Parasite Immunology, 2014, 36, 141–149
DOI: 10.1111/pim.12093
Immunization with Brugia malayi Hsp70 protects mice against Litomosoides sigmodontis challenge infection W. HARTMANN,1,* N. SINGH,2,* S. RATHAUR,2 Y. BRENZ,1 E. LIEBAU,3 B. FLEISCHER1,4 & M. BRELOER1 Department of Immunology and Virology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, 2Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, India, 3Institute of Animal Physiology, University of M€ unster, M€ unster, Germany, 4Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 1
SUMMARY More than 15 billion people are at risk of being infected with filarial nematodes worldwide. Therapy and control of transmission are mainly based on mass drug distribution. As these drugs have to be administered annually or biannually and might be loosing their efficacy, a vaccine against filariae is an alternative approach to chemotherapy. In the current study, we have analysed the potential of Brugia malayi heat shock protein 70 (BmHsp70) as a vaccine candidate in a murine helminth infection. Immunization of BALB/c mice with alum-precipitated recombinant BmHsp70 conferred partial protection against subsequent challenge infection with the rodent parasite Litomosoides sigmodontis. Immunization resulted in reduced numbers of larvae in the pleural cavity as well as reduced numbers of circulating microfilariae. Reduced parasite burden was associated with high titres of BmHsp70specific antibodies and increased production of type I and II cytokines in response to L. sigmodontis antigen and BmHsp70. In summary, the immunization with BmHsp70 induced cellular and humoral immune responses and partially protected against L. sigmodontis in a challenge infection. Therefore, we hypothesize that BmHsp70 might be considered as a potential vaccine candidate for reduction in the incidence of B. malayi infections in future studies. Keywords Brugia malayi, heat shock protein 70, immunization, Litomosoides sigmodontis, lymphatic filariasis Correspondence: Wiebke Hartmann, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany (e-mail: [email protected]). *These authors contributed equally to this work. Disclosures: WH: none; NS and SR were funded by the Alexander von Humboldt foundation for carrying out research at the Bernhard Nocht Institute for Tropical Medicine, Hamburg; YB: none; EL: none; BF: none; and MB: none. Received: 12 July 2013 Accepted for publication: 11 December 2013 © 2013 John Wiley & Sons Ltd
INTRODUCTION It is estimated that worldwide more than 150 million people are infected with filarial nematodes such as Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus (1). In humans, these filariae induce chronic infections with high morbidity especially in the tropical and subtropical regions. The life cycle of filarial worms is similar in humans and in different animal models, starting in an arthropod as intermediate host. The first stage larvae, so called microfilariae (MF), are taken up during a blood meal from a mosquito, blackfly or blood-sucking mite. Within the vector, MF undergo two moults and develop into infective thirdstage larvae (L3). L3 are transmitted during a second blood meal to their final host. Here, L3 migrate depending on the species to different sides of the body. In O. volvulus infection, adult worms reside in nodules in the subcutaneous tissue, while adults from W. bancrofti and B. malayi dwell in lymphatic vessels (2). The development of antifilarial vaccines in addition to effective chemotherapy is a prerequisite to combat diseases caused by filarial nematodes. Despite various strategies to control the transmission or to develop drugs, still an estimated 200 million people remain infected. So far, the existing antifilarial drugs predominantly target MF and remain ineffective towards the adult filarial parasites (3). Ivermectin is used annually or biannually in mass drug administrations to control O. volvulus infection, and it requires continuous administration for at least 4–6 years to interrupt transmission (4–7). However, ivermectin does not kill adult worms and emerging resistance (8–11), and serious adverse effects have been reported in patients treated with this drug (12, 13). Therefore, the development of vaccines that would confer at least partial protection and thus reduce parasite burden and transmission is highly desirable. Here, we analyse the capacity of B. malayi-derived heat shock protein (Hsp) 70 to induce a protective immune
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response against challenge infection with Litomosoides sigmodontis as an established murine model of filarial infection (14). Heat shock proteins are a highly conserved group of proteins known to play an important role as molecular chaperones. Under physiological conditions, Hsps are responsible for proper folding and assembly of polypeptides (15). Under stress conditions such as environmental changes in temperature, pH and pressure, Hsp expression increases rapidly (16). Such conditions are found whenever a pathogen enters a warm-blooded host. In particular, during filarial life cycles, the larvae have to cope with a significant rise in temperature upon transmission from the poikilotherm arthropod vector to the homoiotherm mammalian host (17). Generally, most cells respond to an increase in temperature by upregulating the expression of Hsp that are required either for refolding degraded proteins or for targeting them for degradation. Differential expression of Hsp during transfer of L3 from the vector to the mammalian host has been demonstrated in an early study (18). Due to upregulation of heat shock proteins during transmission, an Hsp-specific immune response occurs in several infectious diseases (19–21). Heat shock protein 70 represents a dominant antigen in various helminth infections such as Schistosoma mansoni, O. volvulus, B. malayi and W. bancrofti (22–25). Furthermore, accumulating evidence suggests that Hsp possesses an intrinsic immune-stimulatory capacity (26). The aim of our study was to test the potential of BmHsp70 immunization in a murine model of filarial infection. Infection of mice with L. sigmodontis is a wellcharacterized murine model system, which shares many biological characteristics with human filarial pathogens (27, 28). Infection of BALB/c mice with L. sigmodontis is currently the only murine model where a filarial nematode develops a patent infection, that is, reaches sexual maturity, mates and releases offspring into the circulation. Laboratory mice can be infected naturally by blood-sucking mites or artificially by injection of L3 (14, 29, 30). Following infection, L3 migrate during the first 3 days via the lymphatic system to the thoracic cavity (27). They moult twice and develop within 30 days into juvenile adults. In the permissive BALB/c strain, L. sigmodontis mate and release MF by day 60 post-infection (p.i.) (31). Thereby, infections of BALB/c mice offer a reliable tool to perform immunization studies against filarial nematodes. In our study, we analysed the impact of immunization with B. malayi Hsp70 on the defence against L. sigmodontis in a challenge infection and humoral and cellular immune responses. Our results indicate that BmHsp70 can be harnessed as a possible vaccine candidate for human filariasis in future studies.
