Normal Cercariae Research Articles

Systems Biology Analysis of the Radiation-Attenuated Schistosome Vaccine Reveals a Role for Growth Factors in Protection and Hemostasis Inhibition in Parasite Survival.

In spite of several decades of research, an effective vaccine against schistosomiasis remains elusive. The radiation-attenuated (RA) cercarial vaccine is still the best model eliciting high protection levels, although the immune mechanisms have not yet been fully characterized. In order to identify genes and pathways underlying protection we investigated patterns of gene expression in PBMC and skin draining Lymph Nodes (LN) from mice using two exposure comparisons: vaccination with 500 attenuated cercariae versus infection with 500 normal cercariae; one versus three doses. Vaccinated mice were challenged with 120 normal parasites. Integration of PBMC and LN data from the infected group revealed early up-regulation of pathways associated with Th2 skewing and polarization of IgG antibody profiles. Additionally, hemostasis pathways were downregulated in infected mice, correlating with platelet reduction, potentially a mechanism to assist parasite migration through capillary beds. Conversely, up regulation of such mechanisms after vaccination may explain parasite blockade in the lungs. In contrast, a single exposure to attenuated parasites revealed early establishment of a Th1 bias (signaling of IL-1, IFN-γ; and Leishmania infection). Genes encoding chemokines and their receptors were more prominent in vaccinated mice, indicating an enhanced capacity for inflammation, potentially augmenting the inhibition of intravascular migration. Increasing the vaccinations from one to three did not dramatically elevate protection, but there was a clear shift towards antibody-mediated effectors. However, elements of the Th1 bias were still evident. Notable features after three vaccinations were markers of cytotoxicity (including IL-6 and NK cells) together with growth factors and their receptors (FGFR/VEGF/EGF) and the apoptosis pathway. Indeed, there is evidence for the development of anergy after three vaccinations, borne out by the limited responses detected in samples after challenge. We infer that persistence of a Th1 response puts a limit on expression of antibody-mediated mechanisms. This feature may explain the failure of multiple doses to drive protection towards sterile immunity. We suggest that the secretions of lung stage parasites would make a novel cohort of antigens for testing in protection experiments.

Molecular cloning and characterization of a HSP70 gene from Schistosoma japonicum

Schistosoma japonicum is the pathogen responsible for schistosomiasis japonica, one of the major infectious diseases targeted for prevention nationally in China. Expression of heat shock proteins (HSPs) following stress plays a very important biological role in many organisms including S. japonicum. Among the HSP family, the 70-kDa HSPs are most responsible for intracellular chaperone and extracellular immunoregulatory functions. Based on the published sequences in GenBank/EMBL (AF044412.1), open reading frame belonging to HSP70 protein corresponds to a full-length cDNA containing an open reading frame of 1,947 bp encoded of 648 amino acids was identified as HSP70 from schistosome. In this study, the coding region that we named rSj648/hsp70 was amplified from S. japonicum adult worm cDNA library, and the recombinant protein was expressed in vector pET32a(+) and purified using a Ni-NTA purification system. The target protein rSj648/hsp70 was determined by matrix-assisted laser desorption/ionization mass spectrometer after thrombin digestion and dialysis. Reverse transcriptase polymerase chain reaction and Western blotting analysis confirmed that Sj648/hsp70 could be expressed in the eggs, normal cercariae, ultraviolet-attenuated cercariae (UVAC), and adult worms of S. japonicum. Real-time quantitative PCR analysis indicated that Sj648/hsp70 was expressed significantly higher in eggs than that in cercariae and adult worms, and the expression in UVAC was higher than that in normal cercariae. A thermotolerance assay showed that rSj648/hsp70 could protect Escherichia coli cells from heat damage. The detection of specific antibody levels by indirect enzyme-linked immunosorbent assay demonstrated that mice immunized with rSj648/hsp70 induced higher level of specific anti-rSj648/hsp70 IgG1 compared with those vaccinated with adjuvant alone, indicating that rSj648/hsp70 was able to elicit Th2-type bias immune response. Our results suggest that Sj648/hsp70 might be an important molecule in parasite-host interaction and display potential roles in mice immunoregulation system.

