Structural and molecular specificity of antibody responses in mice immune to third stage larvae of Onchocerca volvulus

Immunization of mice with irradiated Onchocerca volvulus infective stage larvae (L3) has been demonstrated to confer protection against challenge infections with these larvae. Additionally, cytokine level measurements and cytokine depletion studies have shown that both IL-4 and IL-5 are important in generating a protective immune response against O. volvulus challenge infections, thus suggesting a dependency of protective immunity on IgG1, IgE and/or eosinophils. In the present study, we examined the humoral responses of immunized mice to O. volvulus L3 antigens. ELISA measurements of total serum antibody levels indicated that IgE was the only antibody isotype elevated in mice immunized with O. volvulus L3. IgM from immunized mice was the only isotype that recognized surface antigens on intact O. volvulus L3. IgG1, IgG3, IgE and IgA recognized internal parasite antigens on O. volvulus L3 frozen sections. Western blot analysis of L3 proteins showed that in serum from mice immunized with O. volvulus L3 IgG1, IgG2a/2b, IgA, and IgE, as well as IgM, recognized unique L3 proteins. Antibodies in serum from L3 immunized mice were able to detect O. volvulus adult antigens in a pattern similar to the recognition found in O. volvulus L3. Some L3 antigens were shared by adults, while other antigens were L3 specific. The ELISA, immunohistochemistry and Western blot findings thus demonstrate a complex pattern of antigen recognition of parasite antigens by antibodies found in mice immune to the L3 of O. volvulus     

Stage-specific surface antigens of the cattle lungworm Dictyocaulus viviparus

Immunofluorescence on live Dictyocaulus viviparus parasites revealed a significant antibody response by vaccinated and patently infected bovine hosts to the sheath of infective larvae (L3), a structure which is generally thought to be shed from the parasite surface prior to invasion of host tissue. In contrast, surface-exposed antigens of the adult, egg and pulmonary L1 stages were recognized only by serum antibody from calves exposed to a patent lungworm infection. Radioiodination of sheathed L3 identified a restricted set of components while a more complex pattern of labelled material was observed with adult parasites. Many more components of adult worms were labelled by the Bolton-Hunter than by the lodogen reagent, probably reflecting the more penetrative labelling propensities of the former. Stage-specificity of surface-associated antigens of adult parasites was demonstrated by their immunoprecipitation by antibody from patently-infected, but not from vaccinated, calves. There was no in vitro release of the major iodinatable surface-associated antigens of adult parasites nor any binding of antibody raised against adult excretory-secretory (ES) products to the surface of living adult worms, suggesting that surface components do not contribute to adult ES products in this species. Antibody responses to the surface of adults. L1 and eggs were specific to patently-infected animals and may provide a useful indicator of exposure to patent infection.     

Characterization of excretory-secretory products of adult Dictyocaulus viviparus and the antibody response to them in infection and vaccination

In vitro released products of the adult stage of the bovine lungworm, Dictyocaulus viviparus, were characterized according to their SDS-PAGE profile, glycosylation pattern, in vitro synthesis and antigenicity in the context of infection and vaccination with irradiated larvae. Biosynthetic labelling experiments with ³⁵S-methionine indicated active synthesis of ES throughout this time. There was, however, little incorporation of ³H-glucosamine into ES products, and lectin affinity chromatography and glycopeptidase F digestion identified only one glycosylated component. Immunoprecipitation of ¹²⁵l-labelled ES products with sera from calves patently infected with D. viviparus demonstrated that all of these, with the exception of two components, are antigenic to the bovine host. One of those not immunoprecipitated was shown to be host serum albumin carried over into culture. A limited degree of cross-reactivity between nematode species was observed, with a D. viviparus female-specific antigen of 290 kDa being recognized by serum antibody from calves infected with the gastrointestinal nematodes Cooperia oncophora and Ostertagia ostertagi. Calves vaccinated with irradiated larvae of D. viviparus, despite not being exposed to the adult stage of the parasite, also showed some recognition of adult ES products. This might suggest that vaccination with irradiated larvae operates against both pre-pulmonary and pulmonary stages of the infection.     

