressure elicited by natural infection. Whether or not the immune pressure elicited by vaccination (vaccine pressure) can have a certain effect on drift-related mutations is unclear. Recently, our findings suggested the possible effect of vaccine pressure on HA mutations by directly comparing amino acid differences from the corresponding vaccine strains between isolates from vaccinated and unvaccinated patients. It is possible that influenza vaccine pressure selects variants genetically distant from the vaccine strains. Considering the effect of vaccine pressure on HA mutations would contribute to further understanding the mechanism of antigenic drift, which would be helpful for predicting future epidemic viruses.

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.

Promises and pitfalls for recombinant oligoclonal antibodies-based therapeutics in cancer and infectious disease.

Monoclonal antibodies (mAbs) have revolutionized the diagnosis and treatment of many human diseases and the application of combinations of mAbs has demonstrated improved therapeutic activity in both preclinical and clinical testing. Combinations of antibodies have several advantages such as the capacities to target multiple and mutating antigens in complex pathogens and to engage varied epitopes on multiple disease-related antigens (e.g. receptors) to overcome heterogeneity and plasticity. Oligoclonal antibodies are an emerging therapeutic format in which a novel antibody combination is developed as a single drug product. Here, we will provide historical context on the use of oligoclonal antibodies in oncology and infectious diseases and will highlight practical considerations related to their preclinical and clinical development programs.

Antigenic variation of the human influenza A (H3N2) virus during the 2014-2015 winter season.

The human influenza A (H3N2) virus dominated the 2014-2015 winter season in many countries and caused massive morbidity and mortality because of its antigenic variation. So far, very little is known about the antigenic patterns of the recent H3N2 virus. By systematically mapping the antigenic relationships of H3N2 strains isolated since 2010, we discovered that two groups with obvious antigenic divergence, named SW13 (A/Switzerland/9715293/2013-like strains) and HK14 (A/Hong Kong/5738/2014-like strains), co-circulated during the 2014-2015 winter season. HK14 group co-circulated with SW13 in Europe and the United States during this season, while there were few strains of HK14 in mainland China, where SW13 has dominated since 2012. Furthermore, we found that substitutions near the receptor-binding site on hemagglutinin played an important role in the antigenic variation of both the groups. These findings provide a comprehensive understanding of the recent antigenic evolution of H3N2 virus and will aid in the selection of vaccine strains.

N-terminal disulfide-bridging of Borrelia outer surface protein C increases its diagnostic and vaccine potentials.

OspC is the main target for IgM in early-stage Lyme disease. As such it is employed as its native or recombinant form in routine immunoassays for the determination of Borrelia-specific antibodies. However, recombinant OspC has so far not shown the antigenicity of the native protein. The latter contains an intrinsic signal sequence and an adjacent cysteine residue, the attachment site of the lipid membrane anchor which has been discussed to have an adjuvant effect on the immune reaction. In expression experiments, we have found a recombinant variant, an OspC covalently homodimerized via an N-terminal disulfide bridge, that shows a remarkably enhanced antigenicity without lipid attachment. Three such OspCs derived from different Borrelia strains were subsequently expressed in E. coli and purified under non-reducing conditions. In non-reducing SDS-PAGE, OspC(Δ1-18) exhibited a 48-kDa band of dimeric OspC. When incubated with IgM-OspC-positive human sera, the reaction at 48kDa was always stronger than at 24kDa of monomeric OspC(Δ1-18, C19G). A lineblot with OspC(Δ1-18) also showed a higher diagnostic accuracy than that obtained with OspC(Δ1-18, C19G) based on a higher affinity of IgM for the dimeric form. When used for the immunization of mice, dimeric OspC(Δ1-18) induced consistent high-titre antibodies against OspC, whereas OspC(Δ1-18, C19G) failed to provoke significant titres in some animals. We conclude that the disulfide-bridging of 2 OspC molecules via their N termini forms a complex that is more suitable for the determination of IgM-OspC and is a promising candidate for a monovalent vaccine.

