Tegumental proteins of Schistosoma mansoni: complex biomolecules and potent antigens

The passive transfer of monoclonal antibodies, direct vaccination and in vitro assays have all shown that antigens associated with the tegumental membranes of Schistosoma mansoni are capable of mediating protective immune responses against the parasite in animal models. Furthermore, the principal antigens are highly antigenic during natural infection in man and stimulate strong humoral and cellular responses although, at present, their role in mediating protective immune responses in man remains equivocal. This presentation will review the current state of knowledge of the structure and expression of the major antigenic tegumental proteins of the schistosome and will attempt to relate the relevance of their structural features to possible function both in terms of protective immunity and parasite's ability to survive within the definitive host. A focus will be recent advances that have been made in the identification of means of anchoring of the antigenic proteins to the tegumental membrane. In addition, the implications of the structural complexity of the tegumental proteins in terms of their possible utility in vaccination and diagnosis will be considered.

The role of egg antigens, cytokines in granuloma formation in murine schistosomiasis mansoni

The induction of granuloma formation by soluble egg antigens (SEA) of Schistosoma mansoni is accompanied by T cell-mediated lymphokine production that regulates the intensity of the response. In the present study we have examined the ability of SDS-PAGE fractioned SEA proteins to elicit granulomas and lymphokine production in infected and egg-immunized mice. At the acute stage of infection SEA fractions (<21, 25-30, 32-38, 60-66, 70-90, 93-125, and > 200 kD) that elicited pulmonary granulomas also elicited IL-2, IL-4 lymphokine production. At the chronic stage a diminished number of fractions (60-66, 70-90, 93-125, and > 200 kD) were able to elicit granulomas with an overall decrease in IL-2, IL-4 production. Granulomas were elicited by larval-egg crossreactive and egg-specific fractions at both the acute and chronic stage of the infection. Examination of lymphokine production from egg-immunized mice demonstrated that as early as 4 days IL-2 was produced by spleen cells stimulated with <21, 32-38, 40-46, 93-125, and >200 kD fractions. By 16 days, IL-2production was envoked by 8 of 9 fractions. IL-4 production at 4 days in response to all fractions was minimal while at 16 days IL-4 was elicited with the < 21, 25-30, 50-56, 93-125, and > 200 kD fractions. The present study reveals differences in the range of SEA fractions able to elicit granulomas and IL-2, IL-4 production between acute and chronic stages of infection. Additionally, this study demonstrates sequential (IL-2 followed by IL-4) lymphokine production during the primary egg antigen response.

Identification of schistosome-infected snails by detecting schistosomal antigens and DNA sequences

Cercarial shedding tests do not provide species identification of the shistosomes concerned and cannot detect prepatent schistosomal infections. We have demonstrated that both immunodetection by ELISA of schistosomal antigens in snail hemophlymph, and dot hybridization of snail extracts by DNA probe representing highly repeated sequences, proved suitable for detecting infected snails during prepatnecy as well as patency. A group-specific monoclonal antibody was found to be suitable for detecting Schistosoma mansoni infection in Biomphalaria sp., but not for positive identification of S. haematobium in Blulinus sp. Comparative evaluation of the diagnostic qualities, and technical aspects and cost of these tests, point to the superiority of the immunodetection approach for large scale detection of snails prepatently infected with S. mansoni. This approach is potentially useful for providing extended information on schistosome-snail epidemiology that may facilitate rapid evaluation of the danger of post-control reinfection, and help make decisions on the time and place of supplementary control measures. In this context the potential usefulness of the immunodetection or DNA probing approach for facilitating catalytic model representation of schistosome-snail epidemiology warrants further evaluation. Specific identification of S. haematobium in Bulinus by either of these approaches may be possible depending on the development of suitable antibodies or DNA probes.

Intratypic differentiation of polioviruses isolated from suspected cases of poliomyelitis in Brazil during the period of 1990 to 1993

This study analyzed 3129 fecal samples derived from 1626 patients with sudden onset acute flaccid paralysis clinically compatible with poliomyelitis. The samples were collected in the period ranging from January 1990 to September 1993 in all regions of Brazil. Among the 1626 cases studied, 196 had isolation of poliovirus. Nevertheless, it was observed that some factors influenced the isolation rate and the intratypic characterization of these polioviruses. No cases of acute flaccid paralysis has been found to be etiologically related with wild polioviruses.