Characterization of host responder types after a single Cooperia oncophora infection: kinetics of the systemic immune response

After primary infection with 100,000 third stage larvae of the intestinal nematode Cooperia oncophora in 3-month-old calves, a high variability in egg output and worm counts is observed. Based on this variability, infected animals can be divided in different responder types. The three major phenotypes can be classified as high, intermediate and low responder animals. We investigated whether calves classified into different responder types show different immune responses during infection. Peripheral blood eosinophil counts and flow cytometric analysis of different lymphocyte subsets of the blood did not reveal major differences between infected and control animals, nor between responder types. However, the levels of Cooperia-specific immunoglobulin (Ig)G1 and IgA during primary infection were significantly higher in intermediate responders than in low responders. In the intermediate responders, isotype specific responses were negatively correlated with parasitological parameters expressing worm expulsion and influence on worm fecundity. Total serum IgE levels were elevated in most of the infected animals. A quantitative positive relationship between worm counts and total serum IgE levels was observed. Based on the observed correlations, we propose a role for the humoral response against the maintenance of the infection in the gut.     

Adult worm‐specific IgE/IgG4 balance is associated with low infection levels of Schistosoma mansoni in an endemic area

Field studies have suggested an immune‐mediated mechanism associated with resistance to Schistosoma mansoni infection. Overall, levels of specific IgE have been correlated with resistance to infection, whereas levels of IgG4 have been associated with susceptibility. This study aimed to evaluate serum levels of soluble adult worm antigen preparation (SWAP)‐specific IgE and IgG4 in relation to current infection in a large casuistic of individuals living in an endemic area of schistosomiasis in Bahia, Brazil. The prevalence of S. mansoni infection was 37·7% and the mean parasite burden was 55·4 (0–2100) epg/faeces. There was no significant difference in the levels of SWAP‐specific IgE in individuals with different parasite burden, whereas high producers of parasite‐specific IgG4 presented higher parasite burden when compared to low IgG4 producers. Additionally, S. mansoni parasite load was positively correlated with the levels of specific IgG4 or total IgE. No significant correlation was observed between parasite burden and SWAP‐specific IgE. Nevertheless, SWAP‐specific IgE/IgG4 ratio was higher in uninfected or lightly infected individuals (1–99 epg/faeces) than in heavily infected ones (≥400 epg/feces). These findings highlight the important role of IgE/IgG4 ratio in the resistance to infection, which could be useful for further studies in schistosomiasis vaccine candidates.     

Antibody responses in schistosomiasis haematobium in Somalia. Relation to age and infection intensity

Antibody responses in schistosomiasis haematobium were studied in relation to age and infection intensity in Somalia. The area is highly endemic for Schistosoma haematobium but free of S. mansoni. Antibodies of the IgG class against particulate antigens of S. mansoni adult worms were investigated by immunofluorescence (gut and somatic associated antigens) and against soluble egg and adult worm antigens by ELISA. Total IgE levels were examined by Pharmacia IgE RIA, and specific IgE against soluble adult worm antigen by enzyme immunoassay. The IgG antibody response showed a characteristic pattern with highest reactivity against both gut associated and soluble egg antigens in the age group 10-14 years, when both prevalence and intensity of the infection were highest. Reactivity against somatic associated antigen was also high in this age group, but it increased slightly and remained at high level in the older ages. It is thought that such antigen is exposed mainly after the death of the parasite and that the antigenic stimulation may remain throughout most of the life of infected individuals. On the other hand, the IgG antibody reactivity against soluble adult worm antigen was low during childhood, but it increased significantly with age. It is suggested that repeated booster effects are needed for more potent response against these antigenic components. The finding of high levels of total IgE already in the youngest age groups, together with low specific IgE response, indicates that mainly other antigens are involved in the IgE production. The specific IgE response against soluble adult worm antigen was low but increased significantly with age.     

