A murine macrofilaricide pre-clinical screening model for onchocerciasis and lymphatic filariasis

Abstract

BackgroundNew drugs effective against adult filariae (macrofilaricides) would accelerate the elimination of lymphatic filariasis and onchocerciasis. Anti-Onchocerca drug development is hampered by the lack of a facile model. We postulated that SCID mice could be developed as a fmacrofilaricide screening model.MethodsThe filaricides: albendazole (ABZ), diethylcarbamazine (DEC), flubendazole (FBZ), ivermectin (IVM) and the anti-Wolbachia macrofilaricide, minocycline (MIN) were tested in Brugia malayi (Bm)-parasitized BALB/c SCID mice vs vehicle control (VC). Responses were compared to BALB/c wild type (WT). Onchocerca ochengi male worms or onchocercomata were surgically implanted into BALB/c SCID, CB.17 SCID, BALB/c WT mice or Meriones gerbils. Survival was evaluated at 7–15 days. BALB/c SCID were tested to evaluate the responsiveness of pre-clinical macrofilaricides FBZ and rifapentine (RIFAP) against male Onchocerca.ResultsWT and SCID responded with >95% efficacy following ABZ or DEC treatments against Bm larvae (P < 0.0001). IVM was partially filaricidal against Bm larvae in WT and SCID (WT; 39.8%, P = 0.0356 and SCID; 56.7%, P = 0.026). SCID responded similarly to WT following IVM treatment of microfilaraemias (WT; 79%, P = 0.0194. SCID; 76%, P = 0.0473). FBZ induced a total macrofilaricidal response against adult Bm in WT and SCID (WT; P = 0.0067, SCID; P = 0.0071). MIN induced a >90% reduction in Bm Wolbachia burdens (P < 0.0001) and a blockade of microfilarial release (P = 0.0215) in SCID. Male Onchocerca survival was significantly higher in SCID vs WT mice, but not gerbils, after +15 days (60% vs 22% vs 39% P = 0.0475). Onchocercoma implants had engrafted into host tissues, with evidence of neovascularisation, after +7 days and yielded viable macro/microfilariae ex vivo. FBZ induced a macrofilaricidal effect in Onchocerca male implanted SCID at +5 weeks (FBZ; 1.67% vs VC; 43.81%, P = 0.0089). Wolbachia loads within male Onchocerca were reduced by 99% in implanted SCID receiving RIFAP for +2 weeks.ConclusionsWe have developed a ‘pan-filarial’ small animal research model that is sufficiently robust, with adequate capacity and throughput, to screen existing and future pre-clinical candidate macrofilaricides. Pilot data suggests a murine onchocercoma xenograft model is achievable.

Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis

“…Also, in a pilot study using the related, registered rifamycin, rifapentine, >90% depletion of Wolbachia was measured in O. ochengi males implanted into SCID mice, following two weeks 25 mg/kg bid dosing 56 . However, clinical studies in humans have shown that rifampicin administered for two or four weeks has inferior activity to six-week doxycycline in onchocerciasis 69,70 and neither is combining rifampicin and doxycycline for two weeks sufficient to achieve significant macrofilaricidal activity 54 .…”

“…Between 10–11 male Onchocerca were surgically implanted into the peritoneal cavity of CB.17(BALB/c) SCID mice under anaesthesia as described previously 56 .…”

Comparison of immune responses to Loa loa stage-specific antigen extracts in Loa loa-exposed BALB/c mice upon clearance of infection

“…Thus, to begin deciphering host immunity against L. loa, we analysed immune responses in immune-competent BALB/c mice upon exposure to different L. loa developmental stages. Previously, it was shown that L. loa cannot be maintained in wildtype BALB/c mice probably due to type-2 associated immunity [23,36] that clears L. loa larvae after 1 week [24]. Indeed, we did not isolate any L. loa developmental stages 42 days pi which was reflected in the relatively comparable levels of systemic cytokine, chemokine and immunoglobulins between the infected groups.…”

“…A major reason for the limited understanding of loiasis-associated immune responses and clinical picture [29,30] is the lack of appropriate infection models. Besides the established rodent models of Brugia malayi and Onchocerca volvulus [23,[31][32][33], we recently showed that BALB/c with impaired IL-4, IL-5 and IL-13 signalling as well as a lymphopenic γc-deficient strain allow development of L. loa life stages [22,34]. This was also reflected in our studies with the rodent Litomosoides sigmodontis model since in IL-4Rα/IL-5 −/− BALB/c mice, worm burden and MF counts were significantly higher than in wildtype BALB/c control groups [35], suggesting that principally mice lacking Th2 responses provide a better environment for worm growth.…”

“…Simultaneously, groups of female and male wildtype BALB/c mice were exposed to either: (i) a s.c. injection of 500 L3 in 100 µl RPMI-1640 medium isolated from Chrysops flies; (ii) 10,000 MF via the tail vein in 100 µl RPMI-1640 medium isolated from human peripheral blood [22], or (iii) 10 L4; (iv) 10 L5; and (v) 10 adult worms all isolated from infected-BALB/cRAG2γc −/− mice. Whereas L4 were injected s.c. in 100 µl of RPMI-medium, L5 and adult stages were implanted as previously described [23]. In short, mice were anesthetized with ketamine (Ketaset, 70 mg/kg; Zoetis, Parsippany-Troy Hills Township, New Jersey, USA) and medetomidine (Domitor, 0.8 mg/ kg; Zoetis), flanks were shaved, dosed with betadine and following a small incision, L5 or adult worms were implanted into the mice.…”