Cercarial Challenge Research Articles

Antigenic epitope targets of rhesus macaques self-curing from Schistosoma mansoni infection.

The self-cure of rhesus macaques from a schistosome infection and their subsequent strong immunity to a cercarial challenge should provide novel insights into the way these parasites can be eliminated by immunological attack. High-density arrays comprising overlapping 15-mer peptides from target proteins printed on glass slides can be used to screen sera from host species to determine antibody reactivity at the single epitope level. Careful selection of proteins, based on compositional studies, is crucial to encompass only those exposed on or secreted from the intra-mammalian stages and is intended to focus the analysis solely on targets mediating protection. We report the results of this approach using two pools of sera from hi- and lo-responder macaques undergoing self-cure, to screen arrays comprising tegument, esophageal gland, and gastrodermis proteins. We show that, overall, the target epitopes are the same in both groups, but the intensity of response is twice as strong in the high responders. In addition, apart from Sm25, tegument proteins elicit much weaker responses than those originating in the alimentary tract, as was apparent in IFNγR KO mice. We also highlight the most reactive epitopes in key proteins. Armed with this knowledge, we intend to use multi-epitope constructs in vaccination experiments, which seek to emulate the self-cure process in experimental animals and potentially in humans.

Do schistosome vaccine trials in mice have an intrinsic flaw that generates spurious protection data?

The laboratory mouse has been widely used to test the efficacy of schistosome vaccines and a long list of candidates has emerged from this work, many of them abundant internal proteins. These antigens do not have an additive effect when co-administered, or delivered as SWAP homogenate, a quarter of which comprises multiple candidates; the observed protection has an apparent ceiling of 40–50 %. We contend that the low level of maturation of penetrating cercariae (~32 % for Schistosoma mansoni) is a major limitation of the model since 68/100 parasites fail to mature in naïve mice due to natural causes. The pulmonary capillary bed is the obstacle encountered by schistosomula en route to the portal system. The fragility of pulmonary capillaries and their susceptibility to a cytokine-induced vascular leak syndrome have been documented. During lung transit schistosomula burst into the alveolar spaces, and possess only a limited capacity to re-enter tissues. The acquired immunity elicited by the radiation-attenuated (RA) cercarial vaccine relies on a pulmonary inflammatory response, involving cytokines such as IFNγ and TNFα, to deflect additional parasites into the alveoli. A principal difference between antigen vaccine protocols and the RA vaccine is the short interval between the last antigen boost and cercarial challenge of mice (often two weeks). Thus, after antigen vaccination, challenge parasites will reach the lungs when both activated T cells and cytokine levels are maximal in the circulation. We propose that “protection” in this situation is the result of physiological effects on the pulmonary blood vessels, increasing the proportion of parasites that enter the alveoli. This hypothesis will explain why internal antigens, which are unlikely to interact with the immune response in a living schistosomulum, plus a variety of heterologous proteins, can reduce the level of maturation in a non-antigen-specific way. These proteins are “successful” precisely because they have not been selected for immunological silence. The same arguments apply to vaccine experiments with S. japonicum in the mouse model; this schistosome species seems a more robust parasite, even harder to eliminate by acquired immune responses. We propose a number of ways in which our conclusions may be tested.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1369-9) contains supplementary material, which is available to authorized users.

SjHSP70, a recombinant Schistosoma japonicum heat shock protein 70, is immunostimulatory and induces protective immunity against cercarial challenge in mice.