Protection of calves against Haemonchus placei and Haemonchus contortus after immunization with gut membrane proteins from H. contortus

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in four groups of nine worm-free calves challenged with either 8000 H. contortus or Haemonchus placei infective larvae. Vaccinates received 50 μg of the antigen and 1 mg QuilA adjuvant three times 21 days apart, while the controls got adjuvant alone. The calves were challenged 7 days after the last immunization and killed for worm counts 43 days later. Immunization resulted in high titre antibodies against the vaccine antigens and significant reduction in egg output and worm numbers of both challenge species, compared with control calves. It was concluded that vaccination of calves with native parasite gut membrane glycoproteins obtained from H. contortus conferred protection against both H. placei and H. contortus.     

Detection of circulating parasite antigens in canine dirofilariasis by counterimmunoelectrophoresis

Circulating Dirofilaria immitis antigen was detected in sera from 24 of 24 infected dogs by counterimmunoelectrophoresis (CIE). Parasite antigen was not detected in sera from uninfected dogs or dogs with Dipetalonema reconditum infection. In experimentally infected dogs, the antigen was first detectable 6.5-8.5 months after infection. Preliminary evidence suggests that the antigen is present in male and female adult worms but not in microfilariae. Sera from dogs with microfilaremic and amicrofilaremic infections contained statistically equivalent amounts of D. immitis antigen. However, a significant correlation was observed between serum parasite antigen content and the number of adult worms present in individual dogs at necropsy. Previous studies from several laboratories have shown that microfilarial counts and serum antibody titers are not related to adult worm counts in canine dirofilariasis or other filarial infections. Thus, CIE detection of D. immitis antigenemia represents a significant improvement over previously available diagnostic techniques because it is more sensitive than microfilarial tests, more specific than antibody tests, and the only test that has been related to infection intensity.     

The detection of autoantibodies to IgE in plasma of individuals infected with hookworm (Necator americanus) and the demonstration of a predominant IgG1 anti-lgE autoantibody response

In this study we have demonstrated significantly elevated levels of circulating IgG autoanti-IgE antibody in hookworm infected individuals from Kebasob village on Karkar Island, Papua New Guinea. Although anti-IgE activity was demonstrable in IgG1, IgG3 and IgG4, IgG1 was by far the most important subclass of IgG anti-IgE in terms of frequency of detection (34/39; 87.2%) and magnitude of increase (P = 0.0000); with IgG3 (16/39; 41.0%) and IgG4 (15/39; 38.5%) antibodies being considerably less prevalent. Plasma levels of IgG1 anti-IgE (P = 00019) and IgG3 anti-IgE (P = 0.0034) showed significant correlations with total IgE concentrations, but not with IgE specific to excretory-secretory worm products; thus suggesting that anti-IgE synthesis is more related to polyclonal hyper IgE production than to antigen-specific IgE stimulation. No correlation was seen between IgG subclass anti-IgE levels and faecal egg counts or worm burden. Given that our data failed to show a negative or a positive correlation between anti-IgE and the degree of infection with hookworm, it is tempting to speculate that the main role of autoanti-IgE is to provide the host with protection against immune complex- and IgE-mediated hypersensitivity reactions to parasitic antigens.     

The detection and measurement of coproantibodies to Nippostrongylus brasiliensis in rats following a primary infection

Summary Investigations were initiated to study the possible detection and measurement of coproantibodies in animals infected with a gastrointestinal nematode parasite. Faecal extracts, extracts of small intestinal mucosa and sera of rats infected with intestinal nematode Nippostrongylus brasiliensis were examined for total IgA, IgM and IgG levels and haemagglutinating and precipitating antibodies specific to parasite antigens over a 30-day-period following infection. It was found that in both faecal and mucosal extracts immunoglobulin concentrations increased after a primary infection. In faecal extracts there was a seven-fold increase of IgA, a three to six-fold increase of IgG and about a fifty-fold increase of IgM. Haemagglutinins in faecal extracts detected by adult worm excretory-secretory (ES) products and adult worm and infective larvae somatic extracts were observed from 3 days after infection (DAI). Haemagglutinins detected by ES products reached their highest titres on 11–12 DAI while those reacting with adult worm somatic extracts showed the highest level between IS and 19 DAI. A similar pattern of response was found in the antibody levels of the intestinal mucosa. Haemagglutinins detected in faeces during the first 12 DAI reacted with the same antigens as antibodies present in the sera at that time but coproantibodies from 18, 24 and 30 DAI were different from those circulating in sera at that stage of the infection. The results suggest that measurement of coproantibody levels may provide a convenient and useful index of local immune responses to gastrointestinal helminths.     

