Induction of cell-mediated immunity as a strategy for vaccination against Schistosoma mansoni.
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Animals↗
Biomedical subjects
E J Pearce
Publications and source records attributed to E J Pearce.
Mechanisms of immune evasion in schistosomiasis.
An unusual property of schistosomes is their capacity to survive intravascularly for many years in the face of an ongoing antiparasite immune response by the infected host. This ability to evade the immune system appears to be due to several unusual parasite adaptations occurring soon after infection is initiated. Foremost amongst these are processes which result in reduced surface antigenicity and the development of a tegument intrinsically resistant to immune damage. The unusual ability of the schistosome surface membrane to escape immune recognition and damage suggests that attempts to vaccinate against this important parasite either should be directed against very early schistosomula which have yet to achieve the refractory state, or involve stimulation of effector mechanisms, such as killing by activated macrophages, which do not require recognition or interaction with parasite surface antigens.
Animals↗
Strategies for induction of protective immunity against schistosomes.
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Animals↗
Immunochemical characterization and purification of Sm-97, a Schistosoma mansoni antigen monospecifically recognized by antibodies from mice protectively immunized with a nonliving vaccine.
Mice protected against Schistosoma mansoni infection by intradermal (i.d.) vaccination with nonliving schistosomula or soluble extracts of larval or adult schistosomes (SCHLAP and SWAP, respectively) produce antibodies that react by Western blot analysis with one antigen of Mr (X 10(-3)) 97 in SWAP prepared in the presence of protease inhibitors and two antigens of Mr (X 10(-3)) 95 and 78 in SWAP prepared in their absence. Vaccine antibodies also immunoprecipitated a single 97k molecule, with a pI of 5.5, from detergent extracts of [35S] methionine-labeled schistosomes. Three hybridomas, produced from spleen cells of i.d. immunized mice, all recognized both the 95k/78k doublet and the 97k antigen, indicating that the two lower Mr components are degradation products of the same 97k molecule. The 97k/95k/78k complex (Sm-97) was purified by affinity chromatography and found to constitute 0.5% of the total protein in SWAP. 125I-concanavalin A bound weakly to purified Sm-97, indicating that this antigen is minimally glycosylated. By indirect immunofluorescence, Sm-97 was localized to regions just below the tegumental and gut syncitia of adult worms. Mice protected by i.d. vaccination produced high titers (1:10,240) of anti-Sm-97 antibodies, whereas chronically infected mice responded at a much lower level (titer 1:640). In contrast, mice protectively immunized with irradiated cercariae and mice nonprophylactically inoculated by the i.v. route failed to produce detectable anti-Sm-97 antibodies. Competitive radioimmunoassays performed with 125I-labeled monoclonal antibodies and purified antigen defined at least two distinct epitopes on Sm-97. Antibodies from i.d. vaccinated mice recognized both monoclonal antibody-defined epitopes, whereas anti-Sm-97 antibodies in chronic infection sera recognized neither. Finally, purified Sm-97 was shown to elicit delayed-type hypersensitivity in i.d. vaccinated mice, suggesting that this molecule is also capable of evoking cell-mediated responses, a finding consistent with its proposed function as a vaccine immunogen.
Animals↗
Identification of paramyosin as schistosome antigen recognized by intradermally vaccinated mice.
Mice immunized intradermally with extracts of Schistosoma mansoni in combination with the adjuvant BCG are significantly protected against subsequent infection with living larval forms of the parasite. Remarkably, these vaccinated animals produce antibodies predominantly against a single parasite protein of molecular weight 97 kilodaltons (Sm-97). A complementary DNA that encodes about half of the Sm-97 molecule has now been cloned and sequenced. Analysis of the deduced amino acid sequence reveals a protein containing periodic repeats of hydrophobic amino acids characteristic of an alpha-helical coiled-coil structure. The deduced amino acid composition of the cloned gene and several properties of the native protein are similar to that of paramyosin, an alpha-helical protein that forms the core for myosin filaments in invertebrate muscle. Paramyosin was isolated from Schistosoma mansoni adult worms and antibodies to Sm-97 were shown to react with this molecule as well as with a known paramyosin from molluscan muscle.