Ultraviolet-attenuated cercariae of Schistosoma japonicum fail to effectively induce a Th1 response in spite of up-regulating expression of cytotoxicity-related genes in C57BL/6 mice

Objective To better understand the reason that Schistosoma japonicum ( S. japonicum) ultraviolet (UV)-radiated cercariae could not induce high level of protection in C57BL/6 mice. Methods Microarray technology was performed to investigate the gene transcription profile in skin draining lymph nodes (sdLNs) at 1 w after exposure to attenuated cercariae (AC) or normal cercariae (NC) of S. japonicum in C57BL/6 mice. The expressions of some representative genes were further confirmed by real-time PCR. Subsequently, the expressions of Th1/Th2 cytokine genes, cytotoxicity-related genes, as well as co-stimulator genes in spleens from AC-vaccinated and NC-infected mice were analyzed by real-time PCR at w 3 and 6 post-exposure. Results The gene expressions of Th1 cytokines, including interferon-γ (IFN-γ), interleukin (IL)-12 and tumor necrosis factor-α (TNF-α) in the sdLNs were significantly lower in AC-vaccinated mice than in NC-infected mice. Furthermore, the gene expressions of Th1- and Th2-cytokines, including IFN-γ, IL-12, TNF-α, IL-4 and IL-10, in the spleens from AC-vaccinated mice showed little changes at w 3 and 6 post-vaccination. In addition, cytotoxicity-related molecules including granzyme A, granzyme B, granzyme K, perforin 1 and Fas L were up-regulated from the early stage of vaccination, and peaked at the 3 rd w after vaccination with UV-AC. Conclusion UV-AC of S. japonicum could not effectively induce a Th1 response in C57BL/6 mice, which may be an explanation for the low protection against parasite challenge, and the role played by up-regulated expression of cytotoxicity-related genes in mice needs to be further investigated.

Schistosoma japonicum: proteomics analysis of differentially expressed proteins from ultraviolet-attenuated cercariae compared to normal cercariae

Schistosomiasis is considered the most important human helminthiasis in terms of morbidity and mortality. In this study, comparative soluble proteomic analysis of normal cercariae and ultraviolet-irradiated attenuated cercariae (UVAC) from Schistosoma japonicum were carried out in view of the high efficiency of irradiation-attenuated cercariae vaccine. The results revealed that some proteins showed significant differential expression in the parasite after treatment with ultraviolet light. Total 20 protein spots were identified by mass spectrometry, corresponded to five groups according to their functions in the main that were structural and motor proteins (actin, et al.), energy metabolism associated enzymes (glyceraldehydes-3-phosphage dehydrogenase, et al.), signaling transduction pathway-associated molecules (14-3-3 protein, et al.), heat shock protein families (HSP 70 family, et al.), and other functional proteins (20S proteasome). Furthermore, our results indicated that the differential expression of the proteins by ultraviolet irradiation may be, at least partially, acquired by regulating the mRNA levels of corresponding proteins. These results may provide new clues for further exploring the mechanism of protective immunity induced by UVAC and may shed some light on the development of vaccines against schistosomiasis.

Proteome approach for identification of schistosomiasis japonica vaccine candidate antigen