Detection, quantitation, and specificity of antiparasite IgE antibodies in human schistosomiasis mansoni

Sera from 15 patients with acute or chronic Schistosoma mansoni infection were evaluated for IgE antibodies directed against soluble cercarial, adult worm, and egg antigens. Both the antigen-induced release of histamine from passively sensitized human basophils and specific radioimmunoassays were used to detect these IgE antibodies, and determination of serum IgE levels before and after specific immunosorption permitted their quantification. While chronically infected patients made IgE antibodies to all three stages of the parasite, only egg antigens induced an appreciable IgE antibody response in acutely infected individuals. Despite the fact that patients with chronic infection had significantly greater levels of total serum IgE than patients with acute disease, the percentage of this IgE that was parasite specific was similar for both groups, ranging between 4% and 28%. An ancillary observation was the fact that soluble egg antigen can trigger basophil histamine release through IgE-dependent reactions and through "nonimmunologic" mechanisms that require further characterization.     

Temporal changes in the humoral immune response of cattle during experimental infections with Onchocerca lienalis

In order to gain insights into the immune response in onchocerciasis during early infection, laboratory-reared calves were infected with 1000 Onchocerca lienalis infective larvae and examined serologically over a period of 508 days. Levels of serum antibodies measured by ELISA against adult worm extract revealed a multiphasic response, characterized by a broadly similar profile of peaks in individual animals arising at 15–30, 79 and >266 days after infection. Timings of these changes in responsiveness closely mirrored parasite development, coinciding with larval moults and with the onset of a patent infection. The levels of individual antibody isotypes directed against parasite antigens was strongly skewed. The dominant response was of IgG1, although limited reactivities were also found for IgG2 and IgM: No parasite-specific IgA antibodies were detected. Immuno-blots of adult worms extracts revealed a pattern of antigen recognition over time that matched the results obtained by ELISA. Again, the IgGl response was strongest, although certain lgG2 and IgM specificities were well represented. In general, there was a steady increase in the number of individual antigens recognized as the infection progressed, with a striking expansion of antibody specificities from day 79 following the fourth larval moult. Antibodies to a 16kDa component were a prominent feature of the response following development of a patent infection. These data reveal the strong influence of parasite biology on the development of the immune response in onchocerciasis.     

Humoral immune response of children with chronic schistosomiasis. Isotype recognition of adult worm antigens

In areas of low transmission of schistosomiasis, the evaluation of the success of control depends on reliable diagnostic tests. Under such conditions, some of the serological tests better estimate the real prevalence of this parasitosis than the classical stool examinations. On the search of highly sensitive and specific antigenic fractions for use in serological tests, an immunoblot technique with a luminescent substrate was used in order to evaluate, under dissociating and reducing conditions, the Schistosoma mansoni adult worm antigen (A WA). The sera of 30 infected Venezuelan children were assayed for specific recognition of A WA by IgG, IgM, IgE, IgA, and the four IgG subclasses. Eight highly specific polypeptide molecules from the parasite of 118,114,105, 74, 71,45,36, and 30 kDa were recognizedby total IgG. Additionally, IgGl and IgG2 recognized a molecule of 100 kDa and IgM one of 77 kDa. The present data suggests that certain molecules from the adult worm, specially the 36 kDa, might be relevant in the specific immunodiagnosis of this parasitic disease. The fact that the children antibodies were able to recognize the primary structure of these antigens, will allow us to chemically synthesize the relevant linear epitopes.,     