Borrelia burgdorferi vlsE antigenic variation is not mediated by RecA.

RecA is a key protein linking genetic recombination to DNA replication and repair in bacteria. Previous functional characterization of Borrelia burgdorferi RecA indicated that the protein is mainly involved in genetic recombination rather than DNA repair. Genetic recombination may play a role in B. burgdorferi persistence by generation of antigenic variation. We report here the isolation of a recA null mutant in an infectious B. burgdorferi strain. Comparison of the in vitro growth characteristics of the mutant with those of the wild-type strain under various conditions showed no significant differences. While the RecA mutant was moderately more sensitive to UV irradiation and mitomycin C than the wild-type strain, the lack of RecA abolished allelic exchange in the mutant. Absence of RecA did not affect the ability of the mutant to infect mice. However, the RecA mutant was attenuated for joint infection in competitive-infection assays with the wild-type strain. vlsE sequence variation in mice was observed in both wild-type and RecA mutant spirochetes, indicating that the mechanism of antigenic variation is not homologous genetic recombination.

A Leishmania (L.) amazonensis ATP diphosphohydrolase isoform and potato apyrase share epitopes: antigenicity and correlation with disease progression.

A Leishmania (Leishmania) amazonensis ATP diphosphohydrolase isoform was partially purified from plasma membrane of promastigotes by preparative non-denaturing polyacrylamide gel electrophoresis. SDS-PAGE followed by Western blots developed with polyclonal anti-potato apyrase antibodies identified diffuse bands of about 58-63 kDa, possibly glycosylated forms of this protein. By ELISA technique, a significantly higher total IgG antibody level against potato apyrase was found in serum from promastigote-infected mice, as compared to the uninfected mice, confirming both the existence of shared epitopes between the parasite and vegetable proteins, and the parasite ATP diphosphohydrolase antigenicity. By Western blotting, serum from amastigote-infected BALB/c mice recognizes both potato apyrase and this antigenic ATP diphosphohydrolase isoform isolated from promastigotes, suggesting that it is also expressed in the amastigote stage. The infection monitored along a 90-day period in amastigote-infected mice showed reactivity of IgG2a antibody in early steps of infection, while the disappearance of the IgG2a response and elevation of IgG1 antibody serum levels against that shared epitopes were associated with the progression of experimental leishmaniasis. This is the first observation of the antigenicity of a L. (L.) amazonensis ATP diphosphohydrolase isoform, and of the ability of cross-immunoreactivity with potato apyrase to differentiate serologically stages of leishmaniasis in infected mice.

Rapid acquisition of isolate-specific antibodies to chondroitin sulfate A-adherent plasmodium falciparum isolates in Ghanaian primigravidae.

Recent evidence suggests that pregnancy-associated malaria (PAM), associated with maternal anemia and low birth weight, results from preferential sequestration of parasitized red blood cells (pRBC) in the placenta via binding of variant surface antigens (VSA) expressed on the surface of pRBC to chondroitin sulfate A (CSA). The VSA mediating CSA binding (VSA(CSA)) and thus sequestration of pRBC in the placenta are antigenically distinct from those that mediate pRBC sequestration elsewhere in the body, and it has been suggested that VSA(CSA) are relatively conserved and may thus constitute an attractive target for vaccination against PAM. Using flow cytometry, levels of antibody to VSA and VSA(CSA) expressed on the surface of red blood cells infected with Plasmodium falciparum isolates were measured during pregnancy and lactation in Ghanaian primigravid women enrolled in a trial of maternal vitamin A supplementation. Antibody responses to VSA(CSA) were detected within the first trimester of pregnancy and increased with increasing duration of pregnancy, and they seemed to be isolate specific, indicating that different CSA-adherent parasite lines express antigenically distinct VSA and thus may not be as antigenically conserved as has been previously suggested. Levels of anti-VSA(CSA) were not significantly associated with placental malarial infection determined by histology, indicating that primary immune responses to VSA(CSA) may not be sufficient to eradicate placental parasitemia in primigravidae.

