Allelic Frequencies of HPA-1 to 5 Human Platelet Antigens in Patients Infected With Hepatitis C Virus

Studies have suggested that hepatitis C virus (HCV) may infect not only hepatocytes but may also be carried by platelets. Platelets express more than 20 polymorphic antigenic determinants on their surface, which are called human platelet antigens (HPA), To determine the allele frequency of the HPA-1 to -5 in patients infected with HCV, blood samples were collected from 257 blood donors for the control group and from 191 patients infected with HCV. DNA was isolated and amplified for genes HPA-1 to -4 using PCR Sequence Specific Primers (PCR-SSP) and HPA-5 using PCR-Restriction Fragment Length Polymorphism (PCR-RFLP). The allelic and genotypic frequency of HPA-5a in patients infected with HCV was found to be significantly lower(P < 0.05) than in the controls, and HPA-5b from patients infected with HCV was significantly higher (P < 0.05) than in controls. The increase in HPA5b allelic frequency in HCV infection may indicate a possible association between HCV infection and HPAs. J. Med. Virol. 81:757-759, 2009. (C) 2009 Wiley-Liss, Inc.

Plasmodium falciparum: sequence diversity and antibody recognition of the Merozoite surface protein-2 (MSP-2) in Brazilian Amazonia

The merozoite surface protein-2 (MSP-2) of Plasmodium falciparum comprises repeats flanked by dimorphic domains defining the allelic families FC27 and IC1. Here, we examined sequence diversity at the msp-2 locus in Brazil and its impact on MSP-2 antibody recognition by local patients. Only 25 unique partial sequences of msp-2 were found in 61 isolates examined. The finding of identical msp-2 sequences in unrelated parasites, collected 6-13 years apart, suggests that no major directional selection is exerted by variant-specific immunity in this malaria-endemic area. To examine antibody cross-reactivity, recombinant polypeptides derived from locally prevalent and foreign MSP-2 variants were used in ELISA. Foreign IC1-type variants, such as 3D7 (currently tested for human vaccination), were less frequently recognized than FC27-type and local IC1-type variants. Antibodies discriminated between local and foreign IC1-type variants, but cross-recognized structurally different local IC1-type variants. The use of evolutionary models of MSP-2 is suggested to design vaccines that minimize differences between local parasites and vaccine antigens. (C) 2004 Elsevier B.V. All rights reserved.

Prevalence of Sarcocystis neurona and Neospora spp. infection in horses from Brazil based on presence of serum antibodies to parasite surface antigen

Sera from 961 horses from Brazil were tested for antibodies against the major surface antigens SnSAG4 and NhSAG1 to determine the seroprevalence of Sarcocystis neurona and Neospora hughesi, respectively. Antibodies against SnSAG4 were detected in 669 (69.6%) of the horses, while antibodies against NhSAG1 were detected in only 24 (2.5%) of the horses. These serologic results suggest that there is a high concentration of S. neurona in the environment of Brazil, which results in marked exposure of horses to this parasite. Additionally, the data further confirm that infection with Neospora spp. is relatively uncommon in horses. (c) 2005 Elsevier B.V. All tights reserved.

Origins of sequence diversity in the malaria vaccine candidate merozoite surface protein-2 (MSP-2) in Amazonian isolates of Plasmodium falciparum

The recent evolution of Plasmodium falciparum is at odds with the extensive polymorphism found in most genes coding for antigens. Here, we examined the patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2), a major malarial repetitive surface antigen. We compared the msp-2 gene sequences from closely related clones derived from sympatric parasite isolates from Brazilian Amazonia and used microsatellite typing to examine, in these same clones, the haplotype background of chromosome 2, where msp-2 is located. We found examples of msp-2 sequence rearrangements putatively created by nonreciprocal recombinational events, such as replication slippage and gene conversion, while maintaining the chromosome haplotype. We conclude that these nonreciprocal recombination events may represent a major source of antigenic diversity in MSP-2 in P falciparum populations with low rates of classical meiotic recombination. (c) 2006 Elsevier B.V. All rights reserved.