Total RNA was isolated from B. malayi adult worms using Trizol reagent. The cDNA was synthesized from B. malayi RNA using oligo (dT) primer from first strand cDNA synthesis kit (Thermo Scientific, Schwerte, Germany). A 1935-bp fragment of BmHsp70 was amplified by PCR from cDNA using sense primers 5′-CGCCTCGAGGATGATGTCAAAGAATGCCATTGGTATCG-3′ and antisense primers 5′-GCGCGATATCCTAATCAACTTCTTC AATTGTTGGT-3′ designed using consensus B. malayi mRNA sequence of BmHsp70 gene (Gene accession no. XM_001900162). The amplification was performed as follows: initial denaturation at 94°C for 2 min, 35 cycles of 94°C for 40 s, 52°C for 30 s and 72°C for 2 min and a final extension of 72°C for 8 min. Following amplification, the PCR products and the expression vector pJC40 (32) were digested using appropriate FastDigestâ restriction enzymes (Thermo Scientific) and ligated using T4 DNA ligase (Invitrogen, Darmstadt, Germany). Five microlitres of each ligation was transformed into XL10-gold ultra competent cells according to the suppliers protocol (Stratagene, La Jolla, CA, USA). Positive clones were identified by test digestion and sequencing. After transformation of the respective expression plasmids into Escherichia coli BL21 (DE) cells (Stratagene), expression of the tagged BmHsp70 was initiated by the addition of 05 mM iso-propyl-beta-D-thiogalactopyranoside (IPTG) once the cultures had reached A600 = 05. Following induction, cells were left to grow for additional 3 h at 37°C. Cells were harvested by centrifugation. The resulting bacterial pellet was resuspended in 50 mM Tris buffer (pH 80) containing 500 mM NaCl and 10 mM imidazole and sonicated using a digital sonifier set to 30 watts and 30% amplitude (Branson, Danbury, CT, USA). After centrifugation (10 000 9 g, 30 min) of the lysate, supernatant was purified by affinity column chromatography with profinityTM IMAC Ni2+-nitrilotriacetic acid resin (Bio-Rad Laboratories, München, Germany). Lysate was washed for 2 h at room temperature with 25 bed volumes of washing buffer (lysis buffer containing 20 mM imidazole). Full-length BmHsp70
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MATERIALS AND METHODS Mice, reagents and antibodies All in vivo experiments were carried out at the animal facility of the Bernhard Nocht Institute for Tropical Medicine with permission of the Federal Health Authorities of the State of Hamburg, Germany. BALB/c mice were obtained from Charles River. Animals were kept in individually ventilated cages.
Cloning, expression and purification of BmHsp70
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was eluted with lysis buffer containing 250 mM imidazole and dialysed in PBS. The purity of the dialysed protein was checked by resolution on 125% SDS-PAGE and Coomassie blue staining. Protein concentration was determined using a Bradford assay (33).
Limulus amebocyte lysate Protein preparations were tested in the Limulus amebocyte lysate (LAL) assay (Lonza, Walkersville, MD, USA) for endotoxin concentrations according to the manufacturers instructions. Endotoxin activity was below 3 EU/lg protein.
Litomosoides sigmodontis cycle and experimental infection The life cycle of L. sigmodontis was maintained in cotton rats (Sigmodon hispidus), the natural reservoir of the nematode (34). Microfilariae were counted weekly in the blood of infected cotton rats. To standardize the infectious dose, cotton rats with 500–1500 MF/lL blood were used for infections of mites. Intermediate host mites (Ornithonyssus bacoti) were permitted to feed on infected cotton rats. Fourteen days after this blood meal, 11-week-old BALB/c mice were infected naturally by exposure to infected mites. For infection, BmHsp70-vaccinated and control mice were placed in the same tank to prevent a bias due to batches of mites. Mice were sacrificed at day 30 and 60 p.i., and the number of worms was counted after flushing the thoracic cavity with 10 mL cold PBS. For counting MF, 50 lL blood was collected every 10 days by puncture of the submandibular vein. Erythrocytes were lysed by addition of 200 lL water followed by centrifugation. The pellet was resuspended in 50 lL gentian violet, and MF were counted.
Preparation of Litomosoides sigmodontis antigen Female worms, isolated from infected BALB/c mice, were transferred into sterile phosphate-buffered saline. Homogenization of live female worms with a glass homogenizer was followed by centrifugation at 16 000 g for 30 min at 4°C. The supernatant was passed through a 022-lm filter and then stored at 80°C until used.
Immunizations
Brugia malayi Hsp70 protects against filarial infection
three times with 50 mL PBS by centrifugation at 300 g for 10 min. Alum-precipitated BmHsp70 and PBS were stored at 4°C until used. Eight-week-old BALB/c mice were immunized by i.p. injection of 100 lg BmHsp70 or PBS precipitated in alum. After 7 days, mice received a boost immunization. Fourteen days later, mice were naturally infected with L. sigmodontis by exposure to infected mites.
In vitro stimulation of lymphocytes Mice were sacrificed at indicated time points p.i., and spleens were prepared. 2 9 105 spleen cells were cultivated in 96-well round-bottom plates for 72 h at 37°C and 5% CO2 in RPMI 1640 medium supplemented with 10% foetal calf serum, L-glutamine (2 mg/mL) and gentamicin (50 lg/mL). For stimulation, cells were incubated either with medium alone or with 1 lg/mL anti-CD3 (145– 2C11), or 20 lg/mL Litomosoides sigmodontis antigen (LsAg), or BmHsp70 in quadruplicates. Supernatants were collected and stored at 20°C until analysis.
ELISA For the detection of L. sigmodontis- or BmHsp70-specific Ig, ELISA plates were coated overnight with 4 lg/mL LsAg or BmHsp70 in carbonate buffer, pH 96. Plates were washed with PBS / 001% Tween 20 and blocked by incubation with PBS 1% bovine serum albumin for 2 h at room temperature. Plates were washed again and then incubated for 2 h with either serial dilutions or a 1 : 100 dilution of the serum. Plates were washed and incubated for 1 h with horseradish peroxidase-labelled anti-mouse IgM, IgG1, IgG2b (Invitrogen) to detect antigen-specific isotypes in the serum. Plates were washed five times and developed by incubation with 100 lL 01 mg/mL tetramethylbenzidine, 0003% H2O2 in 100 mM NaH2PO4 pH 55 for 25 min. Reaction was stopped by addition of 50 lL 1 M H2SO4, and OD450 was measured. Titres were calculated by defining the highest serum dilution in a serial dilution (1 : 100 to 1 : 102 400) resulting in an OD450 above the doubled background that was generally below OD450 = 015. Arbitrary units (a.u.) were calculated by subtracting the OD450 of the background from the OD450 of a fixed serum concentration (1 : 1000 for IgG2b). Cytokines were determined in culture supernatants from spleen cells by indirect sandwich ELISA according to the manufacturers instructions (R&D Systems, Wiesbaden, Germany).
BmHsp70 and PBS were mixed (1 : 2) with 9% alum potassium sulphate dodecahydrate (Sigma-Aldrich, Hamburg, Germany). Antigen precipitation was achieved by adjusting the pH to 70 with 4 M NaOH. Precipitate was washed
All statistical tests were performed by Students t-test or 2way ANOVA with Bonferroni post-test using PRISM software
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(GraphPad Software, San Diego, CA, USA). P values below 005 were considered statistically significant.