High levels of protective immunity can be induced in different animals immunized with radiation-attenuated (RA) Schistosoma cercariae or schistosomula. However, the schistosome-derived molecules responsible for the strong protective effect elicited by RA schistosome larvae have not been identified or characterized. The 70-kDa heat shock proteins of schistosomes are considered major immunogens, and may play an important role in stimulating high levels of innate and adaptive immune responses in an RA schistosome vaccine model. Here, we demonstrate the immunobiological functions of Schistosoma japonicum heat shock protein 70 (SjHSP70) by investigating its expression profile in RA-schistosomula-derived cells, evaluating the protection induced by recombinant SjHSP70 (rSjHSP70) against cercarial challenge, and assaying the humoral and cellular immune responses to rSjHSP70 in BALB/c and C57BL/6 mice. The expression of SjHSP70 on the surfaces of cells from RA or normal schistosomula was determined with flow cytometry. Its expression was significantly higher on early RA schistosomula cells than on the cells from normal parasites. The protection afforded both BALB/c and C57BL/6 mice vaccinated with rSjHSP70 alone, rSj22.6 (a membrane-anchoring protein of S. japonicum) alone, or a combination of rSj22.6 and rSjHSP70 without adjuvant was evaluated. rSjHSP70 alone induced the highest protective effect against S. japonicum cercarial challenge, followed by the rSj22.6 plus rSjHSP70 combination and then rSj22.6 alone, in both mouse strains. Like ISA206 adjuvant, rSjHSP70 enhanced the protective efficacy induced by rSj22.6 in the C57BL/6 mouse strain. Antigen-specific IgG1 and IgG2a responses were detected with enzyme-linked immunosorbent assays in mice immunized with rSjHSP70 alone, rSj22.6 alone, or the rSj22.6 plus rSjHSP70 combination. Immunization with rSjHSP70 or the rSj22.6 plus rSjHSP70 combination induced mixed Th1/Th2-type antibody responses in BALB/c mice and a Th2-type antibody response in C57BL/6 mice. The profiles of cytokine production by splenic lymphocytes in both strains of mice immunized with the antigens described above were detected in vitro using a Cytometric Bead Array. The profiles of the proinflammatory cytokines interferon γ, tumor necrosis factor α, interleukin 6 (IL-6), and IL-17A and the regulatory cytokine IL-10 induced by the rSj22.6 plus rSjHSP70 combination were similar to those induced by rSj22.6 emulsified with the ISA206 adjuvant control. Like the ISA206 adjuvant, rSjHSP70 protein enhanced the proinflammatory and Th2-type or regulatory cytokine production induced by the rSj22.6 antigen. These results indicate that SjHSP70 is exposed on the surfaces of cells from RA schistosomula, and that rSjHSP70 protein is a promising protective antigen with a potential adjuvant function. Thus, SjHSP70 protein might play a key role in the protective immunity elicited by the RA schistosome vaccine.

Longevity of Sm-p80-specific antibody responses following vaccination with Sm-p80 vaccine in mice and baboons and transplacental transfer of Sm-p80-specific antibodies in a baboon.

Based on data obtained using vaccine efficacy studies in mice, hamsters, and baboons, the credentials of Sm-p80 as a first tier vaccine candidate for schistosomiasis have been well established. Sm-p80-based vaccine formulation(s) have consistently exhibited potent prophylactic efficacy in reducing adult worm burden following cercarial challenge and induce killing of established adult worms in chronic infection. This vaccine is protective against both intestinal and urinary schistosomiasis. In this study, the longevity of Sm-p80-specific antibody responses was studied in mice and in baboons. Robust antibody titers were detected in mice for up to 60weeks following vaccination with Sm-p80 recombinant vaccine (Sm-p80 + GLA-SE). In the follow-up experiments to our published studies, Sm-p80-specific IgG was also detected in baboons 5-8years following the initial vaccination with an Sm-p80 DNA vaccine. In one baboon, transfer of Sm-p80-specific antibody was detected in umbilical cord blood and in the baby. These long-lasting humoral immune response data coupled with the vaccine efficacy data in rodents and nonhuman primates further strengthens the case for Sm-p80 to be moved forward through development leading to human clinical trials.

Local Immune Responses of the Chinese Water Buffalo, Bubalus bubalis, against Schistosoma japonicum Larvae: Crucial Insights for Vaccine Design

Asian schistosomiasis is a zoonotic parasitic disease infecting up to a million people and threatening tens of millions more. Control of this disease is hindered by the animal reservoirs of the parasite, in particular the water buffalo (Bubalus bubalis), which is responsible for significant levels of human transmission. A transmission-blocking vaccine administered to buffaloes is a realistic option which would aid in the control of schistosomiasis. This will however require a better understanding of the immunobiology of schistosomiasis in naturally exposed buffaloes, particularly the immune response to migrating schistosome larvae, which are the likely targets of an anti-schistosome vaccine. To address this need we investigated the immune response at the major sites of larval migration, the skin and the lungs, in previously exposed and re-challenged water buffaloes. In the skin, a strong allergic-type inflammatory response occurred, characterised by leukocyte and eosinophil infiltration including the formation of granulocytic abscesses. Additionally at the local skin site, interleukin-5 transcript levels were elevated, while interleukin-10 levels decreased. In the skin-draining lymph node (LN) a predominant type-2 profile was seen in stimulated cells, while in contrast a type-1 profile was detected in the lung draining LN, and these responses occurred consecutively, reflecting the timing of parasite migration. The intense type-2 immune response at the site of cercarial penetration is significantly different to that seen in naive and permissive animal models such as mice, and suggests a possible mechanism for immunity. Preliminary data also suggest a reduced and delayed immune response occurred in buffaloes given high cercarial challenge doses compared with moderate infections, particularly in the skin. This study offers a deeper understanding into the immunobiology of schistosomiasis in a natural host, which may aid in the future design of more effective vaccines.