IMMUNOCYTOCHEMICAL DIFFERENTIATION OF REACTIVE HYPERPLASIA FROM FOLLICULAR LYMPHOMA USING MONOCLONAL-ANTIBODIES TO CELL-SURFACE AND PROLIFERATION-RELATED MARKERS

We tested the hypothesis that a panel of antibodies to cell surface, cytoplasmic, and nuclear antigens could reliably distinguish the cells composing reactive germinal centers from those composing follicular lymphoma. Immunocytochemistry was performed on deparaffinized sections of methacarn-fixed lymph node and tonsil (15 cases of reactive hyperplasia and 14 cases of follicular lymphoma) using antibodies to the nerve growth factor receptor (NGFR5), bcl-2 protein (124), proliferating cell nuclear antigen (PCNA; 19A2), and CD45RA (MT2). In 100% of cases of reactive hyperplasia, both MT2 and 124 showed positive immunostaining of mantle zone and scattered interfollicular lymphocytes, but in all cases there was a sharply demarcated absence of immunostaining of germinal center cells. However, diffuse immunostaining of follicular centers with MT2 (64%) and 124 (93%) and scattered intervening cells were seen in follicular lymphoma. The combination of antibodies to CD45RA and bcl-2 yielded positive immunostaining of follicular center cells in 93% of follicular lymphomas. The germinal center cells of reactive hyperplasia showed >75% nuclear positivity with antibodies to PCNA, in contrast to the follicular lymphoma cells, which showed variable PCNA indices ranging from 25 to >75%. A minority of follicular lymphoma cases (29%) showed PCNA indices comparable with those seen in cases of reactive hyperplasia. Antibodies to NGFR were positive in all cases of reactive hyperplasia and in 79% of cases of follicular hyperplasia, although the immunostaining intensity was generally decreased in follicular hyperplasia. In summary, antibodies to bcl-2 appear to be superior to those to CD45RA in distinguishing reactive hyperplasia from follicular lymphoma. Reactive hyperplasia cannot be discriminated from follicular hyperplasia using antibodies to PCNA or to nerve growth factor receptor.

Production of monoclonal antibodies against Streptococcus mutans antigens

Several studies have been conducted in the last decades aiming to obtain an anti-canies vaccine, however some studies have demonstrated cross reactivity between Streptococcus mutans surface antigens and the human cardiac tissue. In this work, the reactivity of five anti-Streptococcus mutans monoclonal antibodies (MoAb) (24A, 56G, G8, E8 and F6) was tested against oral streptococci, cardiac antigens and skeletal and cardiac myosins, aiming to evaluate the specificity of these MoAb. The hybrid producers of immunoglobulins of the IgG 2b class were cloned by limit dilution and expanded in vivo. MoAb were tested by ELISA. The hybrid 24A reacted with S. mutans CCT 1910, S. salivarius CCT 0365 and S. pyogenes T23. No reactivity difference was observed among the tested species. Cross reactivity with heart and cardiac myosin was not confirmed and only reaction with myosin of skeletal muscle was observed (p = 0.0381). The hybrid 56G reacted with all the tested microorganisms and there was statistically significant difference between S. mutans and S. pyogenes T23 (p < 0.001). This hybrid also reacted with myosin of skeletal muscle (p = 0.0095). C8, E8 and F6 presented low reactivity against oral streptococci strains and no reactivity against cardiac antigens. The data of this study showed that the 24A and 56G anti-S. mutans MoAb presented reactivity with S. pyogenes and S. salivarius, reinforcing the occurrence of common antigens between these species. The tested MoAb presented low cross-reactivity with myosin of skeletal muscle, but anti-heart activity could not be confirmed.

Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1

Abstract Background Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable tool for epidemiological studies, for screening blood donors in areas where the malaria is not endemic and for diagnosis of infected individuals. Because P. vivax cannot be easily obtained in vitro, ELISA assays using total or semi-purified antigens are rarely used. Based on this limitation, we tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite surface protein-1 of P. vivax (MSP119) could be useful for serological detection of malaria infection. Methods Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6-MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for their ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 200 serum samples collected from individuals living in the north of Brazil in areas endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection and 177 serum samples from individuals never exposed to malaria. Results Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals was 95%. The proportion of serum samples that reacted with recombinant proteins GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%, respectively. The specificity values of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and His6-MSP119-PADRE) or 100% (yMSP119-PADRE). Conclusions Our study demonstrated that for the Brazilian population, an ELISA using a recombinant protein of the MSP119 can be used as the basis for the development of a valuable serological assay for the detection of P. vivax malaria.