RESULTS AND DISCUSSION Immunization with BmHsp70 confers partial protection against Litomosoides sigmodontis challenge infection The aim of this study was to analyse the impact of B. malayi-derived Hsp70 on the immune response against L. sigmodontis. We cloned the full-length Hsp70 from adult B. malayi worms and expressed it as recombinant protein in E. coli. The recombinant protein was purified via its N-terminal histidine tag. Purification was confirmed by SDS-PAGE followed by Coomassie blue staining (Figure 1). As BmHsp70 is a chaperone and might bind bacterial proteins during expression in E. coli, we cannot exclude contamination of the preparation with bacterial proteins. The faint bands in the SDS gel might represent either degraded BmHsp70 or bacterial contaminants. The latter is unlikely as endotoxin levels of BmHsp70 were below 3 EU/lg protein. BALB/c mice were immunized twice with alum-precipitated BmHsp70 or, as control, with alum/PBS. Subsequently mice were naturally infected with L. sigmodontis by exposure to infected mites. Immunization with BmHsp70 reduced parasite burden in the pleural cavity by approximately 50% at day 30 (Figure 2a) and day 60 p.i. (Figure 2b) compared with the control groups immunized with alum only. Although there was a trend for reduced recovery at day 95 p.i. (Figure 2c),
Parasite Immunology
the differences were not statistically significant at this time point. The numbers of female and male worms were decreased to a similar extent at all time points (data not shown). Numbers of MF in the peripheral circulation were also significantly reduced in BmHsp70-immunized mice between days 60 and 95 p.i. compared with PBS/ alum-treated control mice (Figure 2d). The pronounced reduction in MF could be explained by a number of possibilities: (i) reduction in female worms, (ii) reduced fertility of the remaining female worms, (iii) reduced fertilization of eggs due to reduced numbers of male worms and (iv) enhanced killing of MF.
Immunization with BmHsp70 induces an Hsp70-specific humoral immune response in Litomosoides sigmodontis infected mice
Figure 1 Coomassie blue staining of recombinant BmHsp70. BmHsp70 was expressed in Escherichia coli and purified by affinity chromatography. Following separation by SDS-PAGE, BmHsp70 was stained with Coomassie blue.
To elucidate the underlying mechanism of improved host defence in BmHsp70-treated mice, we compared the immune response of BmHsp70-immunized and PBS control mice. As B cells are required for vaccine-induced protection with irradiated L. sigmodontis larvae (35), we measured BmHsp70-specific and L. sigmodontis-specific antibodies in the serum of infected mice. BmHsp70-specific IgM (Figure 3a, b) and IgG2a (data not shown) were low in PBS/alum- and BmHsp70/alum-treated mice and did not increase between day 30 and 60 p.i. Immunization with BmHsp70 induced BmHsp70-specific IgG1 and IgG2b responses that were already visible at day 30 and slightly increased until day 60 p.i. while control mice failed to generate BmHsp70-specific IgG (Figure 3a, b). Humoral reactivity towards Hsp70 has been described in areas, where lymphatic filariasis is endemic (23, 24). However, in these studies, reactivity of patient sera was analysed by Western blotting that did not distinguish between different isotypes. Our analysis identified the presence of BmHsp70-specific IgM in the serum of infected control mice independent of immunization. In addition, BmHsp70-specific IgM was detectable in the serum from noninfected mice at a low level and increased during infection (data not shown). BmHsp70-specific IgM in infected control mice might represent natural Hsp70specific IgM that cross-reacts with L. sigmodontis-derived Hsp70. In contrast, BmHsp70-specific IgG (IgG1 and IgG2b) were not produced without immunization although the mice had been infected for 60 days. Regarding the L. sigmodontis-specific humoral response, LsAg-specific IgM was detectable by day 30 p.i. while LsAg-specific IgG1, IgG2b (Figure 3c, d) and IgG2a (data not shown) were not detected before day 60 p.i. in infected mice. Thereby, the magnitude of LsAg-specific Ig response was comparable in immunized and
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Figure 2 BmHsp70 vaccination decreases parasite burden. Prior to natural infection with Litomosoides sigmodontis, BALB/c mice were immunized with PBS/alum (white bars) or BmHsp70/alum (black bars). Number of adult worms in the pleural cavity was counted at day 30 p.i. (a), day 60 p.i. (b) and day 95 p.i. (c). (d) Number of microfilariae in the peripheral blood (y-axis) of infected immunized and control mice was counted at the indicated time points (x-axis). (a, b) show combined results of 2 independent experiments (n = 8 for PBS/ alum, n = 9 for BmHsp70). (c, d) represent one experiment with five mice per group. Each dot represents worm numbers from a single mouse (a–c). Results are expressed as mean SEM (d). Asterisks indicate statistically significant differences of the mean (*P < 005, **P < 001). Statistical analysis was performed with Students t-test (a–c) or 2-way ANOVA (d).
nonimmunized mice (Figure 3c, d). Although immunized mice had lower worm burdens during L. sigmodontis challenge infection, this was not associated with increased LsAg-specific humoral responses at day 30 and 60 p.i. In general, immune response in immunized mice might be partially raised against E. coli-derived proteins in our BmHsp70 preparation. We cannot formally exclude immune response against E. coli-derived proteins and cross-reaction with Wolbachia in L. sigmodontis larvae. However, as endotoxin activity of the BmHsp70 preparations was below 3 EU/lg protein and LsAg-specific Ig was comparable in both groups, a major role of bacterial contaminants in the immune response of immunized mice seems to be unlikely. On the other hand, we used a crude extract from adult worms to detect LsAg-specific Ig that might not contain the essential B-cell antigens, for instance, L. sigmodontis-derived Hsp70. In addition, the sensitivity of our ELISA may be too low to detect differences in the L. sigmodontis-specific B cell responses between immunized and control mice. © 2013 John Wiley & Sons Ltd, Parasite Immunology, 36, 141–149
Immunization with BmHsp70 induces antigen-specific production of Th1 and Th2 cytokines As our vaccination with BmHsp70 did not change IgM responses but enhanced T helper cell-dependent IgG responses, we next measured cytokine production by splenic T cells. Spleen cells derived from BmHsp70-immunized and control mice at day 30 and 60 p.i. were restimulated in vitro. We quantified IFN-c (Figure 4a, b) as an indicator of a Th1 immune response, and IL-5 (Figure 4c, d) and IL-13 (Figure 4e, f) as indicators of a Th2 immune response. IL-10 (Figure 4g, h) was measured as a cytokine with regulatory functions. Polyclonal T cell stimulation with anti-CD3 resulted in a comparable cytokine production at day 30 and 60 p.i., revealing no differences between immunized (BmHsp70) and nonimmunized (PBS) mice. Litomosoides sigmodontis antigen-specific cytokine responses were also similar at day 30 p.i in immunized and control mice. A shift towards Th2 cytokine response was visible in spleen cells isolated from
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(a)
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Figure 3 BmHsp70 vaccination increases BmHsp70-specific but not Litomosoides sigmodontis antigen (LsAg)-specific humoral response. BALB/c mice were immunized with PBS (white bars) or BmHsp70 in alum and then infected with L. sigmodontis. BmHsp70-specific (a, b) and L. sigmodontis (c, d) Ig in the serum were measured at day 30 (a, c) or day 60 p.i. (b, d) by ELISA. IgG1 and IgM are shown as titre and IgG2b as arbitrary units. Combined results of two independent experiments (n = 8 for PBS, n = 9 for BmHsp70) are shown. Results are expressed as mean SEM. Asterisks indicate significant differences of the mean (***P < 0001). Statistical analysis was performed with Students t-test.