Antibodies elicited by the secretions from schistosome cercariae and eggs are predominantly against glycan epitopes

The glycoproteins secreted by Schistosoma mansoni cercariae and eggs play a key role in parasite transmission to and from the mammalian host. We used secreted preparations from these two life cycle stages to characterize the reactivity of sera from baboons exposed to normal and/or attenuated cercariae, in comparison with somatic antigen preparations and defined glycan epitopes. Periodate treatment of native antigens revealed that responses to the two secreted preparations were overwhelmingly directed against glycans rather than peptides. Considerable immunological cross-reactivity between glycans in the two preparations was inferred from a comparison of sera from infected-only and vaccinated-only animals, predominantly exposed to egg and cercarial secretions, respectively. In contrast, when somatic antigen preparations derived from adult worms or eggs were used to probe sera, a stronger antipeptide response was seen that accounted for up to 66% of maximum reactivity. Probing of sera with defined glycan structures confirmed the time course of responses and the presence of cross-reactive epitopes. In spite of the intense antiglycan response elicited in mice by administration of live eggs, no protection against a cercarial challenge was observed. Our data further support the hypothesis that antiglycan responses are a smokescreen with negligible protective potential.

Protective immunity induced by phage displayed mitochondrial related peptides of Schistosoma japonicum

New antigens and strategies are necessary for vaccine development against schistosomiasis japonica. Using a pool of 43 high titred anti-SWA sera from individuals residing in an Schistosoma japonicum endemic area of China, we have cloned a S. japonicum gene by cDNA library screening. The recombinant Sj338 protein has 44–46% identity to a mitochondrial precursor receptor protein of humans and rats. Immunization of mice with the recombinant Sj338 conferred 27–32% ( p < 0.01) reduction in worm burdens following cercarial challenge. In an effort to identify protective epitopes in Sj338 and increase the level of protection, we screened a random 12-mer peptide library constructed in M13 using a polyspecific anti-Sj338 rabbit serum. After five rounds of biopanning, we identified 30 reactive clones consisting of 11 distinct peptide sequences. These clones shared limited primary sequence homology with the recombinant Sj338 protein. Anti-sera raised against these phage clones recognized recombinant Sj338 and SWAP by Western blot. In murine vaccination experiments using whole recombinant phage without adjuvant, four of these clones demonstrated worm reductions of 11.6–25.1% ( p = ns − 0.05) compared to M13 vaccinated animals. Animals vaccinated with all four of these phage demonstrated 34.2% ( p < 0.01) worm reduction compared to controls vaccinated with M13 clone. These data suggest that mimotope peptides are potential vaccine candidates for S. japonicum.

Mycobacterial codon optimization of the gene encoding the Sm14 antigen ofSchistosoma mansoniin recombinantMycobacterium bovisBacille Calmette-Guérin enhances protein expression but not protection against cercarial challenge in mice

A mycobacterial codon-optimized gene encoding the Sm14 antigen of Schistosoma mansoni was generated using oligonucleotide assembly. This synthetic gene enhanced approximately fourfold the protein expression level in recombinant Mycobacterium bovis Bacille Calmette-Guérin (rBCG) when compared to that obtained using the native gene in the same expression vector. Immunization of mice with rBCG expressing Sm14 via the synthetic gene induced specific cellular Th1-predominant immune responses, as determined by interferon-gamma production of Sm14-stimulated splenocytes, which were comparable to those recorded in animals immunized with an rBCG strain expressing the native gene. Administration of a single dose of the rBCG-Sm14 construct carrying the synthetic gene conferred protection against cercarial challenge in outbred Swiss mice, at a level equivalent to those provided by either a single dose of rBCG expressing the native gene or three doses of Escherichia coli-derived recombinant Sm14. Our data demonstrated that despite improving the level of antigen expression, the codon optimization strategy did not result in enhanced immunity or protection against cercarial S. mansoni challenge.

