Proteins secreted by the parasitic nematode Nippostrongylus brasiliensis act as adjuvants for Th2 responses.

Infections with parasitic helminths such as Nippostronglyus brasiliensis induce dominant type 2 responses from antigen-specific T helper cells. The potency of the Th2 bias can also drive Th2 responses to bystander antigens introduced at the same time as infection. We now report that the Th2-promoting effect of infection can be reproduced with soluble N. brasiliensis excretory-secretory proteins (NES) released by adult parasites in vitro. Immunization of BALB/c mice with NES results in the production of IL-4 with elevated total serum IgE and specific IgG1 antibodies. NES is also able to stimulate IL-4 and polyclonal IgE production in other mouse strains (C57BL/6, B10.D2, CBA). These features are seen whether NES is administered without adjuvant as soluble protein in phosphate-buffered saline or with complete Freund's adjuvant which normally favors Th1 responses. Thus, NES possesses intrinsic adjuvanticity. Moreover, co-administration of hen egg lysozyme (HEL) with NES in the absence of other adjuvants results in generation of HEL-specific lymphocyte proliferation, IL-4 release and IgG1 antibody responses, documenting that NES can act as an adjuvant for third-party antigens. Proteinase K digestion or heat treatment of NES before immunization abolished the IL-4-stimulating activity, indicating that the factors acting to promote Th2 induction are proteins secreted by the adult parasite.

Nippostrongylus brasiliensis: cytokine responses and nematode expulsion in normal and IL-4-deficient mice.

Infection with the nematode, Nippostrongylus brasiliensis, results in a Th2-dominated immune response. We describe the dynamics of this response in both local and systemic environments. Th2-type responses were evident first in the mesenteric lymph node, with parasite antigen-specific proliferation and IL-4/IL-5 release peaking at Days 7-9 postinfection, shortly before expulsion of the adult worms from the gut. IFN-gamma responses were not observed in the mesenteric lymph node. Responses in the spleen generally followed those in the mesenteric lymph nodes by 2-3 days and showed a greater degree of Th1-type cytokine production. N. brasiliensis was shown to be a powerful inducer of IL-4 responses with as few as six infective N. brasiliensis larvae eliciting IL-4 production in the mesenteric lymph node, but only high doses of larvae (600) elicited IL-4 secretion in the spleen. Similar levels of IL-4 production by lymph node cells stimulated with Con A or parasite antigen postinfection indicated the extent of polyclonal Th2 stimulation by this parasite. Infection of IL-4-deficient mice showed that despite the absence of IL-4-dependent Th2 responses, these mice were able to curtail egg production and expel adult N. brasiliensis in a time frame similar to that of fully immunocompetent animals. These results emphasize the magnitude of the Th2 response to N. brasiliensis and also show that IL-4 is not a prerequisite for the development of immunity to N. brasiliensis.

The contrasting effects of CD8+ T cells on primary, established and Nippostrongylus brasiliensis-induced IgE responses.

Recent data have indicated that CD8+ T cells suppress rodent IgE responses. In this study we investigated the effect of CD8+ T cells on primary and established IgE responses in euthymic and athymic nude rats. Euthymic PVG rats were depleted of CD8+ T cells by intraperitoneal injection of a CD8-specific monoclonal antibody (OX8), which resulted in an apparent loss of 92% of splenic and 98% of peripheral blood CD8+ T cells. The CD8+ T-cell depleted animals failed to mount a significant IgE response compared with control animals given an irrelevant monoclonal antibody (OX21). Furthermore, PVG nude rats reconstituted with purified CD4+ thoracic duct lymphocytes (TDL) alone failed to mount a significant IgE response, while animals given unfractionated TDL (containing CD4+ and CD8+ T cells) did. Depletion of CD8+ T cells 7 days prior to immunization and subsequent reconstitution at the time of immunization restored the IgE response. In contrast, removal of CD8+ T cells 1 month after induction of IgE by immunization with ovalbumin (OVA) and ricin prolonged the IgE response. In all cases IgG antibody responses were unaffected by the presence or absence of CD8+ T cells. This study shows that some CD8+ T cells are required for IgE, but not IgG, production to soluble antigen in a primary immune response. However, later in the immune response CD8+ T cells were shown to inhibit IgE production. These effects were apparently restricted to the immune response to soluble antigen, as Hooded Lister rats infected with 9000 larvae of the nematode Nippostrongylus brasiliensis produced high sustained levels of circulating IgE, in excess of 10 micrograms/ml, regardless of whether CD8+ T cells were depleted before or 1 month after infection.