Evaluation of the acquired immune responses to Plasmodium vivax VIR variant antigens in individuals living in malaria-endemic areas of Brazil

Abstract Background The naturally-acquired immune response to Plasmodium vivax variant antigens (VIR) was evaluated in individuals exposed to malaria and living in different endemic areas for malaria in the north of Brazil. Methods Seven recombinant proteins representing four vir subfamilies (A, B, C, and E) obtained from a single patient from the Amazon Region were expressed in Escherichia coli as soluble glutathione S-transferase fusion proteins. The different recombinant proteins were compared by ELISA with regard to the recognition by IgM, IgG, and IgG subclass of antibodies from 200 individuals with patent infection. Results The frequency of individuals that presented antibodies anti-VIR (IgM plus IgG) during the infection was 49%. The frequencies of individuals that presented IgM or IgG antibodies anti-VIR were 29.6% or 26.0%, respectively. The prevalence of IgG antibodies against recombinant VIR proteins was significantly lower than the prevalence of antibodies against the recombinant proteins representing two surface antigens of merozoites of P. vivax: AMA-1 and MSP119 (57.0% and 90.5%, respectively). The cellular immune response to VIR antigens was evaluated by in vitro proliferative assays in mononuclear cells of the individuals recently exposed to P. vivax. No significant proliferative response to these antigens was observed when comparing malaria-exposed to non-exposed individuals. Conclusion This study provides evidence that there is a low frequency of individuals responding to each VIR antigens in endemic areas of Brazil. This fact may explain the host susceptibility to new episodes of the disease.

Evaluation of glycoconjugate antigens as vaccine candidates against group A Streptococcus and human immunodeficiency virus infections

This PhD thesis discusses the rationale for design and use of synthetic oligosaccharides for the development of glycoconjugate vaccines and the role of physicochemical methods in the characterization of these vaccines. The study concerns two infectious diseases that represent a serious problem for the national healthcare programs: human immunodeficiency virus (HIV) and Group A Streptococcus (GAS) infections. Both pathogens possess distinctive carbohydrate structures that have been described as suitable targets for the vaccine design. The Group A Streptococcus cell membrane polysaccharide (GAS-PS) is an attractive vaccine antigen candidate based on its conserved, constant expression pattern and the ability to confer immunoprotection in a relevant mouse model. Analysis of the immunogenic response within at-risk populations suggests an inverse correlation between high anti-GAS-PS antibody titres and GAS infection cases. Recent studies show that a chemically synthesized core polysaccharide-based antigen may represent an antigenic structural determinant of the large polysaccharide. Based on GAS-PS structural analysis, the study evaluates the potential to exploit a synthetic design approach to GAS vaccine development and compares the efficiency of synthetic antigens with the long isolated GAS polysaccharide. Synthetic GAS-PS structural analogues were specifically designed and generated to explore the impact of antigen length and terminal residue composition. For the HIV-1 glycoantigens, the dense glycan shield on the surface of the envelope protein gp120 was chosen as a target. This shield masks conserved protein epitopes and facilitates virus spread via binding to glycan receptors on susceptible host cells. The broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose oligosaccharides on the gp120 subunit of HIV-1 Env protein. This oligomannose epitope has been a subject to the synthetic vaccine development. The cluster nature of the 2G12 epitope suggested that multivalent antigen presentation was important to develop a carbohydrate based vaccine candidate. I describe the development of neoglycoconjugates displaying clustered HIV-1 related oligomannose carbohydrates and their immunogenic properties.

Synthese von MUC1-Glycopeptid-Konjugaten mit fluorierten Analoga des Thomsen-Friedenreich-Antigens

