Vaccination with Strongyloides ratti heat shock protein 60 increases susceptibility to challenge infection by induction of Th1 response

Abstract

The control of strongyloidiasis affecting approximately 100 million people – caused by the gastrointestinal nematode Strongyloides stercoralis – is still based on anti-helminthic treatment. In the current study we analysed the immune response to Strongyloides ratti heat shock protein 60 (srHSP60) as a possible vaccine candidate in the murine system. We show that srHSP60 is a target of both, humoral and cellular response in S. ratti-infected mice. Strikingly, vaccination with srHSP60 without adjuvant or with CFA induced a S. ratti-specific Th1 response in vivo that did not confer protection but slightly increased larval output during challenge infection. Using in vitro T cell stimulation assays we provide further evidence that srHSP60 skewed activated T cells towards a Th1 response that interfered with efficient clearance of S. ratti infection. Vaccination with alum-precipitated srHSP60, in contrast, overruled the Th1-inducing activity intrinsic to srHSP60, induced a Th2 response, and conferred partial protection against a challenge infection. As srHSP60 is actively secreted by S. ratti during all life stages, our findings strongly suggest that srHSP60 induced polarization towards a Th1 response reflects a mechanism of immune evasion by this pathogenic nematode.

Introduction

Strongyloides stercoralis is a human gastrointestinal nematode that afflicts approximately 100 million people of the world population with high prevalence in tropical and subtropical regions [1], [2]. S. stercoralis infection is generally asymptomatic in normal healthy individuals and can remain undetected for decades. A rare but severe complication in immune compromised individuals is the hyperinfection syndrome that is caused by uncontrolled dissemination of infective larvae and is mortal in 87% of the cases [3], [4], [5], [6]. As the majority of S. stercoralis infections do not induce pathology and thus are never diagnosed, hyperinfection syndrome is often induced accidentally by immune suppressive treatment of patients receiving organ transplantation [7]. Moreover, helminth infections often change the immunological status of their host and therefore the efficient response to vaccinations can be compromised also by mostly asymptomatic infections [8], [9]. Thus, establishment of a vaccination conferring protection to S. stercoralis infection is desirable despite the asymptomatic nature of the majority of infections.

Because S. stercoralis does not reproduce within rodents, the closely related Strongyloides ratti is employed to model this infection in mice and rats [10], [11]. Infective third stage larvae (iL3) actively penetrate the skin of their mammalian host, migrate percutaneously to the mouth, are swallowed and reach the gut. Here, they moult via the fourth larval stage (L4) to parasitic female adults that reproduce by parthenogenesis. Eggs as well as hatched first stage larvae (L1) leave the host with the faeces by day six post infection (p.i.). Female larvae may directly develop to iL3 stage or moult to free-living adults that mate with free-living male adults [12]. The infection is cleared spontaneously within three weeks in the context of a T helper 2 (Th2) response that is characterized by production of S. ratti-specific IgG1, IL-4, IL-5, and IL-13 [10], [13]. Mice and rats that had cleared a primary infection remain semi-resistant to subsequent infections, displaying considerably lower worm burden and larval output [11], [14].

It was shown before that the immunization of mice with soluble protein antigens derived from S. ratti or Strongyloides venezuelensis iL3 conferred partial protection to challenge infection [15], [16], [17]. Although S. stercoralis never proceed beyond the iL3 in mice [18], vaccination with iL3 extract [19], [20] or DNA encoding Na+–K+ ATPase protein (Sseat-6) [21] induced accelerated killing of S. stercoralis iL3 in vaccinated mice.

Searching for more potent vaccine candidates, we analysed the E/S proteins released by S. ratti because they are present at the interface between parasite and host. A recent study of S. ratti derived E/S proteins revealed over 600 proteins produced by different free-living and infective stages [22]. The proteomic characterization by LC–MS/MS mass spectrometry identified these products amongst others as galectins, proteases, and heat shock proteins. Here, heat shock protein 60 (srHSP60) was identified as an abundant constituent of E/S products derived from in vitro-cultured iL3 and parasitic females [23]. Further analysis showed that relative expression of srHSP60 was twofold higher in parasitic females in comparison to free-living females.

Heat shock proteins (HSPs), preferentially called stress proteins, are highly conserved and ubiquitously distributed proteins [24], [25]. In addition to their original role as molecular chaperones, HSPs play a central role in host–parasite interaction as they were described as major immune dominant antigens in several infections [26], [27]. Vaccination with HSP in helminth infection has been recently used. Immunization of rats with srHSP10 against S. ratti infection markedly reduced the larval output in the faeces after challenge infection [23].

Here we analysed the role of srHSP60 in the immune response against S. ratti in mice. Although srHSP60 is a target of both the humoral and cellular response in S. ratti-infected mice, vaccination with srHSP60 induced a Th1 response that aggravated challenge infection with S. ratti iL3. We provide evidence that srHSP60 itself skewed activated T cells towards a Th1 response that interfered with efficient expulsion of Strongyloides parasitic adults. Alum precipitation of srHSP60 in contrast, overruled the Th1-inducing activity intrinsic to srHSP60 and induced protection against challenge infections in the context of a Th2 response.