BmHsp70-immunized mice at day 60 p.i. LsAg-specific IL-5, IL-13 and IL-10 synthesis was higher in spleen cells isolated from immunized mice than in infected control mice (Figure 4d, f, h). We also observed increased BmHsp70-specific IFN-c, IL-5, IL-13, and IL-10 cytokine production in spleen cells derived from BmHsp70-immunized mice at both time points. As immunization with BmHsp70 increased production of Th1-associated IFN-c and Th2-associated IL-5 and
IL-13 as well as IL-10, the immunization obviously did not induce a bias towards type 1 or type 2 immune responses, but enhanced all types of T-cell responses. This was also reflected by the induction of both Th1-associated IgG2 and Th2-associated IgG1 response by BmHsp70 immunization. During L. sigmodontis infection, both Th1 and Th2 cytokines contribute to the parasite control in comparison with other helminthic infections that are predominantly controlled by Th2 cytokine response (36).
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Figure 4 BmHsp70 vaccination increases BmHsp70-specific and Litomosoides sigmodontis antigen (LsAg)-specific cytokine production of spleen cells. BALB/c mice were immunized with PBS (white bars) or BmHsp70 in alum and then infected with L. sigmodontis. Spleen cells were prepared at day 30 (a) and day 60 p.i. and restimulated for 72 h with indicated stimuli (x-axis). Concentrations of IFN-c (a, b), IL-5 (c, d), IL-13 (e, f) and IL-10 (g, h) in the supernatants were measured by ELISA. Results are shown as mean SEM from one individual experiment (n = 5 mice per group) that is representative for two independent experiments. Asterisks indicate significant differences of the mean (*P < 005, **P < 001 and ***P < 0001). Statistical analysis was performed with Students t-test.
Therefore, the enhanced humoral and cellular BmHsp70specific responses might account for increased resistance to parasite infection in BmHsp70-immunized mice. However, the exact mechanism of parasite clearance needs to be further addressed. In two recent studies, different vaccination protocols were analysed to induce protection in a challenge infection
with L. sigmodontis (37, 38). Ziewer et al. (37) administered 1 9 105 viable MF via different routes. Selectively, subcutaneous immunization with MF in alum induced 70– 100% reduction in MF in the peripheral circulation and in the pleural cavity. Here, the number of larvae was unaffected until day 70 p.i. in alum/MF-treated mice (37). In a second study, reduction in L. sigmodontis adults and MF
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was achieved by DNA-based vaccines expressing two immune-modulatory excretory secretory products (ESP), namely abundant larval transcript and cysteine protease inhibitor-2 (38). None of these ESP alone were capable of reducing the parasite burden during a challenge infection with L. sigmodontis. As both ESP had suppressive capacities, additional deletion of immune-modulatory sequences and co-administration of plasmids enhancing antigen presentation was required for improved immune response against the parasite. Although these studies emphasize the difficulty of achieving protection against L. sigmodontis infection by experimental immunization, two doses of alum-precipitated recombinant BmHsp70 were sufficient to significantly reduce the number of L. sigmodontis larvae in the pleural cavity and in the peripheral circulation. The numbers of larvae were already reduced at day 30 p.i. (mean number of worms: 131 27 vs. 316 54), and this phenotype was robust until day 60 p.i. (mean number of worms: 176 3 vs. 31 43). No further reduction in the number of larvae in BmHsp70-immunized mice happened between day 30 and 60 p.i. Thus, the protective capacity of BmHsp70 might occur early during infection, that is, target migrating larvae, L3 or L4 in the pleural cavity. In future studies, protection might be further improved by use of different adjuvants, vaccine doses, as well as routes and time points of administration. In line with our data, three recent studies demonstrate the vaccine potential of small heat shock protein 126 from B. malayi,
either alone or as a multivalent fusion protein in a challenge infection with B. malayi (39–41). Taken together, our study supports the efficacy of developing vaccines based on immune-modulatory molecules or danger signals (38). Heat shock protein 70 seems to be a promising vaccine candidate for the following reasons: (i) Hsp70 is constitutively and abundantly expressed during all developmental stages of nematodes; (ii) Hsp70 expression is further upregulated upon heat shock, which takes place during transmission of L. sigmodontis via the arthropod vector to the mammalian host and; (iii) several lines of evidence suggest that Hsp70 contains an intrinsic immune-stimulatory capacity that induces pro-inflammatory cytokines in professional antigen-presenting cells and enhances T-cell activation (26, 42–45). Due to the partial protection we observed in BmHsp70-immunized mice in a challenge infection with a related rodent parasite, we hypothesize that vaccinations with B. malayi Hsp70 might confer protection against lymphatic filariasis.
ACKNOWLEDGEMENTS NS and SR received funding from Alexander von Humboldt foundation for carrying out the research at Bernhard Nocht Institute for Tropical Medicine, Hamburg. MB, WH, NS, YB conceived and designed the experiments; WH, NS and MB analysed the data; SR, YB, EL, BF contributed essential reagents; WH and MB wrote the manuscript.
