Papain-Based Vaccination Modulates Schistosoma mansoni Infection-Induced Cytokine Signals.

We have previously shown that immunization of outbred rodents with cysteine peptidases-based vaccine elicited type 2-biased immune responses associated with consistent and reproducible protection against challenge Schistosoma mansoni. We herein start to elucidate the molecular basis of C57BL/6 mouse resistance to S. mansoni following treatment with the cysteine peptidase, papain. We evaluated the early cytokine signals delivered by epidermal, dermal, and draining lymph node cells of naïve, and S. mansoni -infected mice treated 1 day earlier with 0 or 50 µg papain, or immunized twice with papain only (10 µg/mouse), papain-free recombinant S. mansoni glyceraldehyde 3-phosphate dehydrogenase and 2-Cys peroxiredoxin peptide (10 and 15 µg/mouse, respectively = antigen Mix), or papain-adjuvanted antigen Mix. Schistosoma mansoni infection induced epidermal and lymph node cells to release type 1, type 2 and type 17 cytokines, known to counteract each other. Expectedly, humoral immune responses were negligible until patency. Papain pretreatment or papain-based vaccination diminished or shut off S. mansoni infection early induction of type 1, type 17 and type 2 cytokines except for thymic stromal lymphopoietin and programmed the immune system towards a polarized type 2 immune milieu, associated with highly significant (P < 0.005 - <0.0001) resistance to S. mansoni infection.

Adjuvant selection for vaccination against murine schistosomiasis.

Schistosoma mansoni cercariae penetrate mouse epidermis, detach the glycocalyx and transform into schistosomula, triggering innate immune responses by host keratinocytes and Langerhans cells. Schistosomula leave the dermis and enter blood capillaries, releasing excretory/secretory products (ESP), which induce readily detectable primary adaptive immunity responses, dominated by T helper (Th) 1 and 17 cytokines. Partial protection against murine schistosomiasis may be achieved using subunit antigens and Th1 cytokine-inducing adjuvants. Conversely, resistance to primary and/or secondary schistosomiasis in rats, mice and humans is associated with production of Th2 cytokines. Accordingly, we reasoned that effective vaccination against murine primary schistosomiasis might be achieved provided selection of an adjuvant capable of skewing the S. mansoni larval ESP-mediated Th1/Th17 immune responses towards a Th2 profile. In an aim to select such an adjuvant, we administered the prototypical Th1 and Th2, respectively, C57BL/6 and BALB/c mice with polyinosinic-polycytidylic acid (Poly I/C), peptidoglycan (PGN), or thymic stromal lymphopoietin (TSLP) before exposure to S. mansoni cercariae. Serum antibody reactivity and ex vivo spleen cells (SC) immune responses to larval ESP, in a recombinant or multiple antigen peptide form, were assessed 1 week after infection. Injection with Poly I/C failed to increase interleukin (IL)-4 and led to elevated gamma interferon (IFN-γ) levels released by unstimulated or ESP-stimulated SC. Treatment with PGN triggered hightened amounts of IL-4, IL-17 and IFN-γ released by unstimulated or ESP-stimulated C57BL/6 SC. In contrast, TSLP succeeded in directing the ESP-mediated immune responses towards a Th2-biased profile in prototypical Th1 and Th2 mice.

Fasciola gigantica excretory-secretory products for immunodiagnosis and prevention of sheep fasciolosis.

Excretory-secretory products (ESP) products of ex vivo Fasciola gigantica adult worms were used for immunodiagnosis of sheep experimental infection with F. gigantica and natural infection with Fasciola spp. by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Specific IgG antibody binding to native or denatured ESP was detected as early as 2 weeks after experimental sheep infection with 100 or 200 metacercariae. No specific IgG antibody binding was displayed by sera obtained from 192 sheep considered to be Fasciola- and other parasite-free by microscopic examination of bile and feces. Additionally, sera from 200 apparently Fasciola-free sheep, yet infected with other parasites, were all negative. The data, thus, indicated that ESP-based ELISA reached nearly 100% sensitivity and specificity in immunodiagnosis of sheep fasciolosis. As expected, the ESP molecules were immunogenic in sheep eliciting interleukin-12p40 mRNA response and considerable amounts of antibodies, which were able to bind to the surface of newly excysted juvenile worms as judged by membrane indirect immunofluorescence, and mediate their attrition via antibody-dependent cell-mediated cytotoxicity. The ESP-induced cellular and humoral immune responses were associated with a modest reduction in worm count, yet with a highly significant (P<0.0001) decrease in size of recovered worms, thus suggesting that ESP immunization might be a safe and cost-effective strategy for reducing transmission of the infection.