Investigation of recombinant Schistosoma japonicum paramyosin fragments for immunogenicity and vaccine efficacy in mice

Schistosoma japonicum paramyosin, a 97 kDa myofibrillar protein, is a recognized vaccine candidate against schistosomiasis. To improve its expression and to identify protective epitopic regions on paramyosin, the published Chinese Schistosoma japonicum paramyosin cDNA sequence was redesigned using Pichia codon usage and divided into four overlapping fragments (fragments 1, 2, 3, 4) of 747, 651, 669 and 678 bp, respectively. These gene fragments were synthesized and expressed in Pichia pastoris (fragments 2 and 3) or E. coli (fragments 1 and 4). The recombinant proteins were produced at high level and purified using a two-step process involving Ni-NTA affinity chromatography and gel filtration. BALB/c mice were immunized subcutaneously three times at 2-week-intervals with the purified proteins formulated in adjuvant Quil A. The protein fragments were highly immunogenic, inducing high, though variable, ELISA antibody titres, and each was shown to resemble native paramyosin in terms of its recognition by the anti-fragment antibodies in Western blotting. The immunized mice were subjected to cercarial challenge 2 weeks after the final injection and promising protective efficacy in terms of significant reductions in worm burdens, worm-pair numbers and liver eggs in the vaccinated mice resulted. There was no apparent correlation between the antibody titres generated and protective efficacy, as all fragments produced effective but similar levels of protection.

Vaccination of mice with a cocktail DNA vaccine induces a Th1-type immune response and partial protection against Schistosoma japonicum infection

Several defined vaccine candidate antigens of Schistosoma japonicum have shown promise in large animal vaccination experiments. However, vaccination of mice in the laboratory with either single recombinant antigens or DNA encoding forms of the individual antigens has so far failed to induce significant protection against S. japonicum cercarial challenge infection as judged by worm reduction, although specific antibodies were generated. This is in contrast to the results achieved using radiation-attenuated vaccines which are highly protective. Even in large animal vaccination experiments, the protection levels obtained with single defined antigens were far below those achieved using the attenuated vaccines. One possible interpretation is that the immune responses induced by single antigen vaccination may not be strong enough to combat the challenging infection. We, therefore, carried out mouse vaccination experiments using a cocktail DNA vaccine comprising four DNA plasmids encoding four different S. japonicum antigens, Sj62, Sj28, Sj23 and Sj14-3-3, respectively. We, also investigated whether co-injection of the mouse IL-12 encoding plasmid with the cocktail DNA vaccine was able to enhance the Th1 responses and hence the protective immunity. Three intramuscular injections of the cocktail DNA vaccine induced a significant Th1-type cellular response with high level of IFN-γ production by splenocytes upon in vitro stimulation with recombinant antigens. Importantly, significant IgG antibody responses were also induced against crude worm antigens. In two out of three experiments, significant resistance (34–37 and 44–45%, respectively) was demonstrated while another experiment did not show any protection against S. japonicum cercarial challenge infection. Co-injection of the IL-12 encoding DNA did not further enhance these responses, nor the level of resistance, compared with the cocktail DNA alone.

Isolation of native, biochemically purified triosephosphate isomerase from a Chinese strain of Schistosoma japonicum and its protective efficacy in mice

The glycolytic enzyme triosephosphate isomerase (TPI) was isolated biochemically from homogenates of a Chinese strain of Schistosoma japonicum (Sjc). Purified SjcTPI migrated on SDS polyacrylamide gels with an apparent molecular size of 28 kDa and had an enzyme activity of 177 IU/mg protein. Immunization of mice with the purified, native SjcTPI resulted in a modest reduction in adult worm burdens relative to adjuvant control mice after the cercarial challenge. No significant reduction in worm burden was noted compared with a soluble extract of adult worm antigen (SWAP). However, mice immunized with SjcTPI exhibited a marked and significant reduction in the number of eggs deposited in the liver relative to both SWAP immunized mice (47–64% reduction) and adjuvant control mice (58–60% reduction). The SjcTPI offers promise as a potential schistosomiasis japonica vaccine antigen, possibly as a `cocktail' with other Sjc recombinant antigens under investigation.

