Expression, immunogenicity and diagnostic value of envelope proteins from an Egyptian hepatitis C virus isolate.

The present work aimed at 1) characterization of the E1 and E2 proteins (HCV-E) from an Egyptian hepatitis C virus genotype 4a (HCV-4a) isolate at the molecular and immunological level, 2) in silico identification of the B- and T-cell epitopes responsible for the immunogenicity of HCV-E, and 3) evaluation of the diagnostic potential of both the recombinant HCV-E and antibodies raised using mammalian expression constructs encoding the protein. The region encoding the E1 and E2 proteins was amplified by RT-PCR from RNA isolated from blood of a human infected with HCV-4 and cloned into the pSC-TA plasmid, and the sequence was verified and used to construct a neighbor-joining phylogenetic tree. The translated nucleotide sequence was used to predict the HCV-E secondary structure using the PREDICT-PROTEIN server and PSI-PRED. A 3D model of HCV-E was generated using the online tool 3Dpro. B- and T-cell epitopes were predicted using the online tools BCPred and Epijen v1.0, respectively. The HCV-E-encoding sequence was later subcloned into the mammalian expression plasmid pQE, and the constructs that were generated were used to immunize mice in the absence and presence of adjuvants of plant origin. The maximum sequence identity obtained by nucleotide and protein BLAST analysis with previously published HCV-E sequences was 85 and 77 %, respectively. The B-cell epitope CFTPSPVVV at position 203 and the T-cell epitope ALSTGLIHL at position 380 were found to be highly conserved among all HCV genotypes. Both ELISA and Western blotting experiments on crude and purified recombinant HCV envelope proteins using mouse antisera raised using the HCV-E mammalian expression construct confirmed the specific antigenicity of the expressed protein. The antibodies raised in mice using the HCV-E-encoding construct could efficiently capture circulating antigens in patients' sera with good sensitivity that correlated with liver enzyme levels (r = 0.4052, P < 0.0001 for ALT; r = -0.5439, P = 0.0019 for AST). Moreover, combining the HCV-E-encoding construct with extracts prepared from Echinacea purpurea and Nigella sativa prior to immunizing mice significantly (P < 0.05) increased both the humoral (14.9- to 20-fold increase in antibodies) and the cellular (CD4(+) and cytotoxic CD8(+)- T lymphocytes) responses compared to mice that received the DNA construct alone or PBS-treated mice. Both recombinant HCV-E protein preparations and antibodies raised using the HCV-E-encoding mammalian expression construct represent useful diagnostic tools that can report on active HCV infection. Also, the immunostimulatory effects induced by the two plant extracts used at the cellular and humoral level highlight the potential of natural products for inducing protection against HCV infection. The neutralizing capacity of the induced antibodies is a subject of future investigations. Furthermore, the predicted B- and T-cell epitopes may be useful for tailoring future diagnostics and candidate vaccines against various HCV genotypes.

Propolis enhances the effectiveness of praziquantel in experimental schistosomiasis: biochemical and histopathological study.

Despite the wide current use of praziquantel (PZQ) in treatment of schistosomiasis, low cure rates have been recorded in many studies. The aim of this study was directed to evaluate the curative effect of propolis (Pps) alone or in combination with PZQ on biochemical, immunological, parasitological, and histological changes associated with experimental schistosomiasis in mice. Schistosoma mansoni-infected mice were divided into two experimental sets, each with four subgroups: (i) untreated, (ii) treated with Pps/day p.o for 4 weeks, (iii) treated with PZQ p.o 2 × 500 mg/kg bd wt, and (iv) treated with Pps + PZQ as in group ii and iii; all treatments started on the 8th week postinfection, in addition to uninfected group as control for the previous groups. Treatment of infected mice with Pps, although failed to eradicate the worm, significantly reduced the hepatic granuloma number, their lymphocytic infiltration and aggregation, hepatic and splenic myeloperoxidase (MPO) activity and plasma, and liver and thymus nitric oxide (NOx) levels together with normalization of plasma proteins and alleviation of oxidative stress in the examined tissues as evidenced by reduction of malondialdehyde (MDA) and normalization of glutathione (GSH). Promising results were obtained when Pps was given in combination with PZQ, where the anti-schistosomal activity of PZQ was markedly potentiated with complete alleviation and amelioration of the histological and biochemical alteration associated with schistosomiasis. This study highlights the potential usefulness of Pps as an adjunct to PZQ in schistosomiasis.

