Pattern Recognition Receptor and Vaccination Studies in the Murine Model of Lymphatic Filariasis

Lymphatic filariasis is a parasitic helminth infection that affects approximately 120 million people. About one third of the infected individuals develop pathological manifestations, e.g. lymphedema. Causative agents of lymphatic filariasis are filarial nematodes, which are transmitted during the blood meal of mosquitoes. Filarial worms contain endosymbiotic bacteria of the genus Wolbachia, which are an additional stimulus for the host’s immune system and have been intensively discussed to promote pathology due to an exacerbated proinflammatory response of the host. Currently, the available treatment used for mass treatment against filariasis is based on chemotherapeutic intervention that reduces the burden of the blood circulating larval stages but has only limited effects on adult worms. Despite major attempts to eradicate filarial diseases, elimination has not been achieved. Also, with resistance against the chemotherapy being observed and high cost and logistics efforts of mass drug administration, a vaccine would be a desirable tool towards the elimination of the disease. However, despite intensive research there are no vaccines against any human filarial infection. Pattern recognition receptors (PRRs) are able to sense structures of pathogens, such as viruses or bacteria. In order to investigate, how Wolbachia may induce such proinflammatory immune responses, the PRRs TLR2, TLR4 and NOD2 were investigated, since they are known to sense bacterial structures. In the first part of this thesis in vitro experiments revealed that proinflammatory responses, measured by the secretion of TNF and IL-6, were induced after in vitro stimulation of antigen-presenting cells with filarial Litomosoides sigmodontis extract. In contrast, L. sigmodontis extract devoid of Wolbachia did not induce the secretion of TNF and IL-6. The recognition of Wolbachia was transduced by the heterodimer TLR2/6 (but not TLR1/2 or TLR4) and the integration of the intracellular adapter molecule MyD88 was mandatory. In contrast, the intracellular receptor NOD2, which is known for his interaction with TLR2, was not necessary for this proinflammatory response. In addition to the in vitro experiments, it was of interest, whether mice deficient for these receptors show an altered course of infection to demonstrate the in vivo importance of these PRRs in filarial infections. While the parasite burden after L. sigmodontis infection was similar between TLR-, MyD88-deficient and the corresponding receptor-competent mice, NOD2-deficient mice showed a higher worm load. In addition, the worms recovered from NOD2-deficient mice were shorter and showed an impaired development. Taken together, these data show that despite the induction of proinflammatory responses, TLR-2 and MyD88 do not influence the infection in vivo. In contrast, the higher worm burden observed in NOD2-deficient mice indicates a role for this PRR in the defense against filarial parasites. Further investigations are needed to identify the molecular mechanism behind these observations. In the second part of this thesis a successful vaccination against the L. sigmodontis microfilarial stage was established. The vaccination presented in the present thesis was performed by subcutaneous injection of microfilariae together with adjuvant alum and led to a strongly reduced microfilarial burden in the blood and at the site of infection. Analysis of filarial embryogenesis revealed that the development of microfilariae was already impaired in the uteri of female worms. The vaccination caused a switch from the Th2 arm of immunity, which is well-known for filarial infections, towards a Th1 milieu, indicated by increased IFN-γ and IgG2 in immunized mice. The results of these experiments not only contribute to the understanding of the immune mechanisms needed to develop a vaccine against filarial parasites, but moreover raise hope for the development of a human vaccination against the transmission stage of lymphatic filariasis

Immunization with L. sigmodontis Microfilariae Reduces Peripheral Microfilaraemia after Challenge Infection by Inhibition of Filarial Embryogenesis

Lymphatic filariasis is caused by parasitic filarial worms that are transmitted by mosquitoes, requiring uptake of larvae and distribution into the blood of the host. More than 120 million people are infected and about 30% of these individuals suffer from clinical symptoms. Reduction in transmission currently depends on mass drug administration, which has significantly reduced transmission rates over the past years. However, despite repetitive rounds of administration, transmission has not been eliminated completely from endemic areas. In some infected individuals the immune system can partially control the parasite, such that a proportion of infected individuals remain microfilaria-negative, despite the presence of adult worms. Therefore mechanisms must exist that are able to combat microfilaraemia. Identifying such mechanisms would help to design vaccines against disease transmitting microfilarial stages. Using the Litomosoides sigmodontis murine model of filariasis research we show a successful immunization against the blood-circulating larval stage that is responsible for arthropod-dependent transmission of the disease. Reduced microfilaraemia was associated with impairment of worm embryogenesis, with systemic and local microfilarial-specific host IgG and with IFN-γ secretion by host cells at the site of infection. These results raise hope for developing a microfilariae-based vaccine, being a pivotal step towards eradicating filariasis