Krebserkrankungen gehen oft mit der Überexpression von mucinartigen Glycoproteinen auf der Zelloberfläche einher. In vielen Krebserkrankungen wird aufgrund der fehlerhaften Expression verschiedener Glycosyltransferasen das transmembranständige Glycoprotein MUC1, mit verkürzten Glycanstrukturen, überexprimiert. Das Auftreten der verschiedenen tumor-assoziierten Antigene (TACA) korreliert meist mit dem Fortschreiten des Krebs und der Metastasierung. Daher stellen TACAs interessante Zielmoleküle für die Entwicklung einer aktiven Tumorimmuntherapie zur spezifischen Behandlung von Adenokarzinomen dar. In dieser Arbeit galt das Interesse dem epithelialen Mucin MUC1, auf Basis dessen ein synthetischer Zugang zu einheitlichen Antitumorvakzinen, welche aus mucinanalogen Glyco-peptid¬konjugaten des MUC1 und Carrierproteinen bestehen, hergestellt werden sollten.rnUm eine tumorspezifische Immunantwort zu erhalten, müssen die selbst schwach immunogenen MUC1-Antigene über einen nicht-immunogenen Spacer mit einem geeigneten Trägerprotein, wie Tetanus Toxoid oder Rinderserumalbumin (BSA), verbunden werden. rnDa ein Einsatz von Glycokonjugaten in Impfstoffen durch die metabolische Labilität der O-glycosidischen Bindungen eingeschränkt ist, wurden hierzu erstmals fluorierte Vetreter von MUC1-analogen Glycopeptiden verwendet, in denen das Kohlenhydrat-Epitop durch den strategischen Einbau von Fluor¬atomen gegenüber einem raschen Abbau durch Glycosidasen geschützt werden soll. Dazu wurden auf Basis des literaturbekannten Thomsen-Friedenreich-Antigens Synthesestrategien zur Herstellung eines 2’F- und eines 2’,6’-bisfluorierten-Analogons erarbeitet. rnSchlüsselschritte in der Synthese stellten neben der elektrophilen Fluorierung eines Galactalvorläufers auch die -selektive 3-Galactosylierung des TN-Antigen-Bausteins zum 2’F- und 2’,6’-bisfluorierten-Analogons des TF-Disaccharids dar. Durch entsprechende Schutzgruppentransformationen wurden die beiden Derivate in entsprechende Glycosyl¬amino-säure-Bausteine für die Festphasensynthese überführt.rnNeben den beiden Analoga des TF-Antigens wurde auch erstmals ein 2F-Analogon des 2,6-Sialyl-T-Antigens hergestellt. Dazu wurde der entsprechende 2’F-TF-Baustein mit Sialinsäure-xanthogenat nach bereits bekannten Syntheseprotokollen umgesetzt. Aufgrund von Substanzmangel konnte die Verbindung nicht zur Synthese eines MUC1-Glycopeptid-Analogons herangezogen werden.rnDer Einbau der hergestellten Glycosylaminosäure-Bausteine erfolgte in die aus 20 Amino-säuren bestehende vollständige Wiederholungseinheit aus der tandem repeat-Sequenz des MUC1, wobei die entsprechenden Glycanseitenketten stets in Position 6 eingeführt wurden. Um die erhaltenen Glycopeptide für immunologische Studien an Carrier-Proteine anbinden zu können und so ggf. zu funktionsfähigen Impfstoff-Konjugaten zu gelangen, wurden diese stets N-terminal mit einem nicht-immunogenen Triethylenglycol-Spacer verknüpft. Die anschließende Funktionalisierung mit Quadratsäurediethylester erlaubte die spätere chemoselektive Konjugation an Trägerproteine, wie Tetanus Toxoid oder BSA.rnIn ersten immunologischen Bindungsstudien wurden die synthetisierten BSA-Glycopeptid-Konjugate mit Serum-Antikörpern aus Vakzinierungsstudien von MUC1-Tetanus Toxoid-Konjugaten, die (i) eine natürliche TF-Antigenstruktur und (ii) ein entsprechendes TF-Antigenderivat mit Fluorsubstituenten an C-6 des Galactosamin-Bausteins und C-6’ des Galactoserests tragen, untersucht.rn

In vitro induction of lymph node cell proliferation by mouse bone marrow dendritic cells following stimulation with different Echinococcus multilocularis antigens