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19 Zugel U & Kaufmann SH. Immune response against heat shock proteins in infectious diseases. Immunobiology 1999; 201: 22–35. 20 Mizzen L. Immune responses to stress proteins: applications to infectious disease and cancer. Biotherapy 1998; 10: 173–189. 21 Falkowska-Podstawka M & Wernicki A. Heat shock proteins in health and disease. Pol J Vet Sci 2003; 6: 61–70. 22 Hedstrom R, Culpepper J, Schinski V, Agabian N & Newport G. Schistosome heatshock proteins are immunologically distinct host-like antigens. Mol Biochem Parasitol 1988; 29: 275–282. 23 Selkirk ME, Denham DA, Partono F & Maizels RM. Heat shock cognate 70 is a prominent immunogen in Brugian filariasis. J Immunol 1989; 143: 299–308. 24 Rothstein NM, Higashi G, Yates J & Rajan TV. Onchocerca volvulus heat shock protein 70 is a major immunogen in amicrofilaremic individuals from a filariasis-endemic area. Mol Biochem Parasitol 1989; 33: 229–235. 25 Ravi V, Kubofcik J, Bandopathyaya S, et al. Wuchereria bancrofti: cloning and characterization of heat shock protein 70 from the human lymphatic filarial parasite. Exp Parasitol 2004; 106: 1–10. 26 Osterloh A & Breloer M. Heat shock proteins: linking danger and pathogen recognition. Med Microbiol Immunol 2008; 197: 1–8. 27 Bain O, Wanji S, Vuong PN, Marechal P, Le Goff L & Petit G. Larval biology of six filariae of the sub-family Onchocercinae in a vertebrate host. Parasite 1994; 1: 241–254. 28 Allen JE, Adjei O, Bain O, et al. Of mice, cattle, and humans: the immunology and treatment of river blindness. PLoS Negl Trop Dis 2008; 2: e217. 29 Al-Qaoud KM, Taubert A, Zahner H, Fleischer B & Hoerauf A. Infection of BALB/c mice with the filarial nematode Litomosoides sigmodontis: role of CD4+ T cells in control-
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38 Babayan SA, Luo H, Gray N, Taylor DW & Allen JE. Deletion of parasite immune modulatory sequences combined with immune activating signals enhances vaccine mediated protection against filarial nematodes. PLoS Negl Trop Dis 2012; 6: e1968. 39 Dakshinamoorthy G, Samykutty AK, Munirathinam G, et al. Biochemical characterization and evaluation of a Brugia malayi small heat shock protein as a vaccine against lymphatic filariasis. PLoS ONE 2012; 7: e34077. 40 Dakshinamoorthy G, Samykutty AK, Munirathinam G, Reddy MV & Kalyanasundaram R. Multivalent fusion protein vaccine for lymphatic filariasis. Vaccine 2013; 31: 1616–1622. 41 Joseph SK & Ramaswamy K. Single multivalent vaccination boosted by trickle larval infection confers protection against experimental lymphatic filariasis. Vaccine 2013; 31: 3320–3326. 42 Breloer M, Fleischer B & von Bonin A. In vivo and in vitro activation of T cells after administration of Ag-negative heat shock proteins. J Immunol 1999; 162: 3141–3147. 43 Asea A, Kraeft SK, Kurt-Jones EA, et al. HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med 2000; 6: 435–442. 44 Asea A, Rehli M, Kabingu E, et al. Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like receptor (TLR) 2 and TLR4. J Biol Chem 2002; 277: 15028–15034. 45 Vabulas RM, Ahmad-Nejad P, Ghose S, Kirschning CJ, Issels RD & Wagner H. HSP70 as endogenous stimulus of the Toll/ interleukin-1 receptor signal pathway. J Biol Chem 2002; 277: 15107–15112.
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DOI: 10.1111/pim.12093
Immunization with Brugia malayi Hsp70 protects mice against Litomosoides sigmodontis challenge infection W. HARTMANN,1,* N. SINGH,2,* S. RATHAUR,2 Y. BRENZ,1 E. LIEBAU,3 B. FLEISCHER1,4 & M. BRELOER1 Department of Immunology and Virology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, 2Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, India, 3Institute of Animal Physiology, University of M€ unster, M€ unster, Germany, 4Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 1
SUMMARY More than 15 billion people are at risk of being infected with filarial nematodes worldwide. Therapy and control of transmission are mainly based on mass drug distribution. As these drugs have to be administered annually or biannually and might be loosing their efficacy, a vaccine against filariae is an alternative approach to chemotherapy. In the current study, we have analysed the potential of Brugia malayi heat shock protein 70 (BmHsp70) as a vaccine candidate in a murine helminth infection. Immunization of BALB/c mice with alum-precipitated recombinant BmHsp70 conferred partial protection against subsequent challenge infection with the rodent parasite Litomosoides sigmodontis. Immunization resulted in reduced numbers of larvae in the pleural cavity as well as reduced numbers of circulating microfilariae. Reduced parasite burden was associated with high titres of BmHsp70specific antibodies and increased production of type I and II cytokines in response to L. sigmodontis antigen and BmHsp70. In summary, the immunization with BmHsp70 induced cellular and humoral immune responses and partially protected against L. sigmodontis in a challenge infection. Therefore, we hypothesize that BmHsp70 might be considered as a potential vaccine candidate for reduction in the incidence of B. malayi infections in future studies. Keywords Brugia malayi, heat shock protein 70, immunization, Litomosoides sigmodontis, lymphatic filariasis Correspondence: Wiebke Hartmann, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany (e-mail: [email protected]). *These authors contributed equally to this work. Disclosures: WH: none; NS and SR were funded by the Alexander von Humboldt foundation for carrying out research at the Bernhard Nocht Institute for Tropical Medicine, Hamburg; YB: none; EL: none; BF: none; and MB: none. Received: 12 July 2013 Accepted for publication: 11 December 2013 © 2013 John Wiley & Sons Ltd
INTRODUCTION It is estimated that worldwide more than 150 million people are infected with filarial nematodes such as Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus (1). In humans, these filariae induce chronic infections with high morbidity especially in the tropical and subtropical regions. The life cycle of filarial worms is similar in humans and in different animal models, starting in an arthropod as intermediate host. The first stage larvae, so called microfilariae (MF), are taken up during a blood meal from a mosquito, blackfly or blood-sucking mite. Within the vector, MF undergo two moults and develop into infective thirdstage larvae (L3). L3 are transmitted during a second blood meal to their final host. Here, L3 migrate depending on the species to different sides of the body. In O. volvulus infection, adult worms reside in nodules in the subcutaneous tissue, while adults from W. bancrofti and B. malayi dwell in lymphatic vessels (2). The development of antifilarial vaccines in addition to effective chemotherapy is a prerequisite to combat diseases caused by filarial nematodes. Despite various strategies to control the transmission or to develop drugs, still an estimated 200 million people remain infected. So far, the existing antifilarial drugs predominantly target MF and remain ineffective towards the adult filarial parasites (3). Ivermectin is used annually or biannually in mass drug administrations to control O. volvulus infection, and it requires continuous administration for at least 4–6 years to interrupt transmission (4–7). However, ivermectin does not kill adult worms and emerging resistance (8–11), and serious adverse effects have been reported in patients treated with this drug (12, 13). Therefore, the development of vaccines that would confer at least partial protection and thus reduce parasite burden and transmission is highly desirable. Here, we analyse the capacity of B. malayi-derived heat shock protein (Hsp) 70 to induce a protective immune
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response against challenge infection with Litomosoides sigmodontis as an established murine model of filarial infection (14). Heat shock proteins are a highly conserved group of proteins known to play an important role as molecular chaperones. Under physiological conditions, Hsps are responsible for proper folding and assembly of polypeptides (15). Under stress conditions such as environmental changes in temperature, pH and pressure, Hsp expression increases rapidly (16). Such conditions are found whenever a pathogen enters a warm-blooded host. In particular, during filarial life cycles, the larvae have to cope with a significant rise in temperature upon transmission from the poikilotherm arthropod vector to the homoiotherm mammalian host (17). Generally, most cells respond to an increase in temperature by upregulating the expression of Hsp that are required either for refolding degraded proteins or for targeting them for degradation. Differential expression of Hsp during transfer of L3 from the vector to the mammalian host has been demonstrated in an early study (18). Due to upregulation of heat shock proteins during transmission, an Hsp-specific immune response occurs in several infectious diseases (19–21). Heat shock protein 70 represents a dominant antigen in various helminth infections such as Schistosoma mansoni, O. volvulus, B. malayi and W. bancrofti (22–25). Furthermore, accumulating evidence suggests that Hsp possesses an intrinsic immune-stimulatory capacity (26). The aim of our study was to test the potential of BmHsp70 immunization in a murine model of filarial infection. Infection of mice with L. sigmodontis is a wellcharacterized murine model system, which shares many biological characteristics with human filarial pathogens (27, 28). Infection of BALB/c mice with L. sigmodontis is currently the only murine model where a filarial nematode develops a patent infection, that is, reaches sexual maturity, mates and releases offspring into the circulation. Laboratory mice can be infected naturally by blood-sucking mites or artificially by injection of L3 (14, 29, 30). Following infection, L3 migrate during the first 3 days via the lymphatic system to the thoracic cavity (27). They moult twice and develop within 30 days into juvenile adults. In the permissive BALB/c strain, L. sigmodontis mate and release MF by day 60 post-infection (p.i.) (31). Thereby, infections of BALB/c mice offer a reliable tool to perform immunization studies against filarial nematodes. In our study, we analysed the impact of immunization with B. malayi Hsp70 on the defence against L. sigmodontis in a challenge infection and humoral and cellular immune responses. Our results indicate that BmHsp70 can be harnessed as a possible vaccine candidate for human filariasis in future studies.
