Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus

Background: Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines. Methods: Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-gamma production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus. Results: We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-alpha and IFN-gamma) in T cells from non-human primates (NHPs) after BCG vaccination. Conclusions: PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

Identification of immuno-dominant antigens of Trypanosoma evansi for detection of chronic trypanosomosis using experimentally infected equines

Trypanosoma evansi is the most extensively distributed trypanosome responsible for disease called surra in livestock in many countries including frequent outbreaks in India. The prevalence of this disease is most commonly reported by standard parasitological detection methods (SPDM); however, antibody ELISA is being in practice by locally produced whole cell lysate (WCL) antigens in many countries. In the present investigation, we attempted to identify and purify immuno dominant, infection specific trypanosome antigens from T. evansi proteome using experimentally infected equine serum by immuno blot. Three immuno dominant clusters of proteins i.e. 62-66 kDa, 52-55 kDa and 41-43 kDa were identified based on their consistent reactivity with donkey sequential serum experimentally infected T. evansi up to 280 days post infection (dpi). The protein cluster of 62-66 kDa was purified in bulk in native form and comparatively evaluated with whole cell lysate antigen (WCL). ELISA and immuno blot showed that polypeptide of this cluster is 100% sensitive in detection of early and chronic infection. Further, this protein cluster was also found immuno reactive against hyper immune serum raised against predominantly 66 kDa exo antigen, revealed that this is a common immunodominant moieties in proteome and secretome of T. evansi.

An evaluation of antigen capture assays for detecting active filarial antigens

Lymphatic filariasis is a parasitic disease of tropical countries. This is a disfiguring and painful disease contracted in childhood, but the symptoms become apparent only in later years. Diagnosis of filarial infection is very crucial for the management of the disease. The main objective of this study was to develop a filarial antigen-based immunological assay for the diagnosis and surveillance of the disease. Monoclonal and polyclonal antibodies were raised to the recombinant protein Brugia malayi vespid allergen homologue (VAH). Capture enzyme-linked immunosorbent assay (ELISA) was standardized utilizing various combinations of antibodies and evaluated with serum samples of endemic normal (EN, n = 110), microfilaraemic (MF, n = 65), chronic pathology (CP, n = 45) and non-endemic normal (NEN, n = 10) individuals. Of the 230 samples tested, VAHcapture assay detected circulating antigen in 97.91% of bancroftian and 100% of brugian microfilaraemic individuals, and 5% of endemic normal individuals, comparable to the earlier reported SXP-1 antigen detection assay. However, the combination of VAH and SXP-1 (VS) capture ELISA was found to be more robust, detecting 100% of microfilaraemic individuals and with higher binding values. Thus an antigen capture immunoassay has been developed, which can differentiate active infection from chronic infection by detecting circulating filarial antigens in clinical groups of endemic areas.

Haloarchaeal gas vesicle nanoparticles displaying Salmonella antigens as a novel approach to vaccine development

A safe, effective, and inexpensive vaccine against typhoid and other Salmonella diseases is urgently needed. In order to address this need, we are developing a novel vaccine platform employing buoyant, self-adjuvanting gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1, bioengineered to display highly conserved Salmonella enterica antigens. As the initial antigen for testing, we selected SopB, a secreted inosine phosphate effector protein injected by pathogenic S. enterica bacteria during infection into the host cells. Two highly conserved sopB gene segments near the 3'- region, named sopB4 and sopB5, were each fused to the grIpC gene, and resulting SopB-GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and SopB5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of SopB-GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ApmrG-H111-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-y, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Thl response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were also found to be stable at elevated temperatures for extended periods without refrigeration. The results show that bioengineered GVNPs are likely to represent a valuable platform for antigen delivery and development of improved vaccines against Salmonella and other diseases.