Experimental vaccination with radiation-attenuated cercariae (RAC) confers possible practical levels of resistance to challenge infection by humoral and by cellular mechanism. Here, we aimed to identify possible vaccine antigens by using specific IgG antibody from RAC vaccinated miniature pig. Two milligrams of soluble egg antigen (SEA) or schistosomal worm antigen preparation (SWAP) was fractionated using two dimensional liquid chromatography (proteome PF 2D) consisted of high performance chromatofocusing (HPCF) and high resolution reversed phase chromatography (HPRP). Of the 42 HPCF fractions of SEA or SWAP, 26 (61.9%) or 15 (35.7%) showed positive dot blot reaction with RAC vaccinated serum respectively. The dot blot positive fractions were applied to the second HPRP column. One hundred and seven out of 26 × 96 of SEA fractions and 18 out of 15 × 96 SWAP fractions reacted with RAC vaccinated serum. From the positive fractions we chose 17 of SEA and 10 of SWAP that had no reactivity with normal cercariae infected (NCI) sera and had single peak of 214 nm; and automated N-terminal amino acid sequence based on in situ Edman Reaction was conducted. Four sequences were obtained and applied to the homology search in NCBI database. A total of eight candidate genes were listed up and their cDNA clones from schistosomula stage were obtained. Two of the recombinant proteins (AAW27472.1 and AXX25883.1) showed strong reactivity with the RAC vaccinated serum but marginal with NCI serum. This protocol using proteome PF 2D could be applicable in identifying immunoreactive proteins from crude extract for the development of vaccines or for diagnostics.

Altered Patterns of Gene Expression Underlying the Enhanced Immunogenicity of Radiation-Attenuated Schistosomes

BackgroundSchistosome cercariae only elicit high levels of protective immunity against a challenge infection if they are optimally attenuated by exposure to ionising radiation that truncates their migration in the lungs. However, the underlying molecular mechanisms responsible for the altered phenotype of the irradiated parasite that primes for protection have yet to be identified.Methodology/Principal FindingsWe have used a custom microarray comprising probes derived from lung-stage parasites to compare patterns of gene expression in schistosomula derived from normal and irradiated cercariae. These were transformed in vitro and cultured for four, seven, and ten days to correspond in development to the priming parasites, before RNA extraction. At these late times after the radiation insult, transcript suppression was the principal feature of the irradiated larvae. Individual gene analysis revealed that only seven were significantly down-regulated in the irradiated versus normal larvae at the three time-points; notably, four of the protein products are present in the tegument or associated with its membranes, perhaps indicating a perturbed function. Grouping of transcripts using Gene Ontology (GO) and subsequent Gene Set Enrichment Analysis (GSEA) proved more informative in teasing out subtle differences. Deficiencies in signalling pathways involving G-protein–coupled receptors suggest the parasite is less able to sense its environment. Reduction of cytoskeleton transcripts could indicate compromised structure which, coupled with a paucity of neuroreceptor transcripts, may mean the parasite is also unable to respond correctly to external stimuli.Conclusions/SignificanceThe transcriptional differences observed are concordant with the known extended transit of attenuated parasites through skin-draining lymph nodes and the lungs: prolonged priming of the immune system by the parasite, rather than over-expression of novel antigens, could thus explain the efficacy of the irradiated vaccine.

CD207+ Langerhans cells constitute a minor population of skin-derived antigen-presenting cells in the draining lymph node following exposure to Schistosoma mansoni

Infectious cercariae of Schistosoma mansoni gain entry to the mammalian host through the skin where they induce a transient inflammatory influx of mononuclear cells. Some of these cells have antigen-presenting cell function (MHCII+) and have been reported to migrate to the skin-draining lymph nodes (sdLN) where they have the potential to prime CD4+ cells of the acquired immune response. Here, in mice exposed to vaccinating radiation-attenuated schistosome larvae, which induce high levels of protective immunity to challenge infection, we describe the parasite-induced migration of Langerhans cells (LCs) from the epidermal site of immunisation to the sdLN using a specific monoclonal antibody that recognises langerin (CD207). CD207+ cells with dendritic morphology were abundant in the epidermis at all times and their migration into the dermis was detected soon after vaccination. All CD207+ LCs were MHCII+ but not all MHCII+ cells in the skin were CD207+. LCs migrated from the dermis in enhanced numbers after vaccination, as detected in dermal exudate populations recovered after in vitro culture of skin biopsies. Elevated numbers of CD207+ LCs were also detected in the sdLN from 24h to 4 days after vaccination. However, compared with other dermal-derived antigen-presenting cells that were CD207−MHCII+ or CD207−CD11c+, the relative numbers of CD207+ cells in the dermal exudate population and in the sdLN were very small. Furthermore, the migration of CD207+ cells after exposure to ‘protective’ radiation-attenuated, compared with ‘non-protective’ normal cercariae, was similar in terms of numbers and kinetics. Together, these studies suggest that CD207+ LCs are only a minor component of the antigen-presenting cell population that migrates from the epidermis and they are unlikely to be important in the priming of protective CD4+ cells in the sdLN.