Total RNA was isolated from B. malayi adult worms using Trizol reagent. The cDNA was synthesized from B. malayi RNA using oligo (dT) primer from first strand cDNA synthesis kit (Thermo Scientific, Schwerte, Germany). A 1935-bp fragment of BmHsp70 was amplified by PCR from cDNA using sense primers 5′-CGCCTCGAGGATGATGTCAAAGAATGCCATTGGTATCG-3′ and antisense primers 5′-GCGCGATATCCTAATCAACTTCTTC AATTGTTGGT-3′ designed using consensus B. malayi mRNA sequence of BmHsp70 gene (Gene accession no. XM_001900162). The amplification was performed as follows: initial denaturation at 94°C for 2 min, 35 cycles of 94°C for 40 s, 52°C for 30 s and 72°C for 2 min and a final extension of 72°C for 8 min. Following amplification, the PCR products and the expression vector pJC40 (32) were digested using appropriate FastDigestâ restriction enzymes (Thermo Scientific) and ligated using T4 DNA ligase (Invitrogen, Darmstadt, Germany). Five microlitres of each ligation was transformed into XL10-gold ultra competent cells according to the suppliers protocol (Stratagene, La Jolla, CA, USA). Positive clones were identified by test digestion and sequencing. After transformation of the respective expression plasmids into Escherichia coli BL21 (DE) cells (Stratagene), expression of the tagged BmHsp70 was initiated by the addition of 05 mM iso-propyl-beta-D-thiogalactopyranoside (IPTG) once the cultures had reached A600 = 05. Following induction, cells were left to grow for additional 3 h at 37°C. Cells were harvested by centrifugation. The resulting bacterial pellet was resuspended in 50 mM Tris buffer (pH 80) containing 500 mM NaCl and 10 mM imidazole and sonicated using a digital sonifier set to 30 watts and 30% amplitude (Branson, Danbury, CT, USA). After centrifugation (10 000 9 g, 30 min) of the lysate, supernatant was purified by affinity column chromatography with profinityTM IMAC Ni2+-nitrilotriacetic acid resin (Bio-Rad Laboratories, München, Germany). Lysate was washed for 2 h at room temperature with 25 bed volumes of washing buffer (lysis buffer containing 20 mM imidazole). Full-length BmHsp70
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MATERIALS AND METHODS Mice, reagents and antibodies All in vivo experiments were carried out at the animal facility of the Bernhard Nocht Institute for Tropical Medicine with permission of the Federal Health Authorities of the State of Hamburg, Germany. BALB/c mice were obtained from Charles River. Animals were kept in individually ventilated cages.
Cloning, expression and purification of BmHsp70
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was eluted with lysis buffer containing 250 mM imidazole and dialysed in PBS. The purity of the dialysed protein was checked by resolution on 125% SDS-PAGE and Coomassie blue staining. Protein concentration was determined using a Bradford assay (33).
Limulus amebocyte lysate Protein preparations were tested in the Limulus amebocyte lysate (LAL) assay (Lonza, Walkersville, MD, USA) for endotoxin concentrations according to the manufacturers instructions. Endotoxin activity was below 3 EU/lg protein.
Litomosoides sigmodontis cycle and experimental infection The life cycle of L. sigmodontis was maintained in cotton rats (Sigmodon hispidus), the natural reservoir of the nematode (34). Microfilariae were counted weekly in the blood of infected cotton rats. To standardize the infectious dose, cotton rats with 500–1500 MF/lL blood were used for infections of mites. Intermediate host mites (Ornithonyssus bacoti) were permitted to feed on infected cotton rats. Fourteen days after this blood meal, 11-week-old BALB/c mice were infected naturally by exposure to infected mites. For infection, BmHsp70-vaccinated and control mice were placed in the same tank to prevent a bias due to batches of mites. Mice were sacrificed at day 30 and 60 p.i., and the number of worms was counted after flushing the thoracic cavity with 10 mL cold PBS. For counting MF, 50 lL blood was collected every 10 days by puncture of the submandibular vein. Erythrocytes were lysed by addition of 200 lL water followed by centrifugation. The pellet was resuspended in 50 lL gentian violet, and MF were counted.
Preparation of Litomosoides sigmodontis antigen Female worms, isolated from infected BALB/c mice, were transferred into sterile phosphate-buffered saline. Homogenization of live female worms with a glass homogenizer was followed by centrifugation at 16 000 g for 30 min at 4°C. The supernatant was passed through a 022-lm filter and then stored at 80°C until used.
Immunizations
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three times with 50 mL PBS by centrifugation at 300 g for 10 min. Alum-precipitated BmHsp70 and PBS were stored at 4°C until used. Eight-week-old BALB/c mice were immunized by i.p. injection of 100 lg BmHsp70 or PBS precipitated in alum. After 7 days, mice received a boost immunization. Fourteen days later, mice were naturally infected with L. sigmodontis by exposure to infected mites.
In vitro stimulation of lymphocytes Mice were sacrificed at indicated time points p.i., and spleens were prepared. 2 9 105 spleen cells were cultivated in 96-well round-bottom plates for 72 h at 37°C and 5% CO2 in RPMI 1640 medium supplemented with 10% foetal calf serum, L-glutamine (2 mg/mL) and gentamicin (50 lg/mL). For stimulation, cells were incubated either with medium alone or with 1 lg/mL anti-CD3 (145– 2C11), or 20 lg/mL Litomosoides sigmodontis antigen (LsAg), or BmHsp70 in quadruplicates. Supernatants were collected and stored at 20°C until analysis.