Juvenile rhesus monkeys have lower type 2 cytokine responses than adults after primary infection with Schistosoma mansoni

Adults and children have differences in their susceptibility to schistosomiasis. The relative influences of age-dependent innate resistance and acquired immunity in the differences between susceptibility to schistosomiasis are difficult to assess in humans. Therefore, we exposed juvenile and adult female rhesus monkeys to primary infection with Schistosoma mansoni. In contrast to the adult animals, the juvenile rhesus monkeys had low levels of interleukin (IL)-4 and IL-5 production by peripheral blood mononuclear cells after schistosome infection, as well as lower levels of parasite-antigen-specific antibody (IgG, IgM, and IgA) responses, and produced limited antigen-specific or total IgE. Juvenile animals had statistically nonsignificant increased worm burdens and tissue or fecal egg counts, compared with that of adults, whereas circulating schistosome antigens were significantly higher in infected juvenile monkeys. These results suggest that juvenile rhesus monkeys have reduced type 2 cytokine responses after primary schistosome infections and perhaps are more susceptible to parasite infection.     

Parasite-specific IgE and IgG levels in the serum and pleural effusion of paragonimiasis westermani patients.

In seven patients with paragonimiasis westermani, parasite-specific IgE and IgG levels in sera and pleural effusion were determined by an enzyme-linked immunosorbent assay (ELISA). The sensitivity to adult excretory-secretory (E-S) antigen was compared with the sensitivity to whole worm extract antigen, and the former was more sensitive in both an IgE-ELISA and IgG-ELISA. Both parasite-specific IgE and IgG could be detected by ELISA at levels much higher than those in control subjects using E-S antigen. When specific IgE and IgG levels in sera and pleural effusion of individual patients were compared, the latter had higher values. The difference between levels of specific IgE in pleural effusion and serum did not correlate with that of specific IgG. These results indicate that specific IgE and IgG antibodies form locally, i.e., in the lung, and that pleural effusions from patients with paragonimiasis are more suitable than serum for immunodiagnosis.     

Immune response in mice immunized with acidic antigenic fractions from Trypanosoma cruzi cytosol.

The humoral and cellular immune responses as well as the resistance to infection with bloodstream forms of T. cruzi were studied in mice immunized with acidic antigenic fractions from parasite cytosol, F III and F IV, plus Bordetella pertussis as adjuvant. The immunization with F III induced positive ITH and DTH responses to homologous antigens. In mice immunized with F IV, the ITH was negative and four out of six animals presented positive DTH reactions. In both groups of mice the analysis of IgG against T. cruzi showed that the major isotype elicited was IgG1. Specific IgE was also detected in sera from F III immunized mice, thus confirming the presence of homocytotropic antibodies. The parasitemias reached by F III and F IV immunized mice after challenge were lower than those of the controls showing in this way a partial protection against the acute infection. The histological studies of heart and skeletal muscle performed two months after the infection revealed variable mononuclear infiltration in all infected mice despite immunization.     

IgE antibody during infection with the ovine abomasal nematode, Teladorsagia circumcincta: primary and secondary responses in serum and gastric lymph of sheep

A monoclonal antibody to ovine IgE was employed in an ELISA to investigate the IgE antibody responses in serum and gastric lymph to a primary infection of Teladorsagia circumcincta, and following challenge in previously infected sheep. During a primary response, IgE antibody to antigens derived from the infective third stage (L3) and adult (L5) worms were negligable, with low levels of IgE antibody detected in serum and lymph. In contrast, there was a pronounced IgE antibody response in 2/4 sheep to L3 antigens during 2–8 days after challenge of previously infected animals but low levels of IgE antibody to L5 antigens. This response was confirmed in a second but similar experiment, where relatively high levels of IgE antibody was detected to antigens from L3. Antibody levels were higher in lymph than in serum from the same animals, and Western blots of L3 antigen following SDS-PAGE under reducing conditions revealed several bands of MW26–96KD which reacted with the IgE antibody from gastric lymph. Immunohistochemical staining indicated that these IgE antibodies may be reacting with allergens associated with the surface cuticle of the worms.     

