A Novel Method for Determining the Accessibility of Vaccine Targets in Live Brugia malayi

Alanna Morris
Smith College

Brugia malayi is a nematode parasite that causes elephantiasis in humans. Currently, treatment of this disease involves various drugs that attack the microfilarial stage of the life cycle but are less effective on adult worms. Therefore, a vaccine may provide better protection from infection and symptoms. B. malayi is only infective as a third stage larvae (L3), thus any vaccine will probably be directed at this stage. Several proteins of B. malayi are being studied as potential vaccine targets. Nematodes are protected by a cuticle, which is relatively impermeable to large molecules, thus any vaccine targets probably need to be on the surface of the organism or antibodies will not be able to bind the target protein. Previous work has localized these targets in sectioned organisms, but this work does not show if the targets are accessible in intact organisms. The purpose of this study was to develop protocols for the localization of different proteins on the surface of live B. malayi.

Live B. malayi third stage larvae were obtained from the Filariasis Repository Research Service. While alive, they were labeled with polyclonal antibodies to either thioredoxin peroxidase-2 (TPX-2), collagen 9 or cuticlins (cut) 1-1, 1-2, or 1-3. The genes for these proteins are upregulated in the L3 larvae, and are localized to the cuticle in sectioned preparations. Following treatment with primary antibody, the worms were fixed in 2% paraformaldehyde and labeled with FITC conjutated goat anti-rabbit IgG secondaries. At the excitation wavelength for FITC (488 nm) B. malayi is autoflourescent, emitting between 500 and 560 nm, the same emission wavelength as FITC. Therefore, prior to mounting, the worms were treated with trypan blue, which shifts the emission wavelength of the worm to above 590. Thus, treatment with trypan blue allows the fluorescence of the worm to be distinguished from the fluorescence of the secondary antibody. Controls included treatment with preimmune sera, and secondary antibody only.

TPX-2 and the cuticlins showed similar localization to the cuticle annulae. TPX2 was primarily localized to the mouth, while the cuticlins localized to 5 rings around the anterior believed to be sensory papillae. Antibodies to collagen-9 did not show any labeling, indicating that the protein was not accessible to antibodies in live B. malayi. This novel method will provide vital information about proteins which may potentially be used in vaccine development.