ELISA For the detection of L. sigmodontis- or BmHsp70-specific Ig, ELISA plates were coated overnight with 4 lg/mL LsAg or BmHsp70 in carbonate buffer, pH 96. Plates were washed with PBS / 001% Tween 20 and blocked by incubation with PBS 1% bovine serum albumin for 2 h at room temperature. Plates were washed again and then incubated for 2 h with either serial dilutions or a 1 : 100 dilution of the serum. Plates were washed and incubated for 1 h with horseradish peroxidase-labelled anti-mouse IgM, IgG1, IgG2b (Invitrogen) to detect antigen-specific isotypes in the serum. Plates were washed five times and developed by incubation with 100 lL 01 mg/mL tetramethylbenzidine, 0003% H2O2 in 100 mM NaH2PO4 pH 55 for 25 min. Reaction was stopped by addition of 50 lL 1 M H2SO4, and OD450 was measured. Titres were calculated by defining the highest serum dilution in a serial dilution (1 : 100 to 1 : 102 400) resulting in an OD450 above the doubled background that was generally below OD450 = 015. Arbitrary units (a.u.) were calculated by subtracting the OD450 of the background from the OD450 of a fixed serum concentration (1 : 1000 for IgG2b). Cytokines were determined in culture supernatants from spleen cells by indirect sandwich ELISA according to the manufacturers instructions (R&D Systems, Wiesbaden, Germany).
BmHsp70 and PBS were mixed (1 : 2) with 9% alum potassium sulphate dodecahydrate (Sigma-Aldrich, Hamburg, Germany). Antigen precipitation was achieved by adjusting the pH to 70 with 4 M NaOH. Precipitate was washed
All statistical tests were performed by Students t-test or 2way ANOVA with Bonferroni post-test using PRISM software
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(GraphPad Software, San Diego, CA, USA). P values below 005 were considered statistically significant.
RESULTS AND DISCUSSION Immunization with BmHsp70 confers partial protection against Litomosoides sigmodontis challenge infection The aim of this study was to analyse the impact of B. malayi-derived Hsp70 on the immune response against L. sigmodontis. We cloned the full-length Hsp70 from adult B. malayi worms and expressed it as recombinant protein in E. coli. The recombinant protein was purified via its N-terminal histidine tag. Purification was confirmed by SDS-PAGE followed by Coomassie blue staining (Figure 1). As BmHsp70 is a chaperone and might bind bacterial proteins during expression in E. coli, we cannot exclude contamination of the preparation with bacterial proteins. The faint bands in the SDS gel might represent either degraded BmHsp70 or bacterial contaminants. The latter is unlikely as endotoxin levels of BmHsp70 were below 3 EU/lg protein. BALB/c mice were immunized twice with alum-precipitated BmHsp70 or, as control, with alum/PBS. Subsequently mice were naturally infected with L. sigmodontis by exposure to infected mites. Immunization with BmHsp70 reduced parasite burden in the pleural cavity by approximately 50% at day 30 (Figure 2a) and day 60 p.i. (Figure 2b) compared with the control groups immunized with alum only. Although there was a trend for reduced recovery at day 95 p.i. (Figure 2c),
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the differences were not statistically significant at this time point. The numbers of female and male worms were decreased to a similar extent at all time points (data not shown). Numbers of MF in the peripheral circulation were also significantly reduced in BmHsp70-immunized mice between days 60 and 95 p.i. compared with PBS/ alum-treated control mice (Figure 2d). The pronounced reduction in MF could be explained by a number of possibilities: (i) reduction in female worms, (ii) reduced fertility of the remaining female worms, (iii) reduced fertilization of eggs due to reduced numbers of male worms and (iv) enhanced killing of MF.
Immunization with BmHsp70 induces an Hsp70-specific humoral immune response in Litomosoides sigmodontis infected mice
Figure 1 Coomassie blue staining of recombinant BmHsp70. BmHsp70 was expressed in Escherichia coli and purified by affinity chromatography. Following separation by SDS-PAGE, BmHsp70 was stained with Coomassie blue.
To elucidate the underlying mechanism of improved host defence in BmHsp70-treated mice, we compared the immune response of BmHsp70-immunized and PBS control mice. As B cells are required for vaccine-induced protection with irradiated L. sigmodontis larvae (35), we measured BmHsp70-specific and L. sigmodontis-specific antibodies in the serum of infected mice. BmHsp70-specific IgM (Figure 3a, b) and IgG2a (data not shown) were low in PBS/alum- and BmHsp70/alum-treated mice and did not increase between day 30 and 60 p.i. Immunization with BmHsp70 induced BmHsp70-specific IgG1 and IgG2b responses that were already visible at day 30 and slightly increased until day 60 p.i. while control mice failed to generate BmHsp70-specific IgG (Figure 3a, b). Humoral reactivity towards Hsp70 has been described in areas, where lymphatic filariasis is endemic (23, 24). However, in these studies, reactivity of patient sera was analysed by Western blotting that did not distinguish between different isotypes. Our analysis identified the presence of BmHsp70-specific IgM in the serum of infected control mice independent of immunization. In addition, BmHsp70-specific IgM was detectable in the serum from noninfected mice at a low level and increased during infection (data not shown). BmHsp70-specific IgM in infected control mice might represent natural Hsp70specific IgM that cross-reacts with L. sigmodontis-derived Hsp70. In contrast, BmHsp70-specific IgG (IgG1 and IgG2b) were not produced without immunization although the mice had been infected for 60 days. Regarding the L. sigmodontis-specific humoral response, LsAg-specific IgM was detectable by day 30 p.i. while LsAg-specific IgG1, IgG2b (Figure 3c, d) and IgG2a (data not shown) were not detected before day 60 p.i. in infected mice. Thereby, the magnitude of LsAg-specific Ig response was comparable in immunized and
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Figure 2 BmHsp70 vaccination decreases parasite burden. Prior to natural infection with Litomosoides sigmodontis, BALB/c mice were immunized with PBS/alum (white bars) or BmHsp70/alum (black bars). Number of adult worms in the pleural cavity was counted at day 30 p.i. (a), day 60 p.i. (b) and day 95 p.i. (c). (d) Number of microfilariae in the peripheral blood (y-axis) of infected immunized and control mice was counted at the indicated time points (x-axis). (a, b) show combined results of 2 independent experiments (n = 8 for PBS/ alum, n = 9 for BmHsp70). (c, d) represent one experiment with five mice per group. Each dot represents worm numbers from a single mouse (a–c). Results are expressed as mean SEM (d). Asterisks indicate statistically significant differences of the mean (*P < 005, **P < 001). Statistical analysis was performed with Students t-test (a–c) or 2-way ANOVA (d).