Gene Transcription Profile in Mice Vaccinated with Ultraviolet-attenuated Cercariae of <italic>Schistosoma japonicum</italic> Reveals Molecules Contributing to Elevated IFN-γ Levels

Vaccination with ultraviolet-attenuated cercariae of Schistosoma japonicum induced protective immunity against challenge infection in experimental animal models. Our preliminary study on the transcription levels of IFN-gamma and IL-4 in splenic CD4+ T cells revealed that attenuated cercariae elicited predominantly a Th1 response in mice at the early stage, whereas normal cercariae stimulated primarily Th2-dependent responses. Further analysis on the gene profile of the skin-draining lymph nodes demonstrated that the levels of IFN-gamma were significantly higher in vaccinated mice than those in infected mice at day 4, 7 and 14 post-vaccination or post-infection. However, for IL-12 and IL-4, the potent inducers of Th1 and Th2 responses, respectively, as well as IL-10, there were no differences over the course of the experiment between the infected and vaccinated mice. To explore the underlying factors that may potentially contribute to elevated IFN-gamma in vaccinated mice, the mRNA profiles of the skin-draining lymph nodes at day 4 post-exposure were compared using oligonucleotide microarrays. Within the 847 probe sets with increased signal values, we focused on chemokines, cytokines and relevant receptors, which were validated by semi-quantitative RT-PCR. A comprehensive understanding of the immune mechanisms of attenuated cercariae-induced protection may contribute to developing efficient vaccination strategies against S. japonicum, especially during the early stage of infection.

TRIGGERING OF HIGH-LEVEL RESISTANCE AGAINST SCHISTOSOMA MANSONI REINFECTION BY ARTEMETHER IN THE MOUSE MODEL

Artemether, a methyl ether derivative of dihydroartemisinin, not only exhibits antimalarial properties, but also possesses strong activity against schistosomula, the immature stages of a parasitic worm that can cause schistosomiasis. To test if the effect would be similar to that of irradiation with respect to the induction of immunologic protective responses, groups of mice were infected with Schistosoma mansoni cercariae and treated with artemether at 1-3 weeks post-infection. Control mice were either infected with normal cercariae or with cercariae exposed to radiation that permitted early development but not maturation of the parasites. The mice were challenged six weeks after the initial infection, and the mean numbers of schistosomes recovered in the various groups were calculated upon dissection eight weeks post-challenge. The administration of artemether two weeks after the initial infection resulted in 58% protection, while giving the drug three weeks post-infection increased the level of protection to 81%. This level of protection is as high as that normally obtained by immunization with irradiated cercariae (84% in the present study) and is superior to the level of resistance obtained with any individual schistosome vaccine candidate antigen thus far reported.

Vaccination with irradiated cercariae of Schistosoma mansoni preferentially induced the accumulation of interferon-γ producing T cells in the skin and skin draining lymph nodes of mice