Evaluation of recombinant protein r140, a polypeptide segment of tegumental glycoprotein Sm25, as a defined antigen vaccine against Schistosoma mansoni.

To investigate the role of tegumental glycoprotein Sm25 in protective immunity against schistosomiasis, codons 43-182 of its gene (GP22) were amplified by PCR and cloned in the pET 15b bacterial expression system. Recombinant protein r140 was inducibly expressed in the presence of rifampicin and purified by Ni-affinity chromatography. In different vaccination trials, Balb/c mice and Fischer rats repeatedly immunized with r140 in combination with one of several adjuvants (alum, cholera toxin or complexed into proteosomes) produced high titre anti-r140 responses. These antibodies detected an N-glycanase sensitive. 25 kDa antigen in a detergent solubilized worm fraction using Western immunoblotting. The choice of adjuvant affected the isotype distribution of the specific anti-r140 antibodies. Despite the presence of high antibody titres and isotypes which have been shown to correlate with protective immunity, protection against subsequent cercarial challenge was not observed. In addition, no appreciable effects on worm sex ratios or liver egg yields were detected in mice. Studies involving biotin labelling of membrane proteins in live worms showed that the majority of anti-r140 reactive molecules present in adult schistosomes are biotinylated after permeabilization of the parasite surface. Several possibilities to account for the lack of protective immunity are analysed.

ICAM-1 and iNOS Expression Increased in the Skin of Mice after Vaccination with γ-Irradiated Cercariae ofSchistosoma mansoni

Host responses to migrating schistosomula ofSchistosoma mansoniwere compared in the skin of naive, multiply infected, or vaccinated (with γ-irradiated cercariae) mice during the first 72 hr after cercarial penetration. Cellular response to the migrating parasite was minimal in the skin of naive mice for up to 72 hr after infection. In sharp contrast, the multiply infected or vaccinated animals exhibited a marked inflammatory response in the skin as early as 8 hr after cutaneous penetration of the challenge cercariae. This early inflammatory response in the skin of sensitized animals was characterized by a significant increase in the number of infiltrating cells, predominantly mononuclear cells and neutrophils. Increased exudation of serum proteins was also present in the skin of sensitized animals in areas of cercarial challenge. A time course of analyses revealed that mononuclear cell numbers increased significantly in the skin of vaccinated animals as early as 60 min after a challenge infection and continued to be present at a significantly higher level up to 72 hr after challenge. Peak neutrophil responses occurred in the skin at 24 hr (in multiply infected animals) and at 48 hr (in vaccinated animals) after a challenge infection. Along with the massive cellular infiltration there was an increased tissue expression of ICAM-1 and mRNA for iNOS in the skin of sensitized animals. Further analysis showed that in sensitized animals increased ICAM-1 expression was predominantly found on endothelial cells lining dermal capillaries, especially in areas around schistosomular migration and on cells that surrounded schistosomula in the dermis. In naive animals, however, a similar infection did not induce any ICAM-1 expression or iNOS production in the skin. Thus, an ICAM-1-mediated early accumulation of mononuclear cells in the skin and local production of nitric oxide may be important for the initial cutaneous inflammatory/immune responses to migrating schistosomula ofS. mansoniin vaccinated animals. On the contrary, in naive animals a potential parasite-induced suppression of ICAM-1 may play an important role in reducing cellular reaction in the skin and consequently help the parasite evade immune responses in the skin.

Calpain is the target antigen of a Th1 clone that transfers protective immunity against Schistosoma mansoni.

A CD4+ clone (clone B), characterized as Th1 based on its selective production of IFN-gamma and IL-2, was established from C57Bl/6 mice protectively immunized against Schistosoma mansoni by intradermal vaccination with soluble worm Ags, plus bacillus Calmette Guerin. In agreement with previous results demonstrating an IFN-gamma-dependent cell-mediated protective mechanism in this vaccination model, Ag-elicited peritoneal macrophages from syngeneic recipients of this clone were activated to kill schistosome larvae (schistosomula) in vitro. Moreover, recipients of clone B displayed significant resistance against cercarial challenge. By screening a battery of lambda(gt11) clones from an adult worm cDNA library, one recombinant (25B) was identified that stimulated clone B specifically. Analysis of the 25B cDNA insert revealed a nucleotide sequence identical with that of the large subunit of schistosome calpain, a Ca2+-activated neutral proteinase. By expressing the products of PCR subcloning, we identified a 146-amino acid region of the 25B gene containing immunologic activity equivalent to the whole polypeptide. Overlapping peptides spanning this region were synthesized, and a core epitope was identified with the sequence EWKGAWCDGS. Since clone B responds to supernatants from cultured schistosomula, we postulate that the recognition of calpain released by invading larvae and resulting induction of Th1 cytokines accounts for the protection mediated by the adoptively transferred clone. Our findings thus implicate calpain as a target of protective immunity in schistosomes and provide the first example of a candidate vaccine Ag for this parasite identified on the basis of T cell reactivity.