Immune responses in mice after immunization with antigens from different stages of the parasite Schistosoma mansoni.

Mice responses to immunization with Schistosoma mansoni antigens were investigated. Priming with cercarial antigen preparation (CAP) induced significant (P < 0.05) IgM, IgG, IgG2a, IgG2b, and IgA increases, while booster caused a significant IgG1 increase. A soluble worm antigen preparation (SWAP) caused significant IgG elevation. Priming with soluble egg antigen (SEA) caused significant IgM and IgG2a increases, while booster induced significant IgM, IgG and IgA increases. CAP-immunized mice sera (IMS) recognized CAP peptides ranging from 23-78 kDa. SWAP-IMS recognized SWAP peptides ranging from 40-75 kDa. SEA-IMS recognized SEA peptides ranging from 33-101 kDa. The cross-reactive peptides among the 3 antigens were identified. CAP caused significant increases in mesenteric lymph nodes (MLNs) CD(4,8)+, B lymphocytes, CD8+ thymocytes, CD4+ T and B splenocytes. SWAP priming caused significant increases in MLNs CD(4,8)+ thymocytes and B splenocytes. SWAP booster caused significant increases in MLNs CD8+ T and B lymphocytes, CD(4,8)+ thymocytes and CD4+ T and B splenocytes. SEA caused significant increase in CD4+ T cells.

Immunostimulatory effects of extract of Pulicaria crispa before and after Schistosoma mansoni infection.

The immunostimulatory effects of methanolic extract from Pulicaria crispa were investigated in mice before and after infection with Schistosoma mansoni. Mice were subjected for daily intra-peritoneal injection by the extract (33 ng/mouse) for 10 successive days followed by infecting every mouse with 100 S. mansoni cercariae. Treatment with the extract induced significant increase (p < 0.05) in sera-IL-2 before and after infection. Upon using soluble worm antigen preparation or cancer bladder homogenates as antigens in ELISA, the detected levels of IgG were significantly (p < 0.05) higher in sera from treated-infected mice than untreated P. crispa infected mice. Using crude Escherichia coli lysate as an antigen in ELISA, it was detected a significant (p < 0.05) increase in IgG levels in sera from the extract-treated mice before and after infection.

Anti-schistosomal activity of colostral and mature camel milk on Schistosoma mansoni infected mice.

The aim of the present study was to investigate the anti-schistosomal activity of colostral and mature camel milk on Schistosoma mansoni infected mice. Six weeks post infection, mean percentage of protection was detected through the hepatic portal vein. Glutathione-s-transferase (GST), alanine, aspartate transaminase (ALT and AST) and immunoglobulin G (IgG) levels were detected in sera of treated mice before and after infection. Antischistosomal activity of colostral and mature camel milk on Schistosoma mansoni infected mice were 12.81% and 31.60% respectively. The results showed that GST levels in sera of mice fed on colostral and mature camel milk were increased with mean values of 0.070, 0.108, 0.128 and 0.120 in colostral milk groups and 0. 072, 0.085, 0.166 and 0.20 in mature camel milk groups compared with the mice fed on basal diet with means values of 0.070, 0.085, 0.078 and 0.069 before infection and after two, four and six weeks of infection, respectively. On the other hand, there were slight differences on ALT and AST activities. Mice treated with colostral and mature milk (200 microl/day) showed an immunostimulatory effect by inducing IgG titers against soluble worm antigen preparation (SWAP) compared with control. Nevertheless, the difference was not considered significant (0.31 +/- 0.1) for colostrum (0.34 +/- 0.1) and for mature milk, as compared to normal control (0.2 +/- 0.04). Two, four and six weeks post infection, IgG level showed no significant change in sera from mice treated with colostral and mature milk as compared to control. In conclusion, colostral and mature camel milk showed an immuno-modualatory effect in normal healthy mice by inducing IgG and GST levels before and after infection with Schistosoma mansoni. Colostral and mature camel milk have a protective response against schistosomiasis.