Cytokine response to schistosomula of Schistosoma mansoni was evaluated in the skin of mice during the initial 72 h following infection. These studies showed a significant increase in the levels of IL-4 and IL-10 message in the skin in areas of cercarial penetration. The IL-4 message was detectable in the skin as early as 8 h after infection and the message for IL-10 appeared from 16 h after infection. However, mRNA for IFN-γ was undetectable in the skin samples for up to 72 h after infection with normal cercariae. In sharp contrast, vaccination with irradiated cercariae induced IFN-γ and IL-2 responses in the skin within 24 h. Analysis of the cytokine profile of cells isolated from the skin during these early time points showed that T cells are probably not a source of IL-4 or IL-10 in the skin of mice infected with normal cercariae. However, in vaccinated animals, the majority of the IFN-γ is derived from skin-residing T cells. In vaccinated animals, responses in the skin were mirrored in the skin-draining lymph nodes as well. Analysis of the CD4/CD8 ratio showed a significant decrease in the skin following vaccination suggesting an increase in CD8 + cells. Interestingly however, when vaccinated animals were challenged with normal cercariae, there was a significant reduction in IFN-γ response in the skin and its draining lymph nodes. These results show that vaccination with irradiated cercariae of S. mansoni, preferentially induce the accumulation of IFN-γ producing T cells in the skin and skin-draining lymph nodes of mice.

Squirrel monkeys as a useful vaccine model for Schistosoma japonicum infection

Young adults of the squirrel monkey, Saimiri sciurea, were immunized with γ-irradiated (20 krad) cercariae of Schistosoma joponicum, and they were examined for the protection against a challenge infection with normal cercariae. In the unimmunized control group, dominant accumulation of neutrophils was observed in egg granulomas in the liver, intestine, and lungs in association with severe hemorrhage in surrounding tissues. In the immunized group, on the other hand, the granulomas were smaller in size and number, and infiltration of lymphocytes and eosinophils within the granulomas was observed as a conspicuous cellular response. Moreover, there was no hemorrhage in the surrounding tissues in immunized animals, suggesting that the vaccination with attenuated cercariae was effective in reducing pathologic damages in the involved tissues, even though there was no significant reduction in the worm recovery in these animals compared with unimmunized controls. After the challenge infection, immunized animals demonstrated earlier and higher IgG antibody responses against egg and adult worm antigens. These results indicate that the squirrel monkey provides a useful vaccine model for S. joponicum infection. As far as we know, this is the first report on S. joponicum infection in this species.

Schistosoma mansoni:Protective Immunity in IL-4-Deficient Mice

Vaccination of mice with radiation-attenuatedSchistosoma mansonilarvae (cercariae) either once or multiple times produces similar levels of resistance to subsequent infection, but by different immunological mechanisms. In singly immunized mice, protection is CD4+ cell mediated and IFN-γ dependent. Resistance in multiply immunized mice is humorally mediated (especially the IgG1 isotype), suggesting a key role of Th2-associated cytokine responses. Since IL-4 has been shown to regulate Th2 development, animals in which the IL-4 gene has been knocked out by homologous recombination (IL-4 −/−) or wild-type mice (IL-4 +/+) were immunized three times with attenuated cercariae and challenged with normal cercariae. In three separate experiments the percentage of reduction of adult worms in immunized compared to unimmunized IL-4 +/+ animals (68 to 82%) was equivalent to IL-4 −/− (52 to 66%), although protection tended to be lower in each experiment. Serum levels of adult worm (SWAP)-specific IgG2a and IgG2b were 10- and 2-fold higher in IL-4 −/− mice while IgA levels were equivalent between the two groups. Serum levels of SWAP-specific IgG1 were slightly lower in IL-4 −/− mice (P= 0.07) compared to wild-type animals and inversely correlated with worm burdens (r= −0.65,P= 0.02), suggesting that the slightly diminished IgG1 accounts for the tendency toward lower worm burdens in IL-4 −/− animals. No relationships between worm numbers and the other isotypes were observed. SWAP-induced IL-5 production by splenocytes from IL-4 −/− animals were 6-fold lower, although present, compared to IL-4 +/+ mice while Ag-induced IFN-γ production was increased by over 4-fold. These results demonstrate that IL-4 is not essential for development of protective immunity in mice multiply vaccinated with irradiated cercariae and that compensatory or alternative pathways exist to generate a Th2-associated response. The limitations of mice with targeted gene deletions in delineating the role of specific cytokines in regulating the immune response to complex infections like schistosomiasis are emphasized.