The immune response of mice experimentally infected with Echinococcus multilocularis metacestodes becomes impaired so as to allow parasite survival and proliferation. Our study tackled the question on how different classes of E. multilocularis antigens (crude vesicular fluid (VF); purified proteinic rec-14-3-3; purified carbohydrate Em2(G11)) are involved in the maturation process of bone-marrow-derived dendritic cells (BMDCs) and subsequent exposure to lymph node (LN) cells. In our experiments, we used BMDCs cultivated from either naïve (control) or alveolar echinococcosis (AE)-infected C57BL/6 mice. We then tested surface markers (CD80, CD86, MHC class II) and cytokine expression levels (interleukin (IL)-10, IL-12p40 and tumour necrosis factor (TNF)-α) of non-stimulated BMDCs versus BMDCs stimulated with different Em-antigens or lipopolysaccharide (LPS). While LPS and rec-14-3-3-antigen were able to induce CD80, CD86 and (to a lower extent) MHC class II surface expression, Em2(G11) and, strikingly, also VF-antigen failed to do so. Similarly, LPS and rec-14-3-3 yielded elevated IL-12, TNF-α and IL-10 expression levels, while Em2(G11) and VF-antigen didn't. When naïve BMDCs were loaded with VF-antigen, they induced a strong non-specific proliferation of uncommitted LN cells. For both, BMDCs or LN cells, isolated from AE-infected mice, proliferation was abrogated. The most striking difference, revealed by comparing naïve with AE-BMDCs, was the complete inability of LPS-stimulated AE-BMDCs to activate lymphocytes from any LN cell group. Overall, the presenting activity of BMDCs from AE-infected mice seemed to trigger unresponsiveness in T cells, especially in the case of VF-antigen stimulation, thus contributing to the suppression of clonal expansion during the chronic phase of AE infection.

Trypanosoma congolense procyclins: unmasking cryptic major surface glycoproteins in procyclic forms

In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant surface molecule (PRS), which is expressed by procyclic forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in procyclic culture forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi procyclic forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah procyclic forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the surface coat of cells that are negative for both PRS and GARP.

Dendritic cells and macrophages form a transepithelial network against foreign particulate antigens

Fine particles (0.1-2.5 microm in diameter) may cause increased pulmonary morbidity and mortality. We demonstrate with a cell culture model of the human epithelial airway wall that dendritic cells extend processes between epithelial cells through the tight junctions to collect particles in the "luminal space" and to transport them through cytoplasmic processes between epithelial cells across the epithelium or to transmigrate through the epithelium to take up particles on the epithelial surface. Furthermore, dendritic cells interacted with particle-loaded macrophages on top of the epithelium and with other dendritic cells within or beneath the epithelium to take over particles. By comparing the cellular interplay of dendritic cells and macrophages across epithelial monolayers of different transepithelial electrical resistance, we found that more dendritic cells were involved in particle uptake in A549 cultures showing a low transepithelial electrical resistance compared with dendritic cells in16HBE14o cultures showing a high transepithelial electrical resistance 10 min (23.9% versus 9.5%) and 4 h (42.1% versus 14.6%) after particle exposition. In contrast, the macrophages in A549 co-cultures showed a significantly lower involvement in particle uptake compared with 16HBE14o co-cultures 10 min (12.8% versus 42.8%) and 4 h (57.4% versus 82.7%) after particle exposition. Hence we postulate that the epithelial integrity influences the particle uptake by dendritic cells, and that these two cell types collaborate as sentinels against foreign particulate antigen by building a transepithelial interacting cellular network.

Host responses to intestinal microbial antigens in gluten-sensitive mice

BACKGROUND AND AIMS: Excessive uptake of commensal bacterial antigens through a permeable intestinal barrier may influence host responses to specific antigen in a genetically predisposed host. The aim of this study was to investigate whether intestinal barrier dysfunction induced by indomethacin treatment affects the host response to intestinal microbiota in gluten-sensitized HLA-DQ8/HCD4 mice. METHODOLOGY/PRINCIPAL FINDINGS: HLA-DQ8/HCD4 mice were sensitized with gluten, and gavaged with indomethacin plus gluten. Intestinal permeability was assessed by Ussing chamber; epithelial cell (EC) ultra-structure by electron microscopy; RNA expression of genes coding for junctional proteins by Q-real-time PCR; immune response by in-vitro antigen-specific T-cell proliferation and cytokine analysis by cytometric bead array; intestinal microbiota by fluorescence in situ hybridization and analysis of systemic antibodies against intestinal microbiota by surface staining of live bacteria with serum followed by FACS analysis. Indomethacin led to a more pronounced increase in intestinal permeability in gluten-sensitized mice. These changes were accompanied by severe EC damage, decreased E-cadherin RNA level, elevated IFN-gamma in splenocyte culture supernatant, and production of significant IgM antibody against intestinal microbiota. CONCLUSION: Indomethacin potentiates barrier dysfunction and EC injury induced by gluten, affects systemic IFN-gamma production and the host response to intestinal microbiota antigens in HLA-DQ8/HCD4 mice. The results suggest that environmental factors that alter the intestinal barrier may predispose individuals to an increased susceptibility to gluten through a bystander immune activation to intestinal microbiota.

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.