Development of a long-term Brugia malayi lymphatic endothelial cell co-culture system and its validation as an alternative to in vivo screening for anti-Wolbachia drug assessment

Tue10  Apr04:45pm(15 mins)
Where:
Stream 3 - Physics 0.15 Main

Authors

A E Marriott1; J Archer1; A Steven1; M J Taylor1; J D Turner1
1 Liverpool School of Tropical Medicine, UK

Discussion

Filarial parasites are the causative agents of lymphatic filariasis (LF) and onchocerciasis. Current elimination programmes rely on up to 12 annual mass drug administrations with standard anti-filarial drugs which target the microfilarial (mf) stage of infection. There is therefore a need to identify new short-course curative drugs (macrofilaricides) in order to accelerate elimination time frames. A promising drug target is the filarial endosymbiotic bacteria, Wolbachia, which can be depleted with antibiotics to initiate blockade of embryogenesis, permanent sterilisation and slow, safe killing of adult parasites.

Reproductively active adult female parasites have a limited life-span in culturetherefore filarial drug screens assessing both Wolbachia depletion and blockade of embryogenesis rely on in vivo models. Here, we have developed a long term in vitro co-culture system to maintain adult female stages of the human lymphatic filariae, Brugia malayi, using primary human lymphatic endothelial cells (LEC).

We examined motility and survival (n≤12), uterine release (n≤6), metabolic activity (n≤10) and Wolbachia titres (n≤10) of 3-6 month old mature female B. malayi following aseptic isolation from infected immunodeficient mice and cultured over periods up to 28 days. Readouts were compared against freshly isolated ex vivo B. malayi or human embryonic kidney (HEK) cell monolayer co-cultures as non-specific feeder cells. When cultured on LEC monolayers, female B. malayi retained full motility on average for 14 days. This compared with 8 days for corresponding cell-free LEC culture medium or HEK monolayers. Survival reduced below 80% at 22 days in LEC co-cultures compared with 16 days for LEC culture medium or HEK monolayers. Uterine release of motile mf was sustained till day 10 in LEC co-cultures compared with 5 days for LEC culture medium. Metabolic activity was not statistically different in 14 day LEC co-cultured B. malayi compared with ex vivo worms, whereas 14 day cell-free LEC culture medium or HEK monolayer cultured worms showed significant (P<0.0098) metabolic decline. Wolbachia titres remained similar in 14 day LEC co-cultured B. malayi compared with ex vivo controls. Having verified LEC co-cultures stably supported B. malayi and Wolbachia viability over 14 days, we assessed Wolbachia reductions and dynamics after treatment with physiologically relevant doxycycline exposures. Doxycycline induced significant (P<0.0001) 79-84% reductions in Wolbachia after continuous treatment for 7 or 14 days whilst drug removal after 7 days led to non-significant 54% reduction after 7 days washout. Thus, we have robustly validated an in vitro co-culture system which may be used to examine anti-Wolbachia agents for superiority compared with doxycycl

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