The role of T cells in vaccine immunity in the murine model of schistosomiasis mansoni

CBA/Ca mice and mice vaccinated with gamma-irradiated cercariae of Schistosoma mansoni were challenged percutaneously with normal cercariae and depleted of L3T4+ T helper cells through the administration of a specific monoclonal antibody. Three regimes were utilized to target known phases of parasite migration. The in vivo depletion of L3T4+ cells resulted in a significant reduction in immunity (up to 65%) in vaccinated/ challenged mice, provided the monoclonal antibody was targeted towards skin-resident schistosomula. When antibody was targeted towards lung phase challenge larvae, however, there was a significant reduction in worm recovery, but no correspondingly significant reduction in vaccine immunity. In contrast, the administration of monoclonal to naive mice, via all three treatment regimes, had no effect on the primary schistosome worm burden. Histopathological studies complemented these worm recovery data. Skin tissue biopsied from vaccinated/challenged mice treated with monoclonal to L3T4+ T cells rarely showed the inflammatory foci which normally characterize untreated vaccinated/challenged mice. This was true when antibody was given either before challenge, or just after challenge, and correlated with the recorded depression in vaccine immunity. Lung tissue collected from monoclonal-treated vaccinated/challenged mice (for all three treatment regimes) exhibited no changes in morphology compared to that from untreated vaccinated/challenged mice. This was not altogether surprising since in the NIMR vaccine mouse model, the lungs represent a poor site for challenge attrition and appear normal in morphology with the exception of a few, small inflammatory reactions. When the monoclonal was given to naive/infected mice, there was no change in the morphology of the pulmonary tissue, as compared to corresponding untreated cohorts. Immunohistochemical studies revealed that Thy-1+ cells dominated the subdermal inflammatory foci of vaccinated/challenged mice. Of the T cells identified, the T helper subset was the most common, with T suppressor cells being only weakly represented, and in some cases not at all. The proportion of macrophages (Mac-1+) varied between reactions.

Schistosoma mansoni: histological analysis of the synergistic interaction between vaccine immunity and praziquantel therapy in the lungs of mice

Naive CBA mice and mice vaccinated 4 weeks previously with gamma-irradiated cercariae of S. mansoni were challenged percutaneously with normal cercariae and then treated with 500 mg/kg body weight of Praziquantel (Pzq). The drug was administered intradermally on day 1 or intramuscularly on day 6, thus targeting against skin stage or lung stage challenge larvae respectively. The skin site of challenge and/or the lungs were removed at various time points to provide samples for histological examination. As reported elsewhere (Flisser, Delgado & McLaren 1989) the efficacy of Pzq was significantly enhanced in vaccinated mice and was influenced by the treatment regime. Histological analysis revealed that when Pzq was administered I/D on day 1 to vaccinated mice, the inflammatory response to challenge differed in extent but not nature from that seen in vaccinated but untreated cohorts. This correlates with worm recovery data showing no (this study), or only marginal synergy between drug treatment and immunity using this regimen of drug treatment (Flisser et al. 1989). Following the day 6 protocol of drug delivery, however, lungs from treated vaccinated mice exhibited many large inflammatory reactions containing trapped challenge larvae. In contrast, lungs from untreated vaccinated mice had only few foci which were small and rarely contained trapped larvae. These data again correlate well with worm recovery data showing that there is a highly significant synergy between vaccination and drug treatment administered at this time (Flisser et al. 1989; this study). It would seem, therefore, that Pzq exacerbates lung phase immunity in the NIMR vaccine mouse model where skin phase immunity predominates and pulmonary attrition is normally minimal. The results are discussed in the light of published data concerning the effector mechanisms thought to characterize skin and lung phase vaccine resistance in the murine model.