nonimmunized mice (Figure 3c, d). Although immunized mice had lower worm burdens during L. sigmodontis challenge infection, this was not associated with increased LsAg-specific humoral responses at day 30 and 60 p.i. In general, immune response in immunized mice might be partially raised against E. coli-derived proteins in our BmHsp70 preparation. We cannot formally exclude immune response against E. coli-derived proteins and cross-reaction with Wolbachia in L. sigmodontis larvae. However, as endotoxin activity of the BmHsp70 preparations was below 3 EU/lg protein and LsAg-specific Ig was comparable in both groups, a major role of bacterial contaminants in the immune response of immunized mice seems to be unlikely. On the other hand, we used a crude extract from adult worms to detect LsAg-specific Ig that might not contain the essential B-cell antigens, for instance, L. sigmodontis-derived Hsp70. In addition, the sensitivity of our ELISA may be too low to detect differences in the L. sigmodontis-specific B cell responses between immunized and control mice. © 2013 John Wiley & Sons Ltd, Parasite Immunology, 36, 141–149
Immunization with BmHsp70 induces antigen-specific production of Th1 and Th2 cytokines As our vaccination with BmHsp70 did not change IgM responses but enhanced T helper cell-dependent IgG responses, we next measured cytokine production by splenic T cells. Spleen cells derived from BmHsp70-immunized and control mice at day 30 and 60 p.i. were restimulated in vitro. We quantified IFN-c (Figure 4a, b) as an indicator of a Th1 immune response, and IL-5 (Figure 4c, d) and IL-13 (Figure 4e, f) as indicators of a Th2 immune response. IL-10 (Figure 4g, h) was measured as a cytokine with regulatory functions. Polyclonal T cell stimulation with anti-CD3 resulted in a comparable cytokine production at day 30 and 60 p.i., revealing no differences between immunized (BmHsp70) and nonimmunized (PBS) mice. Litomosoides sigmodontis antigen-specific cytokine responses were also similar at day 30 p.i in immunized and control mice. A shift towards Th2 cytokine response was visible in spleen cells isolated from
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(a)
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Figure 3 BmHsp70 vaccination increases BmHsp70-specific but not Litomosoides sigmodontis antigen (LsAg)-specific humoral response. BALB/c mice were immunized with PBS (white bars) or BmHsp70 in alum and then infected with L. sigmodontis. BmHsp70-specific (a, b) and L. sigmodontis (c, d) Ig in the serum were measured at day 30 (a, c) or day 60 p.i. (b, d) by ELISA. IgG1 and IgM are shown as titre and IgG2b as arbitrary units. Combined results of two independent experiments (n = 8 for PBS, n = 9 for BmHsp70) are shown. Results are expressed as mean SEM. Asterisks indicate significant differences of the mean (***P < 0001). Statistical analysis was performed with Students t-test.
BmHsp70-immunized mice at day 60 p.i. LsAg-specific IL-5, IL-13 and IL-10 synthesis was higher in spleen cells isolated from immunized mice than in infected control mice (Figure 4d, f, h). We also observed increased BmHsp70-specific IFN-c, IL-5, IL-13, and IL-10 cytokine production in spleen cells derived from BmHsp70-immunized mice at both time points. As immunization with BmHsp70 increased production of Th1-associated IFN-c and Th2-associated IL-5 and
IL-13 as well as IL-10, the immunization obviously did not induce a bias towards type 1 or type 2 immune responses, but enhanced all types of T-cell responses. This was also reflected by the induction of both Th1-associated IgG2 and Th2-associated IgG1 response by BmHsp70 immunization. During L. sigmodontis infection, both Th1 and Th2 cytokines contribute to the parasite control in comparison with other helminthic infections that are predominantly controlled by Th2 cytokine response (36).
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Figure 4 BmHsp70 vaccination increases BmHsp70-specific and Litomosoides sigmodontis antigen (LsAg)-specific cytokine production of spleen cells. BALB/c mice were immunized with PBS (white bars) or BmHsp70 in alum and then infected with L. sigmodontis. Spleen cells were prepared at day 30 (a) and day 60 p.i. and restimulated for 72 h with indicated stimuli (x-axis). Concentrations of IFN-c (a, b), IL-5 (c, d), IL-13 (e, f) and IL-10 (g, h) in the supernatants were measured by ELISA. Results are shown as mean SEM from one individual experiment (n = 5 mice per group) that is representative for two independent experiments. Asterisks indicate significant differences of the mean (*P < 005, **P < 001 and ***P < 0001). Statistical analysis was performed with Students t-test.
Therefore, the enhanced humoral and cellular BmHsp70specific responses might account for increased resistance to parasite infection in BmHsp70-immunized mice. However, the exact mechanism of parasite clearance needs to be further addressed. In two recent studies, different vaccination protocols were analysed to induce protection in a challenge infection
with L. sigmodontis (37, 38). Ziewer et al. (37) administered 1 9 105 viable MF via different routes. Selectively, subcutaneous immunization with MF in alum induced 70– 100% reduction in MF in the peripheral circulation and in the pleural cavity. Here, the number of larvae was unaffected until day 70 p.i. in alum/MF-treated mice (37). In a second study, reduction in L. sigmodontis adults and MF
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was achieved by DNA-based vaccines expressing two immune-modulatory excretory secretory products (ESP), namely abundant larval transcript and cysteine protease inhibitor-2 (38). None of these ESP alone were capable of reducing the parasite burden during a challenge infection with L. sigmodontis. As both ESP had suppressive capacities, additional deletion of immune-modulatory sequences and co-administration of plasmids enhancing antigen presentation was required for improved immune response against the parasite. Although these studies emphasize the difficulty of achieving protection against L. sigmodontis infection by experimental immunization, two doses of alum-precipitated recombinant BmHsp70 were sufficient to significantly reduce the number of L. sigmodontis larvae in the pleural cavity and in the peripheral circulation. The numbers of larvae were already reduced at day 30 p.i. (mean number of worms: 131 27 vs. 316 54), and this phenotype was robust until day 60 p.i. (mean number of worms: 176 3 vs. 31 43). No further reduction in the number of larvae in BmHsp70-immunized mice happened between day 30 and 60 p.i. Thus, the protective capacity of BmHsp70 might occur early during infection, that is, target migrating larvae, L3 or L4 in the pleural cavity. In future studies, protection might be further improved by use of different adjuvants, vaccine doses, as well as routes and time points of administration. In line with our data, three recent studies demonstrate the vaccine potential of small heat shock protein 126 from B. malayi,
either alone or as a multivalent fusion protein in a challenge infection with B. malayi (39–41). Taken together, our study supports the efficacy of developing vaccines based on immune-modulatory molecules or danger signals (38). Heat shock protein 70 seems to be a promising vaccine candidate for the following reasons: (i) Hsp70 is constitutively and abundantly expressed during all developmental stages of nematodes; (ii) Hsp70 expression is further upregulated upon heat shock, which takes place during transmission of L. sigmodontis via the arthropod vector to the mammalian host and; (iii) several lines of evidence suggest that Hsp70 contains an intrinsic immune-stimulatory capacity that induces pro-inflammatory cytokines in professional antigen-presenting cells and enhances T-cell activation (26, 42–45). Due to the partial protection we observed in BmHsp70-immunized mice in a challenge infection with a related rodent parasite, we hypothesize that vaccinations with B. malayi Hsp70 might confer protection against lymphatic filariasis.
ACKNOWLEDGEMENTS NS and SR received funding from Alexander von Humboldt foundation for carrying out the research at Bernhard Nocht Institute for Tropical Medicine, Hamburg. MB, WH, NS, YB conceived and designed the experiments; WH, NS and MB analysed the data; SR, YB, EL, BF contributed essential reagents; WH and MB wrote the manuscript.
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