Fasciola gigantica enolase is a major component of worm tegumental fraction protective against sheep fasciolosis.

Infection of cattle and sheep with the parasite Fasciola gigantica is a cause of important economic losses throughout Asia and Africa. Many of the available anthelmintics have undesirable side effects, and the parasite may acquire drug resistance as a result of mass and repeated treatments of livestock. Accordingly, the need for developing a vaccine is evident. Triton-soluble surface membrane and tegumental proteins (TSMTP) of 60, 32, and 28 kDa previously shown to elicit protective immunity in mice against challenge F. gigantica infection were found to be strongly immunogenic in sheep eliciting vigorous specific antibody responses to a titer>1:16,000 as assessed by enzyme-linked immunosorbent assay. Furthermore, the 60 kDa fraction induced production of antibodies able to bind to the surface membrane of newly excysted juvenile flukes and mediate their attrition in antibody-dependent complement- and cell-mediated cytotoxicity assays, and significant (P<0.05) 40% protection of sheep against F. gigantica challenge infection. Amino acid micro sequencing of the 60 kDa-derived tryptic peptides revealed the fraction predominantly consists of F. gigantica enolase. The cDNA nucleotide and translated amino acid sequences of F. gigantica enolase showed homology of 92% and 95%, respectively to Fasciola hepatica enolase, suggesting that a fasciolosis vaccine might be effective against both tropical and temperate liver flukes.

Evaluation of cholesterol content and impact on antigen exposure in the outer lipid bilayer of adult schistosomes.

Developing and adult Schistosoma mansoni and S. haematobium intact worms do not bind specific antibodies, likely because of structural and biochemical modifications of the outer lipid bilayer. We have estimated the amount of cholesterol in the apical membrane of adult schistosomes via extraction with the membrane-impermeable, cholesterol-binding drug, methyl-beta-cyclodextrin (MBCD), followed by filipin staining of the worms, and evaluation of the amount of cholesterol released in the medium by a commercially available, enzymatic colorimetric assay. Positive correlations between amount of released cholesterol, MBCD concentration, and worm number and age provided evidence for the sensitivity and validity of the newly developed method. Treatment with 40 mm MBCD for 2 h at 37 degrees C led to total loss of cholesterol from the worm outer membrane, as assessed by filipin staining, and the released cholesterol values were used to estimate the amount of cholesterol per worm and per an approximate surface area unit. Additionally, total depletion of outer membrane cholesterol was associated with exposure of surface membrane antigens to specific antibody binding in 50% and 70% of S. haematobium and S. mansoni worms, respectively. These findings together suggest that cholesterol is an essential, but not the sole, factor in sequestration of surface membrane antigens in schistosomes.

Immunogenicity and vaccine potential of dipeptidic multiple antigen peptides from Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase.

Six peptides, A, B1, B, C, D and E, derived from the primary sequence of Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) were selected based on lowest homology to human glyceraldehyde 3-P dehydrogenase (G3PDH), multimerized in dipeptidic multiple antigen peptide (D-MAP) constructs and used for the immunization of BALB/c mice. Tetrabranched D-MAPs A-B, B1-C and D-E and the bis-D-MAP B-E elicited strong cell-mediated and antibody responses against immunogen, unit peptides and their cognate sequences in the native and denatured protein. D-MAP A-B induced protection against challenge infection. Immunization with D-MAP B1-C failed to affect the challenge worm parameters, probably because peptides B1 and C, previously shown to elicit immune responses associated with increase and decrease in challenge worm fertility, respectively, induced immune responses with opposing effects when combined in a D-MAP construct. A similar suggestion may explain the failure of D-MAP D-E to protect the host against challenge infection. In contrast, immunization with D-MAP B-E resulted in robust protection of the host, possibly because it contains peptides known to evoke immune responses associated with a significant decrease in challenge worm burden and fertility. The data together suggest that the specificity, not the quantity, of the induced immune responses is the determining factor for the efficacy of synthetic peptide-based vaccine for schistosomiasis.