Blood-feeding adult hookworms live for up to 18 years in close contact with the host immune system. Thus, in common with other helminths, they are likely to have evolved defences against host immunity. These defences may include suppression of antigen-specific immune responses, modulation of the host immune response from protective to non-protective responses, and inactivation of immune effector mechanisms. The difficulty in demonstrating protective immunity highlights the possibility that hookworms possess molecular strategies to subvert host protective responses, and a repertoire of potentially subversive molecules has been identified, including an eotaxin metalloproteinase (41), calreticulin (42,43), antioxidants (44,45), neutrophil inhibitory factor (46) and pro-apoptotic processes involving activated T cells (47). It has been difficult to define a precise role for each of these molecules in vivo in humans. However, evidence is accumulating from immunoepidemiological studies that immunomodulation occurs during hookworm infection. Several immunoepidemiological patterns can indicate immunosuppression during infection. These include a decline in immune responsiveness during infection, and an increase in responsiveness after removal of parasites by chemotherapy. Negative correlations between immune responses and worm burdens are consistent with both immunosuppression and protective immunity, and are discussed later. Such field studies have clearly demonstrated the suppression of specific immune responses in infections with tissue-dwelling helminths such as filarial worms and schistosomes (48).
Dynamics of immune responses during infection
Simple mathematical models of anti-helminth immune responses, assuming constant exposure with age, predict that immune responses will generally increase with host age to a plateau in adults, though declines across some age-classes are possible (49). Consistent with this, monotonic increases in antibody levels through time were seen in experimental infections (16). In contrast, eosinophil levels typically reach a peak in early experimental infection but then decline, even after repeated infection (18), and lymphoproliferative responses declined sharply from an early peak in A. ceylanicum infection, though egg counts also declined during this period (20). Results from natural infections are more variable. A number of antibody responses, and eosinophilia, in the Papua New Guinea and Zimbabwe studies did increase with host age, in a manner similar to worm burden (21,22,50,51). These included the overall IgG responses, some IgE responses and eosinophilia. However, levels of anti-larval IgA, and anti-adult IgD and IgE, in Papua New Guinea, and IgG1 and IgM in Zimbabwe, decreased with age. The dynamics of cytokine responses have only been assessed in one study, in which no relationship was seen between cytokine responses and host age (38). The declines in some immune responses during the course of infection are consistent with immunosuppression. There is also evidence that intestinal pathology declines with repeated infection and is less severe in chronic than acute infection (5,6). However, there are potential confounding factors in field studies, and studies which compare communities with different intensities of infection would be useful (49).
Changes in immune response after chemotherapy
Increases in immune responses after chemotherapy-induced loss of parasites provide stronger evidence for immunosuppression. Such studies are experimental rather than observational, so bias and the influence of confounding factors can be removed by randomization and placebo-controls (though this has yet to be done in hookworm studies). Changes in antibody responses after chemotherapy have been assessed in several studies. In general, hookworm-specific antibody levels decline sharply after chemotherapy, even in the presence of continued reinfection (Table 2a). However, a significant increase in anti-adult IgD was seen in one study (21). Total IgE levels were reduced after chemotherapy in four of five studies (26,32,38,52,53), and eosinophilia fell by 45–50% 6 weeks after treatment (32,52,53). In contrast, intestinal nonspecific IgA levels increased significantly after treatment (32). Only one study has investigated changes in cytokine responses after chemotherapy (38). There was a significant increase in IFN-γ responses to hookworm antigen, whereas IL-4 and IL-5 responses were unchanged (Table 2b). Proliferative responses increased slightly but not significantly after treatment, whereas both IFN-γ and proliferative responses fell significantly with increasing worm burden, consistent with immunosuppression. These data suggest that parasite-mediated immunosuppression occurs for some immune responses. An alternative explanation, that rises in responsiveness result from increased antigenic exposure after worm death, is unlikely for hookworm infection, though is important in schistosome infection. However, host-mediated immunoregulatory changes are also possible, where the removal of antigen might reduce cross-regulatory mechanisms.