Evaluation of live-attenuated Salmonella vaccines expressing Campylobacter antigens for control of C. jejuni in poultry

Campylobacter jejuni is a zoonotic bacterial pathogen of worldwide importance. It is estimated that 460,000 human infections occur in the United Kingdom per annum and these involve acute enteritis and may be complicated by severe systemic sequelae. Such infections are frequently associated with the consumption of contaminated poultry meat and strategies to control C. jejuni in poultry are expected to limit pathogen entry into the food chain and the incidence of human disease. Toward this aim, a total of 840 Light Sussex chickens were used to evaluate a Salmonella enterica serovar Typhimurium ΔaroA vaccine expressing the C. jejuni amino acid binding protein CjaA as a plasmid-borne fusion to the C-terminus of fragment C of tetanus toxin. Chickens were given the vaccine at 1-day-old and two weeks later by oral gavage, then challenged after a further two weeks with C. jejuni. Across six biological replicates, statistically significant reductions in caecal C. jejuni of c. 1.4 log10 colony-forming units/g were observed at three and four weeks post-challenge relative to age-matched unvaccinated birds. Protection was associated with the induction of CjaA-specific serum IgY and biliary IgA. Protection was not observed using a vaccine strain containing the empty plasmid. Vaccination with recombinant CjaA subcutaneously at the same intervals significantly reduced the caecal load of C. jejuni at three and four weeks post-challenge. Taken together these data imply that responses directed against CjaA, rather than competitive or cross-protective effects mediated by the carrier, confer protection. The impact of varying parameters on the efficacy of the S. Typhimurium ΔaroA vaccine expressing TetC-CjaA was also tested. Delaying the age at primary vaccination had little impact on protection or humoral responses to CjaA. The use of the parent strain as carrier or changing the attenuating mutation of the carrier to ΔspaS or ΔssaU enhanced the protective effect, consistent with increased invasion and persistence of the vaccine strains relative to the ΔaroA mutant. Expression in the ΔaroA strain of a TetC fusion to Peb1A, but not TetC fusions to GlnH or ChuA, elicited protection against intestinal colonisation by C. jejuni that was comparable to that observed with the TetC-CjaA fusion. Our data are rendered highly relevant by use of the target host in large numbers and support the potential of CjaA- and Peb1A-based vaccines for control of C. jejuni in poultry. © 2009 Elsevier Ltd. All rights reserved.

A preliminary survey of the helminth fish parasites in Imo River

A preliminary survey of the helminth fish parasites was carried out in Imo River (Nigeria). A total of 191 fishes belonging to 15 genera were examined and only 13.6% were infested with various species of helminth parasites which is low percentage of infection. The following helminth parasites were recorded. Wenyonia virilis, kainji; Wenyonia sp., Procamallanus laeviconchus; Procamallanus sp., Sprionoura congolense; Spironoura sp., Cucullanus sp. and Serradacnitis serrata. It was found that helminth parasites showed some degree of specificity in their distribution within their fish hosts. This specificity could be as a result of the physiochemical and physiological factors operating in the gut such as pH, osmotic tension, oxygen tension and nutrient levels. The results also revealed that age and sex influence helminth parasites infections in fishes

Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys

We transplanted kidneys from alpha 1,3-galactosyltransferase knockout (GalT-KO) pigs into six baboons using two different immunosuppressive regimens, but most of the baboons died from severe acute humoral xenograft rejection. Circulating induced antibodies to non-Gal antigens were markedly elevated at rejection, which mediated strong complement-dependent cytotoxicity against GaIT-KO porcine target cells. These data suggest that antibodies to non-Gal antigens will present an additional barrier to transplantation of organs from GaIT-KO pigs to humans.

Co-expression of CD173 (H2) and CD174 (Lewis Y) with CD44 suggests that fucosylated histo-blood group antigens are markers of breast cancer-initiating cells

Histo-blood group antigens CD173 (H2) and CD174 (Lewis Y) are known to be developmentally regulated carbohydrate antigens which are expressed to a varying degree on many human carcinomas. We hypothesized that they might represent markers of cancer-initiating cells (or cancer stem cells, CSC). In order to test this hypothesis, we examined the co-expression of CD173 and CD174 with stem cell markers CD44 and CD133 by flow cytometry analysis, immunocytochemistry, and immunohistochemistry on cell lines and tissue sections from breast cancer. In three breast cancer cell lines, the percentage of CD173(+)/CD44(+) cells ranged from 17% to > 60% and of CD174(+)/CD44(+) from 21% to 57%. In breast cancer tissue sections from 15 patients, up to 50% of tumor cells simultaneously expressed CD173, CD174, and CD44 antigens. Co-expression of CD173 and CD174 with CD133 was also observed, but to a lesser percentage. Co-immunoprecipitation and sandwich ELISA experiments on breast cancer cell lines suggested that CD173 and CD174 are carried on the CD44 molecule. The results show that in these tissues CD173 (H2) and CD174 (LeY) are associated with CD44 expression, suggesting that these carbohydrate antigens are markers of cancer-initiating cells or of early progenitors of breast carcinomas.