Heterogeneity of IgG antibody responses to cloned Onchocerca volvulus antigens in microfiladermia positive individuals from Esmeraldas Province, Ecuador.

The prevalence of IgG antibodies to three recombinant O. volvulus antigens, OvMBP/10, OvMBP/11 and OvMBP/29 was determined in a group of 94 microfilaria positive (mf+) individuals resident in the hyperendemic onchocercal area of Esmeraldas Province, Ecuador. Clone OvMBP/11 was the antigen most frequently recognized by patients sera followed by OvMBP/10 and OvMBP/29. When a cocktail of the three recombinant antigens was used the proportion of positive sera increased to 100%. Antibody responses to the fusion partner maltose binding protein (MBP) were low in comparison with those to the cloned antigens and no correlation of responses between individual antigens was observed. The relative level of antibody response to each of the clones in the cocktail varied between individuals. The distribution of IgG responses to OvMBP/11 was bimodal and those to OvMBP/29 and OvMBP/10 were positively and negatively skewed, respectively. When the three recombinant antigens were used in combination this variation was minimized and the pattern of responses showed a normal distribution as was also seen to crude O. volvulus antigen. The cocktail of recombinants thus offers excellent diagnostic sensitivity in combination with the parasite specificity demonstrated previously.

Characterization of proteolytic enzymes from larval and adult Nippostrongylus brasiliensis.

Proteases from infective larval (L3) and adult stages of Nippostrongylus brasiliensis were investigated with a combination of techniques involving gelatin degradation and cleavage of fluorogenic substrates. Analysis of L3 excretory-secretory (ES) products revealed enzymes of Mr 51, 58, 79, approximately 150 and approximately 250 kDa. Inhibition profiles indicate that the major 51 kDa protease is a metallo-enzyme. Significantly, little activity was present in larval somatic extracts, suggesting the synthesis of zymogens or precursor forms prior to secretion. Adult ES contained a distinct enzyme, of 50 kDa, and a number of other proteases were detected in somatic extracts of this stage, ranging from 51 to greater than 300 kDa. The largest of these adult somatic enzymes is also a putative metallo-protease. While nearly all enzymes from both L3 and adult are heat labile, incubation at 100 degrees C generated a previously unobserved activity at 20 kDa. Furthermore, a protease of similar size may be found in uninfected rat intestinal tissue, suggesting specific uptake of a host-associated enzyme by the parasite in the form of an inactive, heat-labile complex.

Restricted sets of parasite antigens from the surface of different stages and sexes of the nematode parasite Nippostrongylus brasiliensis.

Surface molecules of parasitic stages of the nematode Nippostrongylus brasiliensis can be readily iodinated by the chloramine T technique, and assessed for antigenic reactivity with humoral antibody from infected animals. Free-living infective larvae are less amenable to analysis by this, or similar methods, but within 18 hr of larvae entering the host, new macromolecular surface antigens can be detected. The parasites change their surface antigens twice more in the course of the maturation to the adult stage. Surface antigens are stage-specific: lung larvae (L3), intestinal larvae (L4) and gut-living adults each possess characteristic sets of cuticular molecules. Single stage infections result in antibody reactive only to the antigens from the homologous stage. The adult surface appears to bear the greatest number of antigens, one of which is found only on the male worm. The composition of these antigens does not differ grossly between adult worms from a naive or immune host, or worms established after the adaptation of a 'trickle' (multiple low dose) infection. There appears to be an interesting contrast between the rapidity and extent of changes in surface antigens in the early phases of infection, and the stability of adult antigens analysed at different points in the host immune response.