Amino Acid Sequence↗
Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae.
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Animals↗
Kinetic correlation of the acquisition of resistance to immune attack in schistosomula of Schistosoma mansoni with a developmental change in membrane potential.
When Schistosoma mansoni cercariae penetrate the skin of the mammalian host they rapidly pass from fresh water to a high salt physiologic environment and transform into schistosomula. Following this transition, the parasites migrate from the skin to the lungs during which time they change from being highly susceptible to immune attack to being refractory, as measured by in vitro cytotoxicity assays. In this study, in vivo or in vitro schistosomula of different ages were examined for developmentally linked changes in membrane function which might correlate with the attainment of the resistant state. In particular, alterations in the distribution of tetraphenylphosphonium (TPP+), a synthetic lipophilic cation which shows a potential dependent partition across membranes, were followed. Three-hour-old schistosomula, which are greater than 75% susceptible to antibody-dependent complement-mediated attack or lymphokine-activated macrophage-mediated cytotoxicity, acquired TPP+ at a similar rate and steady state level to 5-day-old lung worms, which were completely resistant to both these effector mechanisms. The addition of ouabain, a Na+/K+-ATPase inhibitor, caused a 50% decrease in both the rate and steady state of TPP+ uptake by 3 h parasites but had little effect on these parameters in lung worms. Valinomycin, a K+-ionophore, completely inhibited TPP+ influx in both stages. The characteristics of TPP+ efflux from 3-h and 5-day-old parasites preloaded with the cation were found to be dissimilar. Whereas 30% of acquired TPP+ was lost from lung worms within 2 h, only 10% of acquired cation was released from 3-h schistosomula during the same period.(ABSTRACT TRUNCATED AT 250 WORDS)
Adaptation, Physiological↗
Evidence that the reduced surface antigenicity of developing Schistosoma mansoni schistosomula is due to antigen shedding rather than host molecule acquisition.
Antibody and lectin binding characteristics of Schistosoma mansoni schistosomula maturing in vivo and in vitro were quantitatively assessed and compared in order to investigate the basis of the reduced surface antigenicity of host derived larval schistosomes. Quantitative indirect immunofluorescence assays showed that schistosomula recovered from mice at 24 h and 5-10 days post infection bound low or insignificant amounts of a variety of anti-schistosome antibodies including those from chronically infected and radiation attenuated cercariae-vaccinated mice, a vaccinated rabbit and rabbits hyper-immunized with non-living larval and adult schistosome antigen preparations. In contrast, parasites maturing in vitro continued to bind highly significant levels of each of these antibody preparations until at least 10 days post transformation. To investigate the basis of the decreased surface antigenicity of parasites maturing in vivo, 6-day-cultured parasites were injected intravenously into mice and recovered from the lungs at various times thereafter and examined for their ability to bind both anti-parasite and anti-host antibodies. After 30 min in vivo, cultured schistosomula exhibited a significantly decreased capacity to bind anti-parasite antibodies and concanavalin A (Con A), and by 16 h had lost their binding sites for fucose binding protein (FBP) as well. That this reduction in antigenicity was due to shedding of surface antigens was suggested by the observation that the reduced ability of these parasites to bind anti-parasite antibodies coincided closely with the loss of 125I-labelled surface proteins. Furthermore unlike 6 day schistosomula which had developed wholly in vivo, 6-day-cultured parasites recovered after 30 min in vivo failed to bind anti-host antibodies suggesting that in these organisms parasite antigens were not masked by host molecules. These data argue that surface antigen shedding may explain the reduced surface antigenicity of schistosomula developing in vivo. While this surface modulation apparently occurs independently of host antigen uptake, it is dependent upon an as yet unidentified host factor.
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Post lung-stage schistosomula of Schistosoma mansoni exhibit transient susceptibility to macrophage-mediated cytotoxicity in vitro that may relate to late phase killing in vivo.