Helminth parasites of Caspian kilka (genus Clupeonella) as bioindicators of heavy metals

In the present study, intestinal helminth parasite fauna of 398 specimens of three species of kilkas, C. engrauliformis (N= 92), C. grimmi (N= 136) and C. cultriventris (N= 170) from Babolsar harbor were investigated. Five parasite species were found including: Corynosoma strumosum (Acanthocephala), Pronoprymna ventricosa (Trematoda), Contracaecum sp. (Nematoda), Raphidascaris sp. (Nematoda) and Anisakis sp. (Nematoda). The highest prevalence and abundance were observed in C. strumosum and P. ventricosa. The prevalence and abundance of C. strumosum in C. grimmi was significantly higher than C. engrauliformis. The prevalence and abundance of P. ventricosa in C. cultriventris was significantly higher than C. engrauliformis. The prevalence, mean intensity and mean abundance of the parasites were compared according to the sex, length group and season also effect of parasite on host growth parameters was considered. The diversity, equability, similarity, species richness, dominance Indices and specificity indices of helminth parasites of the three host species were studied. Also the lead and cadmium concentrations in the intestine, muscle, liver, kidney and gonad of kilkas and their parasites C. strumosum and P. ventricosa were measured and compared. The results revealed that lead and cadmium concentrations in C. strumosum and P. ventricosa were significantly higher than kidney, liver, intestine, gonad and muscle. The lead and cadmium concentrations of the parasites were compared according to the sex, parasitism and season.

Comparative study of seasonal changes in some blood parameters and genetic polymorphisms of serum antibodies and antigens on red blood cells surface in immature Caspian salmon

Seasonal sampling from 40 immature Caspian salmon were performed in summer, autumn, winter and spring. The maximum ranges of RBC counts, Hct, Hb, WBC count and clotting times were observed in spring, summer, spring, spring and winter, respectively. The minimum amounts of these factors were counted in summer, winter, winter, winter and winter, respectively. Blood Samples were taken from healthy smolt, immature and adult Caspian salmon in spawning time. Hematological determinations and biochemical serum analysis were performed in 101 fish in the three samples. The ranges of hematological values for sample mean were counted. Red blood cell counts were 866600 mm3 and 1259400 mm3 in smolt and adult respectively. Hematocrit was 48.39% in smolt and 44.29% in adult. Hemoglobin was 8.85 gr/dl in smolt and 10.91 gr/dl in adult. White blood cell count was 8781.58 mm3 in smolt and 5217.55 mm3 in adult and mean were differential of WBC, Lymphocyte 90.57%in smolt and73.22% in adult. Neutrophil was 5.12% in smolt and 16.92% in adult, Monocyte were 1.27% in smolt and 4.24% in adult, Clotting time was 282.34 Seconds in smolt and 291.47 seconds in adult MCV, MCH and MCHC also meagered in smolt and adult. Biochemical parameter in immature and mature Caspian salmon meagered .Glucose concentration was 2.97 mmol.l- in immature and 1.99 mmol.l- in mature .Cholesterol concentration was 4.26 mmol.l- in immature and 7.06 mmol.l- in mature. Triglyceride amount was 2.35 mmol.l- in immature and 2.47 mmol.l- in mature and Calcium was 2.47 in immature and 2.61 mmol.l- in mature. An in situ study was made on erythrocytic isoantigens and hetero-antigen and their corresponding iso-and hetero-antibodies of sera by means of hemoagglutination tests on the blood sample, of 450 immature and 50 mature Caspian salmon. The absence of erythrocyte iso-antigens and hetero-antigen and their corresponding iso-and hetero-antibodies were shown by the experimental. It could be indicated an intra-specific variation and differences in species for kelardasht hatchery.

Helminth fauna of Oreochromis leucostictus (Pisces: Cichlidae) from a tropical lake, Lake Naivasha, Kenya

The helminth fauna of Oreochromis leucosticus (Trewavas) from Lake Naivasha is described. The study was carried out on 1,050 gillnetted fish caught over a period of 24 months at different sites within the lake. Oreochromis leucosticus which is a commercially important fish was observed to be a host of four helminth parasites. These were an acanthocephalan polyacanthorhynchus kenyensis (Schmidt and Canaris), a nematode contracaecum sp.. a trematode clinostomum sp. and a dilepidid cestode Amirthalingamia sp. Seasonal variation in prevalence, intensity of infection, organs infected and the variation in infection with the size, sex and body condition of the host are described. There was no seasonal variation in prevalence but intensity of infection was observed to increase with the age of the fish. Although male fish were observed to be more heavily infected, the parasites did not seem to have any significant effect on the body condition of the fish.