Heightened anti-filarial immune responsiveness in a Haitian pediatric population

The immunological consequences of exposure to filarial infection were examined by cross-sectional serological studies. Serum samples from 121 pediatric patients (18 months-15 years of age) were analyzed in parallel with a panel of sera from adults residing in the same area of Haiti. Parasite antigen specific IgG and IgE levels were determined by ELISA. IgG levels in children were significantly elevated in humoral immunoreactivity to Brugia pahangi extracts compared to adults. In addition, anti-filarial IgG levels in amicrofilaremic children were significantly greater than in microfilaremic children. In contrast, IgG levels in adults were equivalent independent of microfilaremic status. Anti-filarial IgE levels in sera from both children and adults were low in comparison to that of a subject with tropical pulmonary eosinophilia and were unrelated to clinical status. No correlations were found between humoral responses and age, sex, or degree of parasitemia. Sera from amicrofilaremic children and, to a lesser extent, adults recognize more antigens, particularly those of high molecular weight (greater than 55 kDa), than sera from microfilaremic patients.     

Differential recognition patterns of Schistosoma haematobium adult worm antigens by the human antibodies IgA, IgE, IgG1 and IgG4

Schistosoma haematobium antigen recognition profiles of the human isotypes IgA, IgE, IgG1 and IgG4 were compared by image analysis of western blots . Adult worm antigens separated by two‐dimensional gel electrophoresis were probed with pooled sera from Zimbabweans resident in a S. haematobium endemic area, followed by the identification of individual antigenic parasite proteins using mass spectrometry. Overall, IgG1 reacted with the largest number of antigens, followed by IgE and IgA which detected the same number, while IgG4 detected the fewest antigens. IgE recognized all antigens reactive with IgG4 as well as an additional four antigens, an isoform of 28‐kDa GST, phosphoglycerate kinase, actin 1 and calreticulin. IgG1 additionally recognized fatty acid–binding protein, triose‐phosphate isomerase and heat shock protein 70, which were not recognized by IgA. Recognition patterns varied between some isoforms, e.g. the two fructose 1‐6‐bis‐phosphate aldolase isoforms were differentially recognized by IgA and IgG1. Although the majority of S. haematobium adult worm antigens are recognized by all of the four isotypes, there are clear restrictions in antibody recognition for some antigens. This may partly explain differences observed in isotype dynamics at a population level. Differential recognition patterns for some isoforms indicated in the study have potential importance for vaccine development.     

Immunoblot analysis of Schistosoma japonicum egg antigens with sera from patients with acute and chronic schistosomiasis japonica

Humoral immune responses of IgG, IgM, IgA, IgE and IgG subclass antibodies to Schistosoma japonicum egg antigens were determined by immunoblotting with serum samples from individuals in China with acute (n=24) or chronic (n=35) schistosomiasis. In general, IgM, IgA, and IgE in sera from acute patients exhibited strong binding to antigens but binding was much weaker in chronic cases. Reaction of IgG4 of chronic cases was stronger than that of IgG4 of acute cases. The recognition profile of each antibody isotype in sera was analyzed for 11 major antigen molecules (antigens with apparent molecular weights of 82, 76, 61, 57, 53, 46, 40, 32, 27, 10 and less than 6.5 kDa). Except for the 10 kDa molecule, they were well-recognized by IgA and IgE in sera of acute cases. In other combinations of antibody class and clinical phase, recognition patterns against these molecules differed among individuals. Notably, the 10 kDa molecule was specifically recognized by total IgG and IgG4 in sera from most of the chronic patients, but in sera from only one acute case. This result suggests that the 10 kDa molecule is one of the major target antigens of IgG4 and may be useful as a marker antigen to characterize the clinical phases of S. japonicum infection.