In situ Pulmonary Responses of T Cell and Macrophage Subpopulations to a Challenge Infection in Mice Vaccinated with Irradiated Cercariae of Schistosoma mansoni

Cellular events related to the resistance induced by radiation-attenuated cercariae of Schistosoma mansoni were determined immunocytochemically in the lung tissues of mice. Thy-1, CD4, CD8, Mac-1 MOMA-1, MOMA-2, and Ia antigens were identified on cryostat sections by the immunogold-silver staining technique with specific monoclonal antibodies. In mice vaccinated with irradiated cercariae and challenged with normal cercariae, the number of Thy-1+ and CD4+ lymphocytes was increased dramatically relative to the normal numbers both in perivascular tissues and in focal cellular aggregates in the parenchyma of the lungs. A high ratio of CD4+/CD8+ T cells was noted in the aggregates, both in perivascular tissues and in the foci. Macrophages showing positive reactions for Mac-1, MOMA-1, MOMA-2, and Ia also infiltrated the foci. In control mice that were unvaccinated and challenged, foci showing positive reactions for the lymphocyte subpopulations barely were detectable in the lungs by day 14. The numbers of Thy-1+, CD4+, and CD8+ cells and the CD4+/CD8+ ratio in controls were considerably less than those in vaccinated/challenged mice over the period of observation. In conclusion, pulmonary cellular aggregates in vaccinated and challenged mice were composed mainly of Thy-1+ and CD4+ cell populations characteristic of delayed-type hypersensitivity (DTH) reactions. Thus, Thy-1+ and CD4+ cells in the lungs of vaccinated mice may be involved in the elimination of challenge parasites through DTH reactions.

Evidence that radio-sensitive cells are central to skin-phase protective immunity in CBA/Ca mice vaccinated with radiation-attenuated cercariae ofSchistosoma mansonias well as in naive mice protected with vaccine serum

Naive CBA/Ca mice and CBA/Ca mice vaccinated 4 weeks previously with radiation-attenuated cercariae of Schistosoma mansoni were subjected to 550 rad of whole body (gamma) irradiation and then challenged 3 days later with normal cercariae. The perfusion recovery data showed that this procedure reduced the primary worm burden in naive mice by 22% and the challenge worm burden in vaccinated mice by 82%. Irradiation also ablated the peripheral blood leucocytes of both mouse groups by 90-100% at the time of challenge. Histological data revealed that such treatment caused a dramatic change in number, size and leucocyte composition of cutaneous inflammatory skin reactions that characterize challenged vaccinated mice and are known to entrap invading larvae; cutaneous eosinophils were preferentially abolished by this treatment. Polyvaccine mouse serum that conferred protection passively upon naive recipient mice, failed to protect naive/irradiated mice when administered by the same protocol. Distraction of macrophages by treatment of mice with silica did not affect the establishment of a primary worm burden and reduced the protection exhibited by vaccinated mice by only 16%. These data indicate that radio-sensitive cells are important to both innate and specific acquired resistance in this mouse model and that macrophages contribute only marginally to the expression of vaccine immunity.

Variable generation of specific acquired resistance in CBA/Ca mice chronically infected with schistosomiasis mansoni

Naive CBA/Ca mice and CBA/Ca mice infected 12 weeks previously with 20 normal cercariae of Schistosoma mansoni were challenged percutaneously with isotopically labelled parasites. Challenge worm migration was followed, in 5 separate experiments, by means of compressed organ autoradiography. In three experiments, 43.1% of challenge parasites did not arrive in the lungs of infected mice on day 6 as compared to 7.7% in naive controls, thereby indicating that pre-lung sites constitute the first barrier in resistance to reinfection. A further 15% of the challenge worm burden was lost in the lungs or en route to the liver in the immune animals, and portal perfusion revealed that 25.4% of the challenge was lost in the liver. Two other experiments revealed no comparable phases of pre-liver attrition however; instead resistance was only evident at final perfusion on days 28 or 35. These data reveal the variable generation of specific acquired immunity in mice harbouring a chronic schistosome infection and thus clarify current discrepancies in the literature. The results are discussed in relation to documented evidence for the nature of specific and non-specific immune mechanisms reported to operate at different sites in the infection model of schistosomiasis mansoni.