Studies of protective immunity against Schistosoma mansoni in immunized mice suggest that a proportion of challenge parasites may be eliminated after they have passed through the lungs of the host several days after infection; however, no potential immune effector mechanism of resistance against this stage of the parasite has yet been identified, since schistosomes have been shown to rapidly become resistant to antibody-dependent killing mechanisms. In this study, different development stages of S. mansoni were examined for their susceptibility to in vitro cytotoxicity by lymphokine-activated macrophages. As previously shown, newly transformed larvae were readily killed by lymphokine-treated peritoneal macrophages or the macrophage cell line IC-21 (80% mortality over 48 h in vitro), whereas 7 and 10 day old lung-stage parasites had become refractory to macrophage effects. However, after 2 to 2 1/2 weeks of development in vivo, juvenile parasites recovered from the liver were again susceptible to activated macrophage-mediated cytotoxicity (25-65% mortality). Ultrastructural studies of 2 1/2 week old parasites co-cultured with activated IC-21 cells revealed that damage was largely restricted to the areas beneath the parasite surface and gut syncitia; surface membrane disruption was not evident. This late stage of susceptibility was transient and by 4 to 6 weeks liver-stage worms had again become refractory to macrophage killing. The interaction of post lung-stage parasites with activated macrophages was antibody independent. Furthermore, schistosomes isolated from the portal circulation 2 1/2 weeks after infection showed no evidence of surface-bound immunoglobulin in a quantitative immunofluorescence assay, nor did antisera from chronically infected mice (CIS) or mice vaccinated with irradiated cercariae (VS) react with the surface of these parasites in vitro, making the possibility of direct antibody-dependent killing mechanisms unlikely. However, both CIS and VS did recognize excretory/secretory proteins synthesized by 2 1/2 week old liver-stage schistosomes, including a major antigen of approximate Mr (X 10(-3] 220 (220K). It is therefore possible that such antigens might participate in protective immunity, for example via immune complex formation or activation of sensitized T cells. These observations support the role of macrophages as immune effector cells in mice immunized against Schistosoma mansoni, and provide the first physiologically relevant mechanism whereby the immune system might recognize and kill post-lung stage schistosomes.
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Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice.
Two clones which contain genes encoding Schistosoma mansoni proteins recognized by immune mouse sera were chosen from cDNA lambda gt11 expression vector library by preselecting clones from the library with rabbit antisera against adult worm phosphate-buffered saline (PBS)-soluble antigens. One clone, MAC 182, codes for part of a Mr 70 000 protein; the other clone, MAC 184, codes for a Mr 27 000 protein. The insert sizes of MAC 182 and MAC 184 are 400 bp and 800 bp, respectively. Both clones express S. mansoni beta-galactosidase fusion proteins as products of the construct. Antibodies from either chronically infected mice or mice vaccinated with irradiated cercariae recognize the MAC 182 fusion protein (MAC 182fp) but not the MAC 184 fusion protein (MAC 184fp). Rabbit antibodies prepared against MAC 182fp immunoprecipitate a Mr 70 000 in vitro translation product from adult mRNA and react in Western blot with a corresponding Mr 70 000 protein present in eggs, cercariae and adult worms but absent in schistosomula. Although the MAC 184fp is not recognized directly by chronic infection or vaccinated mouse antibodies, antisera prepared against the purified fusion protein immunoprecipitate a Mr 27 000 in vitro translation product which also reacts with mouse chronic infection sera. The same Mr 27 000 protein appears to be present in eggs, cercariae, schistosomula and adults as determined by Western blots with rabbit antisera against the MAC 184fp. These results suggest that the S. mansoni polypeptide encoded by the MAC 184 gene, when expressed within a fusion protein, fails to present epitopes normally recognized during natural infection. We propose that these epitopes are conformationally determined and are destroyed when the MAC 184 protein is expressed within beta-galactosidase. This abrogation of conformational epitopes may explain the failure of antibodies from chronically infected or vaccinated mice and rabbits to effectively recognize gene products of certain lambda gt11-fusion protein clones.
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Site potential for challenge attrition in mice, rats and guinea pigs vaccinated with irradiated cercariae of Schistosoma mansoni.