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.

Allergenic diversity of the olive pollen.

A great number of allergenic proteins have been detected in olive pollen extracts. To date, nine allergens have been isolated and characterized, which have been called Ole e 1 to Ole e 9. The most prevalent olive allergen is Ole e 1, which affects more than 70% of patients hypersensitive to olive pollen, but others, such as Ole e 2, Ole e 8, and Ole e 9, have been demonstrated to be major allergens, and Ole e 6 or Ole e 7 reach high values of clinical incidence. Many of these allergens, such as Ole e 2 (profilin) and Ole e 3 (polcalcin), are involved in cross-reactivities, which agrees with their adscription to panallergenic families. Among the many olive allergens of high molecular mass, only Ole e 9 (46 kDa) has been characterized. The allergen is a polymorphic and glycosylated beta-1,3-glucanase, which belongs to a pathogenesis-related (PR-2) protein family. In addition to the polypeptide epitopes, Ole e 1 also exhibits IgE-binding determinants in the carbohydrate, which are recognized by more than 60% of the sera from patients sensitive to the whole allergen, although the level of such glycan-specific IgE seems not to be clinically relevant in the overall content of the sera. Recent advances in the elucidation of the structure of the Ole e 1-oligosaccharide component allows us to explain the antigenicity of the molecule. Finally, the recombinant production of several allergens from olive pollen in both bacterial and eukaryotic cells has allowed us to resolve problems derived from the polymorphism and scarcity of the natural forms of these allergens. The biological equivalence between the natural and recombinant forms lets us initiate studies on the design of mixtures for clinical purposes, in which hypoallergenic derivatives of these allergens could play a definitive role.

Heterogeneity of pollen proteins within individual Betula pendula trees.

To determine the variation of antigenic water soluble proteins in white birch (Betula pendula) pollen, extracts of pollen from different sides of individual trees were analyzed by isoelectric focusing (IEF), crossed immunoelectrophoresis, and crossed radioimmunoelectrophoresis. IgE-antibody-binding patterns were also studied in samples analyzed by IEF by probing with serum pooled from patients with birch pollen allergy, followed by radiolabelled anti-IgE. Antigenic proteins and allergens per unit weight of extracted protein were greatest in pollen extracts from the south side of the trees. Allergens decreased progressively in pollen from west-through east- to north- facing branches. Proteins with high isoelectric points (pI > 8.5) and proteins between pI 4.5 and 5.6 were infrequent in extracts from the north side. Extracts from branches facing north were poor in allergens: in general, only one or two precipitin lines were found, and in some cases they did not bind to IgE antibodies. Differences between numbers of proteins and allergens found in extracts from south, west and north branches were statistically significant for all methods used. The results indicate the need to collect birch pollen for allergen extract manufacture from south-facing branches.

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.

Variability of crossreactivity of IgE antibodies to group I and V allergens in eight grass pollen species.

Crossreactivity to Dactylis glomerata, Festuca rubra, Phleum pratense, Anthoxanthum odoratum, Secale cereale, Zea mays, and Phragmites communis of IgE antibodies against Lol p I or Lol p V was investigated by means of RAST-inhibition. Within a group of sera the degree of crossreactivity was demonstrated to be highly variable. Individual sera were not always equally crossreactive to all pollen species. A high degree of crossreactivity for Group I allergens did not necessarily implicate the same for Group V. Group I and Group V representatives were found to be present in all eight species. It was demonstrated that within this group of grass species significant quantitative and qualitative differences exist, with respect to Group I and Group V allergens. Species with a low phylogenetic affinity to Lolium perenne, like Zea mays and Phragmites communis showed a very low degree of reactivity, even when measured with the most crossreactive sera. A higher taxonomic relationship however, did not always implicate a closer antigenic resemblance. Antigenically both allergens from Zea mays are more similar to Lol p I and Lol p V, than the analogues in Secale cereale.