Table 2. Changes in anti-hookworm immune responses after anthelminthic treatment. The sample size (n), hookworm species, time after initiation of chemotherapy that post-treatment samples were taken, worm burden at time of post-treatment sampling (expressed as a percentage of pre-treatment epg), and mean levels of each response pre- and post-treatment. Significant increases after chemotherapy are highlighted in bold
|(a) Antibody responses (OD units)|
|Country (reference)||n||Hookworm species||Time post- treatment||Worm burden post-treatment||Antigen||Antibody||Mean level pre-treatment||Mean level post-treatment|
|India (26)|| 25||nd||1 month|| 0%||L3 ES||IgE||1·510||0·476***|
|Papua New Guinea (21)||172||N. americanus||1 year||47%||Adult ES||IgG||0·842||0·524***|
| || || || || ||IgM||0·870||0·442***|
| || || || || ||IgA||0·468||0·429|
| || || || || ||IgD||0·088||0·202***|
| || || || || ||IgE||0·296||0·304|
| || || || ||L3 SOM||IgG||0·522||0·460*|
| || || || || ||IgM||0·810||0·561***|
| || || || || ||IgA||0·290||0·093***|
| || || || || ||IgD||0·115||0·144|
| || || || || ||IgE||0·400||0·099***|
|Brazil (37)|| 12||N. americanus||6 months|| 0%||Adult SOM||IgG||1·01||0·65|
| || || || || ||IgG1||0·35||0·18**|
| || || || || ||IgG4||1·19||0·53**|
| || || || || ||IgA||0·93||0·35**|
| || || || || ||IgE||0·33||0·18|
|(b) lymphoproliferative (cpm) and cytokine responses (pg/mL)|
|Country (reference)||n||Hookworm species||Time post- treatment||Worm burden post-treatment||Antigen||Response||Mean level pre-treatment||Mean level post-treatment|
|Papua New Guinea (38)||54||N. americanus||1 month||17%||Adult ES||Proliferative||10339||13888|
|42|| || || || ||IFN-γ|| 166|| 322**|
|42|| || || || ||IL-4|| 20|| 17|
|37|| || || || ||IL-5|| 163|| 151|
Thus the available evidence suggests that there is worm-associated suppression of IFN-γ responses. Suppression was not antigen-specific, as there was a worm burden-dependent decline in IFN-γ responses to mycobacterial PPD (38), and suppression of nonspecific cellular responses has also been reported (54). Such bystander suppression is also seen in other helminth infections. In contrast, there is little evidence for suppression of antibody responses or eosinophilia, which typically fall after chemotherapy. However, anti-adult IgD levels increased significantly after treatment, and declined with age pre-treatment (21). Similarly, total intestinal IgA levels increased after treatment, and were negatively related to serum IgE levels pre-treatment (32). The mechanisms of immunosuppression are unknown, though the high levels of IL-10 in hookworm infection suggest a role for regulatory Treg cells (48), and high levels of IL-10 were associated with reduced levels of IFN-γ, IL-5 and IL-13 in Brazilian patients (37). Levels of another down-regulatory cytokine, TGF-β, have not been assessed in hookworm infection. Recent data from Brazil implicate a block in entry of cells to the G1 phase of the cell cycle in immunosuppressed individuals. Stimulation with larval or adult hookworm antigens failed to activate extracellular-signal-regulated-kinase (ERK) in PBMC from hookworm-infected individuals, indicating that the cells remained blocked in G0. In contrast, activated ERK was produced in response to PPD, and in response to specific stimulation in S. mansoni and A. lumbricoides infected patients (Bethony and Bottazzi, unpublished observations).
A number of other immunomodulatory changes are apparent in helminth infections. Most obvious is the Th2 bias induced by hookworms and other helminths. Various hookworm products may contribute to this Th2 bias (55). Whether the Th2 response reflects a host response to control worms, or is partly parasite-driven to aid parasite survival, remains controversial (48). However, the increasing evidence for Th2-mediated protective immunity (see below) suggests the former. As cytokine studies have shown, there is also a significant Th1 response in hookworm infection. Observations from other helminth systems and allergic disease suggest that complex interactions occur between Th1 and Th2 responses. Individuals with schistosomiasis or filariasis can have a modified Th2 response, with high IL-10 and IgG4, a balanced Th1/Th2 response, with high IgE, or a predominantly Th1 response (48). These different responses may reflect the course of infection, variation in exposure, or host differences. The identification of such variation in hookworm infection will require more detailed immunoepidemiological studies, particularly of changes in antibody and cytokine production with age, and between communities with different intensities of infection. Both Th2 and Th1 stimulatory products have been described from hookworms (55,56).
Further understanding of the mechanisms of immune modulation by hookworms, and identification of the molecules responsible, may lead to the development of novel, much needed therapeutics for immunopathological diseases. The existence of hookworm-mediated immunosuppression also has some practical implications for hookworm vaccine development (7,8,57). Vaccine-induced immunity may itself be susceptible to immunosuppression, so that protective responses may not develop unless pre-existing infections are removed by chemotherapy. More importantly, individuals vaccinated with a partially protective vaccine will be subject to reinfection, which might then suppress vaccine efficacy. The use of immunomodulatory molecules as vaccine candidates could potentially avoid such problems, since successful vaccination might prevent future immunosuppression. It is thus of interest that vaccination with immunomodulatory hookworm calreticulin induced successful protection in a murine model (58), reinforcing the belief that hookworm defence molecules may make good vaccines.