The potential sites of attrition of a challenge population of schistosomes have been investigated in mice, rats and guinea pigs vaccinated with irradiated cercariae of Schistosoma mansoni, by the use of challenge regimens that permit sequential site elimination. Vaccinated mice showed significant immunity to a percutaneous cercarial challenge, but were only marginally resistant to an i.v. challenge with healthy lung stage worms. Vaccinated rats and guinea pigs differed from mice, in that they were able to mediate significant challenge attrition at post-skin sites. Healthy lung worms were subject to immune elimination by rats in the lungs, or perhaps en route to the liver, but not in the liver itself. In contrast, guinea pigs had the capacity to kill challenge lung worms injected into either the lungs or the liver. Interestingly, lung worms harvested by extended incubation were shown to be sub-optimal in terms of viability, since they were eliminated in significant numbers when injected i.v. into vaccinated mice. These data show that different hosts vaccinated in essentially the same manner differ in terms of their site potential for challenge attrition. It is emphasised however, that sites implicated by these experiments as having the capacity to mediate immune elimination are not necessarily the sites at which challenge attrition occurs under normal circumstances.
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Induction of protective immunity against Schistosoma mansoni by a non living vaccine. I. Partial characterization of antigens recognized by antibodies from mice immunized with soluble schistosome extracts.
A single intradermal injection of frozen and thawed schistosomula in conjunction with the bacterial adjuvant Mycobacterium bovis strain Bacille Calmette Guerin, Phipps substrain (BCG) induced significant levels of resistance to challenge Schistosoma mansoni infection in C57BL/6 mice. Immunization with the aqueous fraction remaining after 100,000 X G centrifugation of the larval lysate was also protective under these conditions, suggesting that some immunogenic determinants may not be membrane associated. Frozen-thawed cercariae and soluble components of adult worms also protected against challenge infection in these experiments. These observations indicate that soluble immunogens are present in both early and late developmental stages of the parasite, and therefore may be good candidate antigens for an immunochemically defined vaccine against schistosomiasis. Induction of humoral reactivity against soluble or membrane antigens was examined in mice protected against cercarial challenge by prior exposure to frozen-thawed larvae, soluble larval, or soluble adult antigens plus BCG. Animals that were immunized with frozen-thawed larvae produced low but significant levels of antibodies against larval surface antigens when examined by indirect immunofluorescence or by immunoprecipitation of surface-labeled schistosomula. Mice immunized with soluble antigens, however, showed negligible antibody reactivity against surface membrane antigens. Because mice immunized with soluble antigens were resistant to challenge infection, these results strongly suggest that anti-surface membrane reactivity is not required in the mechanism of protective immunity in this model. Sera from mice immunized with either total freeze-thaw larval lysate or soluble schistosome extracts all showed strong reactivity against soluble antigens, as detected by ELISA. Western blot analysis showed these antisera to react with a restricted number of high m.w. antigens that were present both in schistosomula and in adult worms. These antigens are therefore likely to play a major role in the development of resistance in this model as immunogens and/or as targets of protective immune response.
Adaptation, Physiological↗
Reappraisal of the guinea-pig as an experimental host for studies of schistosomiasis mansoni.
The guinea-pig has been reassessed as a potential laboratory host for Schistosoma mansoni. Twenty-six per cent of an infective cercarial population survive to maturity in this rodent and there are no gross fluctuations in worm burden subsequent to pairing of male and female parasites. Five day, 4-week and 6-week-old schistosomes grown in guinea-pigs have topographical features that are identical to those exhibited by similarly aged mouse worms, but different from those exhibited by rat worms. Schistosome eggs are never detected in the faeces of infected guinea-pigs, but they can be observed in the pulmonary, hepatic and intestinal tissues. Only 55% of the eggs that can be recovered from the intestinal tissues are viable, and some of these can be hatched to release miracidia that penetrate the intermediate snail host. Cercariae are sometimes liberated from infected snails, but in insufficient numbers to permit infection of naive guinea-pigs. The schistosome cycle cannot, therefore, be completed in this host. Collaterals are sometimes observed in the vasculature serving the rectum and kidneys of infected guinea-pigs and the fact that schistosome eggs are deposited in the lungs of these animals indicates that portal systemic anastomosis is a feature of the model. Pathology associated with cercarial invasion or egg deposition is not dissimilar to that described for other laboratory animals infected with S. mansoni, except that basophils participate in the inflammatory response observed in the skin and intestine.