In vivo differentiation of Trypanosoma cruzi - 1. Experimental evidence of the influence of vector species on metacyclogenesis

Vector species has not hitherto been studied as influencing metacyclogenesis of Trypanosoma cruzi, while the role of the parasite strain has been frequently stressed as of dominant importance in this process. In order to fill this gap in our knowledge, metacyclogenesis was monitored in nine triatomine species. The first part of this paper presents photographs of the main and intermediate parasite stages in each vector species studied. In the second part of the study the proportional distribution of all these forms, as seen in Giemsa stained smears is summarized, thus providing an opportunity to analyze both: the length of time between the ingestion of the blood trypomastigotes and the appearance of metacyclic forms and the rates of developmental stages leading to these latter. The most remarkable observation was that metacyclogenesis rates in vivo appear to be vector dependent, reaching 50 in Rhodnius neglectus, 37 in its congener R. prolixus and being dramatically lower in the majority of Triatoma species (5 in T. sordida, 3 in T. brasiliensis and 0 in T. pseudomaculata) at the 120th day of infection. These observations suggest that through screening of different vector species it is possible to find some that are capable of minimizing or maximizing metacyclic production.

Optimization of a mouse immunization protocol with Paracoccidioides brasiliensis antigens

The objectives of the present study were to optimize the protocol of mouse immunization with Paracoccidioides brasiliensis antigens (Rifkind's protocol) and to test the modulation effect of cyclophosphamide (Cy) on the delayed hypersensitivity response (DHR) of immunized animals. Experiments were carried out using one to four immunizing doses of either crude particulate P. brasiliensis antigen or yeast-cell antigen, followed by DHR test four or seven days after the last immunizing dose. The data demonstrated that an immunizing dose already elicited response; higher DHR indices were obtained with two or three immunizing doses; there were no differences between DHR indices of animals challenged four or seven days after the last dose. Overall the inoculation of two or three doses of the yeast-cell antigen, which is easier to prepare, and DHR test at day 4 simplify the original Rifkind's immunization protocol and shorten the duration of the experiments. The modulation effect of Cy on DHR was assayed with administration of 2.5, 20 and 100 mg/kg weight at seven day intervals starting from day 4 prior to the first immunizing dose. Only the treatment with 2.5 mg Cy increased the DHR indices. Treatment with 100 mg Cy inhibited the DHR, whereas 20 mg Cy did not affect the DHR indices. Results suggest an immunostimulating effect of low dose of Cy on the DHR of mice immunized with P. brasiliensis antigens.

Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein

Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44) identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger) than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

Genetic variability and differentiation between populations of Rhodnius prolixus and R. pallescens, vectors of Chagas' Disease in Colombia

Enzyme polymorphism in Rhodnius prolixus and R. pallescens (Hemiptera, Reduviidae), principal vectors of Chagas' disease in Colombia, was analyzed using starch gel electrophoresis. Three geographic locations were sampled in order to determine gene flow between populations and to characterize intra- and interspecific differences. Of 25 enzymes assayed 10 were successfully resolved and then used to score the genetic variation. The enzymes PEPD, GPI, PGM and ICD were useful to differentiate these species and PGD, PGM and MDH distinguished between sylvatic and domiciliary populations of R. prolixus. Both polymorphism and heterozygosity indicated greater genetic variability in sylvatic habitats (H = 0.021) compared to domiciliary habitats (H = 0.006) in both species. Gene flow between sylvatic and domiciliary populations in R. prolixus was found to be minimal. This fact and the genetic distance between them suggest a process of genetic isolation in the domiciliary population.

Trypanosoma cruzi defined antigens in the serological evaluation of an outbreak of acute Chagas disease in Brazil (Catolé do Rocha, Paraíba)

Immunoglobulin G and M humoral response to recombinant protein B13 and glycoconjugate LPPG Trypanosoma cruzi defined antigens was evaluated by ELISA in 18 patients in the acute phase of Chagas disease, who were contaminated on the same occasion. LPPG showed 100% positivity detecting both IgM and IgG antibodies, while positivity of 55-65% was observed for B13. An epimastigote alkaline extract (EPI) also showed high sensitivity for acute IgM (100%) and IgG (90%) antibodies. However LPPG had better discriminatory reactivity since with EPI two patients showed negative IgG and several other sera presented OD values for IgG and IgM antibodies very close to the cutoff. Thus, it is suggested that detection of IgM antibodies by LPPG may be used for diagnosis of the acute phase of Chagas disease. An intense decline of IgG and IgM antibodies to the three antigens was observed in response to anti-T. cruzi chemotherapy in all acute phase patients. After treatment, six (30%) individuals maintained IgG positivity to EPI, LPPG, and B13 with lower reactivity than that measured at the acute phase. For comparison, serology of a group of 22 patients in the chronic phase of Chagas disease and also submitted to chemotherapy was determined. Positive IgM antibodies to EPI, LPPG and B13 were detected in only 5-9% cases. In all chronic-phase patients IgG antibodies highly reactive to the three antigens were present and no significant decrease resulted after benznidazole administration. These observations reinforce previous reports that treatment in the acute phase may reduce or eliminate the parasite.