Molecular modeling on human CCR5 receptors and complex with CD4 antigens and HIV-1 envelope glycoprotein gp120

AIM: To investigate the interaction between human CCR5 receptors (CCR5) and HIV-1 envelope glycoprotein gp120 (HIV-1 gp120) and HIV-1 receptor CD4 antigens (CD4). METHODS: The structurally con served regions (SCR) of human CCR5 was built by the SYBYL/Biopolymer module using the corresponding transmembrane (TM) domain of bacteriorhodopsin (bR) as the template. The coordinates for amino-ter minal residue sequence, and carboxyl-terminal residue sequence, extracellular and cytoplasmic loops were generated using LOOP SEARCH algorithm. Subsequently the structural model was merged into the complex with HIV-1 gp120 and CD4. RESULTS: Human CCR5 interacted with both an HIV-1 gp120 and CD4. The N-terminal residues (especially Met1 and Gln4) of human CCR5, contacted with CD4 residues, mainly 7Nith one span (56 - 59) of CD4 in electrostatic interaction and hydrogen-bonds. The binding sites of human CCR5 were buried in a hydrophobic center surrounded by a highly basic periphery. On the other hand, direct interatomic contacts were made between ? CCR5 residues and 6 gp120 amino-acid residues, which included van der Waals contacts, hydrophobic interaction, and hydrogen bonds. CONCLUSION: The interaction model should be helpful for rational design of novel anti-HIV drugs.

Diversity of intestinal helminth communities of carp from six lakes in the flood plain of the Yangtze river, China

The infra- and component communities of intestinal helminths of carp Cyprinus carpio were investigated in six lakes in the flood plain of the lower and middle reaches of the Yangtze River, China. Eight species of helminth parasites were recorded. The intestinal helminth communities were species rich in Niushan and Tonghu lakes where the digenean Asymphylodora japonica was the dominant species, whereas in Qinggang and Yanglan lakes a species-poor helminth community had only one species, Khawia sinensis. The degree of similarity within localities was highest in Qinggang and Yanglan lakes, and was high between communities where K. sinensis was the dominant species. The rich composition of these helminth communities may be because China is the heartland for carp while the poor helminth composition of those in Qinggang and Yanglan lakes may reflect the poor fauna there. It is suggested that species compositions of intestinal helminth communities of carp may be diversified in lakes in the hood plain of the Yangtze River. (C) 1999 The Fisheries Society of the British Isles.

Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens

Edwardsiella tarda is an opportunistic pathogen that can infect humans, animal, and fish. Two E. tarda antigens, Eta6 and FliC, which are homologues to an ecotin precursor and the FliC flagellin, respectively, were identified by in vivo-induced antigen technology from a pathogenic E. tarda strain isolated from diseased fish. When used as a subunit vaccine, purified recombinant Eta6 was moderately protective against lethal challenge of E. tarda in a Japanese flounder model, whereas purified recombinant FliC showed no apparent immunciprotectivity. Similarly, DNA vaccines based on eta6 and fliC in the form of plasmids pEta6 and pFliC induced, respectively, moderate and marginal protection against E. tarda infection. To improve the vaccine efficacy of eta6, a chimeric DNA vaccine, pCE6, was constructed, which encodes Eta6 fused in-frame to FliC. pCE6 was found to induce significantly higher level of protection than pEta6. Likewise, another chimeric DNA vaccine, pCE18, which expresses FliC fused to a previously identified E. tarda antigen Et18, elicited significantly stronger protective immunity than the DNA vaccine based on et18 alone. Fish immunized with pEta6 and pCE6 produced specific serum antibodies and exhibited significantly enhanced expression of the genes encoding elements that are involved in both innate and adaptive immune responses. Furthermore, the induction magnitudes of most of these genes were significantly higher in pCE6-vaccinated fish than in pEta6-vaccinated fish. (C) 2009 Elsevier Ltd. All rights reserved.