Schistosoma mansoni: enhanced efficacy of Praziquantel treatment in immune mice

Naive CBA/Ca mice and CBA/Ca mice vaccinated 4 weeks previously with radiation-attenuated cercariae of Schistosoma mansoni were challenged with normal cercariae and then treated with 500 mg/kg body weight of Praziquantel (Pzq). The drug was administered intramuscularly or intradermally on day 1, but intramuscularly only on day 6. The results show clearly that in naive mice skin-stage larvae were susceptible to Pzq provided the drug was given intradermally on day 1. Lung worms were susceptible to Pzq given intramuscularly on day 6. Drug efficacy in naive mice is thus dependent upon treatment being given at the correct time and via the optimal route. The efficiency of Pzq treatment was enhanced in vaccinated mice, but was again affected by the treatment regime. Analysis of the data revealed a highly significant synergistic effect between drug treatment and vaccination when Pzq was given intramuscularly on day 6. Synergy was detectable but only marginally significant when the drug was administered intradermally on day 1, and could not be demonstrated when Pzq was given via the intramuscular route on day 1. These findings are discussed in the light of known sites and mechanisms of vaccine resistance in mice, as well as in relation to the mode of action of Pzq against schistosome parasites.

Schistosoma mansoni: evidence that eosinophils and/or macrophages contribute to skin-phase challenge attrition in vaccinated CBA/Ca mice.

Naive CBA mice and mice vaccinated 4 weeks previously with gamma-irradiated cercariae were challenged percutaneously with normal cercariae and the skin site of challenge removed at various times to provide samples for histological examination. Neutrophil abscesses surrounding schistosomula as well as cast cercarial tails were identified at the skin surface in both naive/challenged and vaccinated/challenged mice. The dermis in both groups of animals became infiltrated with mononuclear cells and granulocytes. Neutrophils were the predominant granulocyte in naive/challenged skin but eosinophils replaced neutrophils by 48 h post-challenge in skin from vaccinated mice. Schistosomula with adherent mononuclear cells and granulocytes were identified preferentially in the dermis of vaccinated mice; such larvae frequently exhibited subtegumental vacuolation. A further major characteristic of vaccinated/challenged skin was the formation, from 18 h, of extensive subdermal inflammatory foci, consisting of up to 50% mononuclear cells and 50% eosinophils. These foci invariably surrounded one or more larval parasites which were seen by both optical and scanning electron microscopy to have a morphology typical of lung stage rather than skin stage schistosomula. Dead, infiltrated parasites were also recognized within the subdermal foci. Our data support the view that vaccine immunity in this mouse model is mediated primarily in the skin and indicate that challenge attrition involves immobilization and killing of larval parasites by effector cells within subdermal inflammatory foci.

Analysis and comparison of immune reactivity in guinea-pigs immunized with equivalent numbers of normal or radiation-attenuated cercariae ofSchistosoma mansoni

Guinea-pigs immunized with equivalent numbers of normal or radiation-attenuated cercariae of Schistosoma mansoni are known to develop close to complete resistance to reinfection at weeks 12 and 4.5 respectively. We here analyse and compare the immune responses induced by the two populations of cercariae. Both radiation-attenuated and normal parasites of S. mansoni elicited an extensive germinal centre response in guinea-pigs by week 4.5 post-immunization. The anti-parasite antibody titre and cytotoxic activity of serum from 4.5-week-vaccinated, or 4.5-week-infected guinea-pigs were approximately equal, but sera from 12-week-infected individuals had high titres of anti-parasite antibody, which promoted significant larvicidal activity in vitro. In all cases, larvicidal activity was mediated by the IgG2 fraction of the immune serum. Lymphocyte transformation tests conducted on splenic lymphocytes from 4.5-week vaccinated guinea-pigs revealed maximal stimulation against cercarial, 2-week and 3-week worm antigens, whereas spleen cells from 4.5-week-infected guinea-pigs were maximally stimulated by cercarial and 6-week worm antigens. The splenic lymphocyte responses of 12-week infected animals were dramatic against antigens prepared from all life-stages of the parasite.