The serological differentiation of acute and chronic schistosomiasis japonica using IgA antibody to egg antigen

Two groups of Schistosoma japonicum infected patients (acute and chronic ) and non-infected individuals were studied using IgA antibody to egg antigen (SEA) and IgG and IgM antibodies to keyhole limpet haemocyanin (KLH). The means and standard deviation of the optical density in ELISA of acute, chronic and negative groups for IgA anti-SEA were 583±124.7, 98.2±78.8 and 82.2±39. 3, respectively. There was a statistically significance between acute patients and chronic patients (P<0.01). The means and standard deviation of IgG and IgM antibodies to KLH were 501.5±150.6, 113.0±79.1, 28.8±56.3 and 413.6±148.5, 70.2±14.8, 65.3±45.3, respectively. The detection results of IgA to SEA compared with the IgG and IgM to KLH did not demonstrate a significant difference (P>0.01). The sensitivities of IgA to SEA and IgG and IgM antibodies to KLH for the detection of acute infection were 95.24%, 90.48% and 85.71%, respectively. Therefore, this study showed that the detection of IgA to SEA is also a useful new method for the serological differentiation of acute and chronic schistosomiasis japonica in humans.

Antibodies anti Bloodstream and Circumsporozoite Antigens (Plasmodium vivax and Plasmodium malariae/P. brasilianum) in Areas of Very Low Malaria Endemicity in Brazil

During 1992-1994, 33 malaria cases were reported in two regions in Brazil where few sporadic atypical cases occur, most of them in home owners, who are weekenders, while home caretakers live there permanently. Indirect Fluorescent Antibody Test (IFAT), with Plasmodium vivax, and Enzime Linked Immunosorbent Assay (ELISA) with repeat peptides of the circumsporozoite (CS) proteins of the 3 known P. vivax variants and P. malarie/P. brasilianum, were performed on 277 sera, obtained within a 5 to 10 km range of malaria cases. Very rarely did any of these donors recall typical malaria episodes. Blood smears of all but 5 were negative. One of the 5 malaria cases included in our serology was of a home owner, 1 of a permanent resident, 3 from Superintendência de Controle de Endemias employees who went there to capture mosquitoes. In Region 1 the prevalence of IFAT positive sera was 73% and 28% among caretakers, 18% and 9.6% among home owners. In Region 2 (3 localities) no distinction was possible between caretakers and home owners, IFAT positivity being 38%, 28% and 7%. The relative percentage of positive anti-CS repeats ELISA, differed for each of the peptides among localities. Dwellings are in the vicinity of woods, where monkeys are frequently seen. The origin of these malaria cases, geographical differences and high seropositivity is discussed

Expression of Recombinant Antigens in Escherichia coli: Application on Immunochemical Studies of Schistosoma mansoni Tegumental Antigens

Sm15 and Sm13 are recognized by antibodies from mice protectively vaccinated with tegumental membranes, suggesting a potential role in protective immunity. In order to raise antibodies for immunochemical investigations, the genes for these antigens were expressed in pGEX and pMal vectors so that comparisons could be made among different expression systems and different genes. The fusion proteins corresponding to several parts of the gene for the precursor of Sm15 failed in producing antibodies recognizing the parasite counterpart. On the other hand, antibodies raised against Sm13 MBP-fusion proteins recognized the 13 kDa tegumental protein. Thus the peculiarities of the gene of interest are important and the choice of the expression system must sometimes be decided on an empirical basis

Age-related worm load and worm fecundity patterns in human populations, as indicated by schistosome circulating antigens

Recently, our group determined the relationship between serum CAA levels and fecal egg counts in two foci with very intense Schistosoma mansoni transmission: Maniema (Zaire), an area endemic for S. mansoni since several decades, and Ndombo (Senegal), where transmission has only been established since a few years. The objective was to study and compare age-related worm load and worm fecundity patterns in these two different endemic settings. Here, we will summarize the most important findings and conclusions of this study.