Brossard, Michel

2008, Decrem, Yves, Beaufays, Jérôme, Blasioli, Virginie, Brossard, Michel, Vanhamme, Luc, Godfroid, Edmond

Ticks are obligate blood-feeding arachnids. During their long-lasting blood meal, they have to counteract the protective barriers and defense mechanisms of their host. These include tissue integrity, pain, hemostasis, and the inflammatory and immune reactions. Here, we describe a multigene family coding for five putative salivary metalloproteases induced during the blood meal of Ixodes ricinus. The evolutionary divergence inside the family was driven by positive Darwinian selection. This came together with individual variation of expression, functional heterogeneity, and antigenic diversification. Inhibition of the expression of some of these genes by RNA interference prevented completion of the tick blood meal and affected the ability of the tick saliva to interfere with host fibrinolysis. This family of proteins could therefore participate in the inhibition of wound healing after the tick bite, thereby facilitating the completion of the blood meal.

2007, Prevot, Pierre-Paul, Couvreur, B., Denis, V., Brossard, Michel, Vanhamme, L., Godfroid, E.

Iris is a specific elastase inhibitor expressed in the salivary glands of the hard tick Ixodes ricinus. It belongs to the superfamily of serpins and interferes with both haemostasis and the immune response of the host. In this study, we first show that Iris is expressed in nymphs but not in the female midgut nor in males. We also show that Iris is present in the saliva. To examine its potency as anti-tick vaccine candidate, we set up three models of I. ricinus infestation on immunized animals: nymphs on mice, and adults and nymphs on rabbits. We report the rise of neutralizing antibodies following immunization of rabbits and mice. This comes with a significant protective immunity against ticks in rabbits only, resulting in a 30% mortality rate and a diminution of weight gain in both nymphs and adults and a prolongation of blood feeding time in adults. This is the first report on an anti-tick vaccine trial on I. ricinus using a protein able to interact with both host immunity and haemostasis, as a vaccinating antigen.

1981, Brossard, Michel, Monneron, J.-P., Papatheodorou, V.

The sensitivity of rabbit basophils to antigens from Ixodes ricinus females has been studied by a degranulation test. Observations of basophil numbers and degranulation were made on the 6th day of each of four sequential infestations. Maximal degranulation of cells was observed after challenge of cells with antigen at a concentration of 10⁶ and 10⁷ pg/ml. At these concentrations, during a 1st infestation, 21.8 and 23.6% of cells degranulated. During a 2nd infestation, these percentages increased (34.8 and 33.8%) and reached 59.8 and 63.8% by the 4th infestation. A plasma factor which partially blocks basophil degranulation, is described. This was already present during the 1st infestation, since in its presence the percentage of degranulation was reduced by 2.8 and 4.0% respectively on challenge with 10⁶ and 10⁷ pg antigen/ml. Inhibition was maximal at the 4th infestation (difference: 16.5 and 20.5%). Basophil sensitization and inhibition of the degranulation are thus both progressive phenomena. After 10–15 infestations on four other rabbits, 75. 0 and 79. 8% degranulation was obtained. The inhibition of degranulation by plasma was also greater (difference: 25. 5 and 27. 4%). IgG specific anti-I. ricinus antibodies were identified by indirect immunofluorescence. In two animals, they were detected at the 6th day of the 1st infestation. Subsequently, they were generally present for all the animals.

2008, Decrem, Yves, Mariller, Marcel, Lahaye, Kathia, Blasioli, Virginie, Beaufays, Jérôme, Boudjeltia, Karim Zouaoui, Vanhaeverbeek, Michel, Cérutti, Martine, Brossard, Michel, Vanhamme, Luc, Godfroid, Edmond

Two cDNAs coding homologous putative metalloproteases (Metis 1 and Metis 2, expected molecular weights of 55.6 and 56.0 kDa, respectively) were identified from the hard tick Ixodes ricinus. The expression of Metis genes was induced in salivary glands during tick blood meal. RNA interference was used to assess the role of both Metis 1 and Metis 2 in tick feeding. It was found that salivary gland extracts lacking Metis 1–2 had a restricted ability to interfere with fibrinolysis. RNAi against Metis 1–2 also induced a high mortality rate. An immune reaction was raised in repeatedly bitten animals against Metis 1 and 2. Vaccination of hosts with the recombinant Metis 1 protein produced in a eukaryotic system partially interfered with completion of the blood meal. Although vaccination did not alter the survival rate or feeding time of ticks, their weight gain and oviposition rate were reduced. This will affect their reproductive fitness in the field. We believe this is the first report of an anti-tick vaccine trial using a metalloprotease derived from I. ricinus.