Mice immunized with Mf in alum have reduced numbers of Mf.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum. Control mice received alum alone. <i>L. sigmodontis</i> infection was performed one week after the last immunization. Microfilaraemia was monitored twice a week throughout patency. (A) Kinetics of Mf load of sham-treated (dashed line) and immunized (black line) mice in the peripheral blood. One representative of three independent experiments with ten mice per group is shown (2-way ANOVA, mean ± SEM), including both Mf⁻ and Mf⁺ mice. For additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s003" target="_blank">figure S3A</a>, B. (B) Percentage of Mf⁺ mice of three independent experiments was analyzed using Student's t-test. Each mouse with peripheral Mf at any given time point was defined as Mf⁺. (C, D) Mf burden in the pleural space days 70 (C) and 90 (D) p.i.. Graphs show one representative of three (C) and two (D) independent experiments (at least seven mice each group, see also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s003" target="_blank">Figure S3C</a>–E) and were analyzed with Welch-corrected t-test. Numbers below the symbols indicate the number of Mf⁺ mice (median, * <i>P</i><0.05, ** <i>P</i><0.005).</p

Immunization inhibits embryogenesis in female worms.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum. Control mice received alum alone. <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization. Seventy days after infection female worms were analyzed for their embryonic stages. Representative pictures of oocyte (A; micron bar 10 µm), divided egg (B; 10 µm), pretzel stage (C; 15 µm) and stretched Mf (D; 30 µm) are shown. (E) Embryogram illustrating the composition of embryonic stages in female worms. If present, three female worms of each mouse were investigated (27 females in the control group, 28 females from the immunized group, additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s004" target="_blank">Figure S4</a>). Statistical analysis was performed with Mann-Whitney U-test (mean ± SEM, ** <i>P</i><0.01, *** <i>P</i><0.001).</p

Immunization induces Mf-specific IgG1 and IgG2.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum (Al-Mf/naïve, Al-Mf/Inf). Control mice received alum alone (Al/naïve, Al/Inf). <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization (Al/Inf, Al-Mf/Inf) or left uninfected (Al/naïve, Al-Mf/naïve). Plasma levels of Mf-specific IgG1 (A) and IgG2a/b (B) were measured. Two-way ANOVA was used for statistical analysis, day 0 indicates day of challenge infection. Asterisks indicate significant differences between the immunized and infected, and the corresponding control group (*** <i>P</i><0.001) and pound signs between the immunized but uninfected, and the corresponding control group (^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001). (C–F) Pleural space lavage was analyzed for specific IgG1 and IgG2a/b on days 22 (C, D) and 70 p.i. (E, F). Data analyzed with Welch-corrected t-test (mean, *** <i>P</i><0.001). Graphs show representatives of three independent experiments with eight to ten mice each group (additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s006" target="_blank">Figure S6A, B, E–J</a>).</p

Immunization reduces adult worm burden, but does not affect their development.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum. Control mice received alum alone. <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization. Numbers of worms on days 15 (A), 56 (B), 70 (C) and 90 (D) p.i. (additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s005" target="_blank">Figure S5A</a>–C), gender balance (E) (individual experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s005" target="_blank">Figure S5D</a>, E), as well as length of males (F) and females (G) at day 90 p.i. (10/90 percentile, outliers are indicated, individual experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s005" target="_blank">Figure S5F</a>–I) were analyzed with Student's t-test (** <i>P</i><0.01, *** <i>P</i><0.001).</p

Immunization strategies that failed to protect mice from peripheral microfilaraemia.

<p>Mice were immunized with 100,000 Mf either three times i.v. (A, B) or first s.c. followed by an i.p. and i.v. immunization (C, D). All control mice received PBS. <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization. (B) After immunization mice were treated i.v. with IVM. (D) Mice were immunized with irradiated (400 Gy) Mf. Microfilaraemia was monitored throughout patency. Data obtained from single experiments with at least six mice per group are shown. Two-way ANOVA (mean ± SEM) was used for statistical analysis including both Mf⁻ and Mf⁺ mice.</p

Immunization enhances IFN-γ responses.

<p>(A) At day 22 p.i. the pleural lavage was analyzed for IL-5 and IFN-γ. Combined data of three independent experiments with five mice each group are shown. (B, C) At day 22 p.i. cells from the site of infection were restimulated for 72 h with 5 µg/ml Concanavalin A (ConA), 100 µg/ml complete adult (Ls) or microfilarial (Mf) crude extract of <i>L. sigmodontis</i> and IFN-γ (B) and IL-5 (C) secretion were measured (mean ± SEM). Representative data of two independent experiments with five mice each group. Analysis was done using the 2-way ANOVA, for significances see text.</p