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Schistosoma mansoni: in vivo and in vitro studies of immunity using the guinea-pig model.
In vivo and in vitro parameters of immunity have been assessed in guinea-pigs sensitized with 500 normal or 500 radiation-attenuated cercariae of Schistosoma mansoni. High levels of resistance to a challenge infection developed in both the chronic and irradiated vaccine model, but immunity was expressed earlier (week 4) and reached higher levels (90%) in the latter case. Vaccinated guinea-pigs have thus been shown to achieve greater resistance than the more commonly used rodent hosts. In vitro cytotoxicity assays have demonstrated that antibodies capable of participating in complement-dependent (lethal antibody) or eosinophil-mediated schistosomular killing, develop in the serum of guinea-pigs immunized with either normal or irradiated cercariae. The time course of development of the eosinophil adherence promoting antibody approximated in both models, the development of immunity in vivo, but the lethal antibody response paralleled the immune status of the animal only in the irradiated vaccine model.
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The human immune response to defined immunogens of Schistosoma mansoni: elevated antibody levels to paramyosin in stool-negative individuals from two endemic areas in Brazil.
Sera from individuals living in 2 areas endemic for Schistosoma mansoni in Minas Gerais, Brazil were assayed for the presence of antibodies against paramyosin and glutathione-S-transferase (GST), molecules previously implicated as vaccine immunogens from studies in laboratory hosts. A group was identified consisting of subjects who were stool-negative and had no record of previous infection but who were seropositive by enzyme-linked immunosorbent assay against crude adult worm antigen (SWAP). These individuals had anti-paramyosin antibody levels which were dramatically elevated with respect to those measured in infected (stool-positive) individuals living in the same endemic area. In contrast, the same 2 groups of stool-positive and stool-negative subjects could not be distinguished on the basis of their seroreactivity to either GST or SWAP. After chemotherapy, anti-paramyosin antibodies rose above pre-treatment levels and remained elevated in those individuals who became stool-negative. In contrast, anti-paramyosin antibodies decreased to pretreatment values in drug-treated individuals who failed to show complete parasitological cure. These results suggest that the immune response of humans to paramyosin may play a role in natural resistance to schistosome infection, and that an elevated antibody level against this antigen may be a useful correlate of drug-induced cure.
Adolescent↗
Interferon-gamma production by peripheral blood mononuclear cells from residents of an area endemic for Schistosoma mansoni.
During human schistosomiasis host responses to antigens of various parasite life-cycle stages may contribute to whether the severe, hepatosplenic state develops or the patient remains relatively asymptomatic throughout infection, and may play a role in resistance. This study evaluated production of interferon gamma (IFN-gamma) in vitro by schistosome antigen-stimulated peripheral blood mononuclear cells (PBMCs) from asymptomatic patients, and by PBMCs from apparently uninfected, untreated persons living in areas endemic for Schistosoma mansoni ('endemic normals'). IFN-gamma production parallels PBMC proliferation in that schistosomal egg antigens stimulate patent patients' cells poorly, but strongly stimulate PBMCs from 'endemic normals'. This is proportionally true for antigens from adult worms and cercariae. Although asymptomatic patent patients' cells produced little or no IFN-gamma in response to the 3 schistosomal antigenic extracts, their PBMCs, and PBMCs from 'endemic normals', produced expected amounts of IFN-gamma when exposed to phytohaemagglutinin. This implies that persons with patent infections have schistosome antigen-specific defects in their ability to respond to IFN-gamma production that are not exhibited by putatively resistant 'endemic normals'.
Adult↗
Detecting a history of childhood sexual experiences among women substance abusers.
This article examines a means of detecting a history of childhood sexual experience (CSE) to improve substance abuse treatment outcomes. The symptom profile of women with a history of CSE is discussed in relation to the difficulties it presents in detection of a history of CSE, especially in women who also have a past or current history of substance abuse. A symptom checklist is described, and its validity and reliability are reported as satisfactory. The use of the checklist in client assessment and treatment planning is discussed.
Adolescent↗