Paramyosin: a candidate vaccine antigen against Schistosoma japonicum.

Paramyosin, a 97 kDa myofibrillar protein, is a candidate vaccine antigen for prevention of infection with the human parasite Schistosoma mansoni. To determine if paramyosin would also induce protection against Schistosoma japonicum, paramyosin was biochemically purified from S. japonicum adult worms. SDS-PAGE demonstrated a single protein with a molecular weight of 97 kDa. In four separate experiments, vaccination of mice with S. japonicum paramyosin without adjuvant induced significant resistance (62%-86%, P < 0.001) against cercarial challenge as compared to controls. These data suggest that S. japonicum paramyosin may represent a candidate vaccine for immunization against schistosomiasis japonica.

Schistosoma mansoni Hexokinase: CDNA Cloning and Immunogenicity Studies

DNA encoding a Schistosoma mansoni hexokinase (SHEX) was amplified from cDNA by the polymerase chain reaction using opposing oligonucleotide primers designed to hybridize with two short segments of hexokinase coding sequences that an well-conserved through evolution. The resulting DNA fragment was then used as a probe to identify a full-length hexokinase cDNA clone. SHEX cDNA encodes a 50-kDa protein that is approximately 46% homologous to rat hexokinase, 40% to rat glucokinase, and 34% to yeast hexokinase A. SHEX coding DNA was expressed within Escherichia coli cells and the 50-kDa recombinant product (rSHEX) was partially purified. Mice repeatedly immunized with rSHEX produced antibodies which recognize rSHEX but this offered no significant protection against subsequent cercarial challenge. On Western blots, rSHEX is weakly recognized by antisera against rat brain hexokinase but not by sera from three strains of mice experimentally infected with S. mansoni parasites or from numerous human schistosomiasis patients. Thus, unlike other reported S. mansoni glycolytic enzymes, hexokinase appears to be poorly immunogenic during schistosome infection and of limited potential as a vaccine candidate.

Attenuation ofSchistosoma mansonicercariae with a molluscicide derived fromMillettia thonningii

A chloroform extract of molluscicidal plant secondary compounds from the seeds of a West African legume Millettia thonningii was used to attenuate cercariae of Schistosoma mansoni prior to infection of NMRI female mice. Exposure of cercariae to 0.3, 0.6, 1.3, 2.5, 10 or 20 mg/l concentrations of extract for 30 min, immediately before standardized infection, was associated with a concentration-dependent decline in worm establishment at 55 days post-infection. The mean numbers of adult worms established declined from about 17 worms/mouse with control cercariae and those exposed to 0.3 mg/l Millettia to 0.1 worms/mouse after 10 mg/l pre-treatment. Mice exposed to cercariae after 20 mg/l pre-treatment had no adult worms at 55 days post-infection. The activities of cercariae 30 min after exposure to Millettia extract at concentrations up to 2.5 mg/l were similar to those of control larvae: none was immobile. Exposure to higher concentrations of Millettia progressively reduced swimming activity and increased the proportion of immobile cercariae. After pretreatment with 20 mg/l Millettia the majority of larvae were immobile. Levels of anti-parasite antibodies (estimated by ELISA assay using an adult worm-based antigen preparation) rose between 21 and 55 days p.i. with control cercariae and those pre-treated with 2.5 mg/l Millettia. After 5 mg/l pre-treatment, cercariae induced a reduced antibody rise, while mice exposed to cercariae after 10 mg/l pre-treatment showed no rise in anti-parasite antibody levels. These results are discussed in the context of protocols which could possibly use Millettia-attenuated cercariae to induce useful levels of protection in mice towards further cercarial challenge.

Isotype responses to candidate vaccine antigens in protective sera obtained from mice vaccinated with irradiated cercariae of Schistosoma mansoni.