Immunization of mice with crude extract of Saccharomyces boulardii yeast induces cross-reactive immune responses with antigenic preparations from different developmental stages of the Schistosoma mansoni and reduces the parasite worm burden.

Immunization of mice with Saccharomyces boulardii crude extract caused a significant reduction in Schistosoma mansoni worm burden (23.7%; P = 0.02) in comparison to control mice. Mice sera collected after the 1st immunization showed a significant increase (P < 0.05) in both IgM and IgG against parasite soluble cercarial antigenic preparation (CAP) than unimmunized mice sera (UMS). Upon 2nd immunization, sera from yeast extract immunized mice (YIM) showed increase of IgM and IgG (1.6 and 1 fold) against CAP than UMS. Sera collected 2, 4 and 6 weeks (W) after infection of YIM with S. mansoni cercariae showed higher IgM (1.4, 1.5 and 1 folds) and IgG (0.9, 1.2 and 1.1 folds) levels against CAP than sera of infected UM (IUM). When compared to sera from UM, a week post 1st and 2nd immunization sera showed a significant elevation (P < 0.05) of both IgM and IgG to parasite soluble adult worm antigenic preparation (SWAP). Sera collected at 2, 4 and 6W after infection of YIM with parasite cercariae showed higher IgM (1.1, 1.3 and 1.2 folds) and IgG (1.2, 1.1 and 1.3 folds) reactivities against SWAP than sera of IUM. One week post 1st and 2nd immunization with the yeast extract, sera showed a significant increase (P < 0.05) of both IgM and IgG levels against parasite soluble egg antigen (SEA) when compared with sera from UM. At 2 and 4W post infection, sera from immunized infected mice showed a significant decrease in IgM (P < 0.05) but the decrease of IgG (0.7 fold) was not significant when compared to IUM. Six weeks post infection the decrease in both antibodies remained but it was significant for IgG (P < 0.05) but not for IgM (2.1 folds) when compared with IUM. Immunization with S. boulardii extract caused a non-significant increase (1.2 folds) in mesenteric lymph node (MLN)-CD4+T cells, significant elevation of MLN-CD8+T cells (P < 0.05) and a non-significant increase (0.9 folds) of B-cells when compared to that of UM. Also, Immunization with S. boulardii extract caused 1 fold elevation in both CD4+ and CD8(+)-T lymphocytes in thymus of YIM when compared with their counts in thymus of UM.

Humoral and cellular immune responses induced in mice by purified iridoid mixture that inhibits penetration of Schistosoma mansoni cercariae upon topical treatment of mice tails.

When tested for possible blocking effect on the cercarial, serine proteinase, elastase (CE) activity, an iridoid mixture extracted from leaves of Citharexylum quadrangular abolished 31% of the enzyme activity at final concentration 15 microg. When formulated in jojoba oil and applied to mice tails followed by infection with Schistosoma mansoni cercariae, the iridoid mixture blocked cercarial penetration and caused significant reducetion (94%; P < 0.05) in worm burden in treated mice in comparison to controls. Also, immunomodulatory effects of iridoid mixture, iridoid-treated S. mansoni worm homogenate on mice were studied by measuring IgG and IgM levels against E. coli lysates (ECL), solube S. mansoni worm antigenic preparation (SWAP) and cancer bladder homogenates (CBH) as antigens by ELISA. Cellular immune responses were studied by calculating mean percent of CD4+, CD8(+)-T, B-mesenteric lymph node cells (MLNC) and CD4+, CD8(+)-T thymocytes by direct immunofluorescence staining in treated mice as compared to untreated homogenate given mice or untreated mice. Injecting mice with serial dilutions of iridoid mixture resulted in fluctuation, peaks and troughs, in both IgG and IgM responses against the above mentioned antigens. 1st and 2nd immunizations with iridoid mixture treated homogenate resulted in significantly elevated (P < 0.05). IgM and IgG levels against the 3 used antigens in comparison with sera from control mice. Immunized mice with homogenate treated with iridoid mixture showed a significant increase (P < 0.05) in CD4+T thymocytes, a non significant increase in CD8+T thymocytes, a significant increase (P < 0.05) in CD4+T lymphocytes (MLNC) and a non significant increase in CD8+ T- and B-lymphocytes (MLNC) compared with mice immunized with untreated homogenate or non-injected normal mice.