2001, Mejri, Naceur, Franscini, Nicola, Rutti, Bernard, Brossard, Michel

BALB/c mice were infested with Ixodes ricinus larvae, nymphs or adults. Expression of IL-4 and IFN-γ mRNA in axillary and brachial draining lymph node cells were measured by competitive quantitative reverse transcription-polymerase chain reaction 9 days after the beginning of primary-infestation. IL-4 mRNA was always higher than that of IFN-γ mRNA for all tick instars. Moreover, IL-4 mRNA expression progressively increased during nymphal primary-infestation with a high burst of expression 7 days after the beginning of infestation. No evolution of IFN-γ mRNA expression was detected. Draining lymph node cells of infested BALB/c produced higher level of IL-4 than IFN-γ following in vitro restimulation with adult tick saliva, salivary gland extract (SGE) or with five selected different chromatographic fractions of SGE. Anti-tick IgG1 antibodies but no IgG2a were detected in BALB/c pluri-infested with I. ricinus nymphs, which confirmed the Th2 polarization of the immune response.

2007, Mejri, Naceur, Brossard, Michel

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) in priming naive T cells. Using an in vitro priming system, we show that DCs incubated with Ixodes ricinus tick saliva initiate the T_h2 differentiation of CD4⁺T cells. As determined with reverse transcription–PCR, the expression of IL-4 mRNA by these cells is higher than IFN-γ mRNA. Early endogenous production of IL-4 is thought to be important during the in vitro interaction of saliva-pulsed DCs with CD4⁺T cells. Its neutralization with specific mAbs inhibits the development of IL-4-secreting T_h2 cells. Moreover, differentiated T_h2 cells proliferate only when saliva-pulsed DCs and IL-1ß are added together early in the primary culture. As demonstrated by FACS analysis, the treatment in vitro of saliva-pulsed DCs by IL-1ß enhanced the expression of B7 and mainly CD40 co-stimulatory molecules, which provide sufficient signals to stimulate sensitized CD4⁺T cell proliferation. On the other hand, DCs treated with tick saliva only up-regulated mostly B7-2 co-stimulator expression and this was associated with differentiation of naive CD4⁺T cells into T_h2 type of cells. The in vitro priming system is suitable to investigate the major elements implicated in the anti-tick immune response such as naive CD4⁺T cells, whole DCs population and tick saliva, and it can provide the possibility to delimit further the saliva molecules, the DC subsets and the type of host cells involved in the T_h2 polarization. Corresponding in vivo experiments involving subcutaneous injection of tick saliva-pulsed DCs into BALB/c mice also elicited a T_h2 immune response. Ex vivo cultures of draining lymph node T cells stimulated with tick saliva produced higher IL-4 : IFN-γ ratios compared with controls, confirming the relevance obtained in the in vitro priming model. These experiments demonstrate the importance of tick saliva in priming DCs to initiate a T_h2-biased immune response in vitro and in vivo.

1997, Ganapamo, F., Rutti, Bernard, Brossard, Michel

BALB/c mice infested with larvae or nymphs of Ixodes ricinus develop in their lymph nodes a T cell-specific immune response triggered by salivary gland soluble antigens (SGA). SGA are apparently conserved in the 3 biological stages of I. ricinus ticks and are species specific. SGA derived from partially fed females I. ricinus stimulate lymph node T cells from mice infested with I. ricinus larvae or nymphs. In contrast, lymph node cells from mice infested with Amblyomma hebraeum nymphs do not respond. A chromatographic fraction enriched with a 65 kDa protein (IrSG65) isolated from salivary glands of I. ricinus partially fed females induces in vitro a specific T cell proliferation of lymph node cells from mice infested with I. ricinus nymphs. The depletion of CD4+ T cells drastically reduces the ability of lymphocytes from infested mice to proliferate after IrSG65 stimulation.