In experimental schistosomiasis, sera of mice multiply vaccinated with radiation-attenuated cercariae of Schistosoma mansoni passively transfer resistance against cercarial challenge to naive mice. To further characterize these sera, we tested their protective capacities in two mouse strains (C57BL/6J and CBA/J) and compared the antigen-specific isotype compositions of the different protective sera by means of the enzyme-linked immunosorbent assay. By using an array of purified schistosomal antigens, the patterns of antibody titers and isotypes differed for each experimental group and antigen. In the most-protective C57BL/6J sera, high levels of immunoglobulin G1 (IgG1), IgG2a, and IgG2b bound to heat shock protein 70 and the integral membrane protein Sm23, whereas recognition of these antigens by less-protective CBA/J sera was lower. Glutathione S-transferase (GST) was recognized predominantly by IgM antibodies of all vaccinated groups, and a significant portion of this response was directed against carbohydrate epitopes. Antibodies specific for triosephosphate isomerase, paramyosin, and Sm32 (hemoglobinase) were present in less-protective sera and thus seem less relevant for passive transfer of resistance. The results of this study suggest a contribution of IgG antibodies specific for heat shock protein 70 and Sm23, and possibly a contribution of GST-specific IgM antibodies, to the protective effect of sera from C57BL/6J mice vaccinated with irradiated cercariae.

Candidate vaccine antigens identified by antibodies from mice vaccinated with 15- or 50-kilorad-irradiated cercariae of Schistosoma mansoni

In murine schistosomiasis, the highest levels of resistance to cercarial challenge are obtained by vaccination with radiation-attenuated cercariae. To identify candidate vaccine antigens relevant to the vaccine model, we examined parasite antigens recognized by antibodies from mice vaccinated with irradiated cercariae of Schistosoma mansoni. To optimize recognition of a wide spectrum of antigens, several factors that influence the level of protection in this model were varied; specifically, we examined the effect of (i) single versus multiple vaccinations with irradiated cercariae, (ii) the dose of irradiation (15 or 50 kilorads) administered to the cercariae, and (iii) the genetic background of mouse strains, high-responder (C57BL/6J) versus moderate-responder (CBA/J) mice. We found that the number of vaccinations did not alter antibody specificity but modified the relative antibody titers against particular antigens. The dose of irradiation used to attenuate the immunizing cercariae had a similar effect on antibody titers but in addition influenced antibody specificity. Only mice that had been vaccinated with moderately irradiated cercariae recognized cathepsin B (Sm31) and Sm32. Interestingly, when vaccinated mice of the two strains, C57BL/6J and CBA/J, were compared, differences in antibody responses to particular antigens were observed. Both strains recognized the integral membrane protein Sm23, glutathione S-transferase, and cathepsin B, whereas Sm32 and paramyosin were recognized only by CBA/J mice, and heat shock protein 70 was recognized exclusively by C57BL/6J mice. In this study, we conclusively identified six distinct antigens that are specifically recognized by the humoral immune response of vaccinated mice.

Detection of Schistosoma mansoni Circulating Cathodic Antigen for Evaluation of Resistance Induced by Irradiated Cercariae

The appearance of serum levels of circulating cathodic antigen (CCA) detectable by a monoclonal antibody (mAb) (5H11) antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) system was evaluated during acute Schistosoma mansoni infections in female CF1 mice exposed to either 100 or 25 cercariae. Measurable CCA levels occurred in these groups at 5 and 7 wk after infection, respectively. The kinetics of appearance of CCA were thus related to the intensity of infection. The level of resistance developed by female C57BL/6 mice upon immunization with irradiated cercariae, as expressed by both worm burden and CCA levels after cercarial challenge was evaluated. Immunization conferred 44% protection against the challenge infection, and the level of CCA detected in the sera of the control group was significantly (P less than 0.02) higher than that found in the sera of the immunized group, 6 wk after challenge. These results demonstrate that CCA detection by the 5H11 mAb antigen-capture sandwich ELISA can reflect vaccine-induced resistance against S. mansoni. Localization studies showed that 5H11 reacts with a CCA epitope in the adult worm gut and to a lesser extent with the male tegument. Adaptations of this and other antigen detection systems may prove useful in monitoring the efficacy of developmental vaccines, an ability that may be essential for the extension of such studies to humans.