Onchocerca volvulus glycolytic enzyme fructose-1,6-bisphosphate aldolase as a target for a protective immune response in humans

Abstract

To identify potential vaccine candidates for the prevention of infection with the filarial nematode Onchocerca volvulus, we screened an O. volvulus L3 stage cDNA library with sera from putatively immune (PI) subjects, and a prominent immunogenic clone of 1,184 nucleotides was identified. It contained an open reading frame of 363 amino acids encoding the glycolytic enzyme fructose 1,6 bisphosphate aldolase (Ov-fba-1). Immunolocalization experiments demonstrated that the protein was most abundantly expressed in metabolically active tissues, including body wall muscle and the reproductive tract of adult female worms. Immunoelectron microscopy of L3 demonstrated binding in the region where the cuticle separates during molting, in the channels connecting the esophagus to the cuticle, and in the basal lamina surrounding the esophagus and the body cavity. Among subjects from areas where this organism is endemic specific humoral and cellular immune responses to recombinant protein were observed in both PI and infected subjects, whereas responses were not observed among subjects who had not been exposed to O. volvulus. Despite the absence of differential responsiveness in parasite-exposed human populations, when the recombinant was tested for protective efficacy in a mouse chamber model, a reduction in survival of larvae by ca. 50% was seen. This observation provides support for the further study of this parasite enzyme as a vaccine candidate in larger animal models.

FIG. 1.

(A) CLUSTAL IV multiple sequence alignment of deduced amino acid sequence of aldolase from O. volvulus, human aldolase A (pdb1ALD), Drosophila (pirADFF), C. elegans (spP54216), S. mansoni (spP53442), and P. falciparum (gbM28881). The amino acid residues associated with enzymatic activity (Asp³³, Lys⁴¹, Lys⁴², Lys¹⁴⁶, Arg¹⁴⁸, Lys²²⁹, His³⁶¹, and the terminal Tyr) are highlighted. The sequence divergence at residue 42 from other eukaryotes (where Lys residue replaced the highly conserved Arg) is underlined. Amino acid residues where significant sequence diversity exists between human and O. volvulus and where the structural study indicates that these residues are located on the external surface of the protein are shaded. (B) Projection of three-dimensional structure of human aldolase. The amino acid residues at positions 238 to 244 and positions 345 to 350, where there is significant sequence divergence between the O. volvulus and human sequences, are highlighted.

FIG. 2.

(A) Genomic DNA from filarial parasites, the free-living nematode C. elegans, arthropod vectors of filariasis, and humans was subjected to dot blot analysis with a labeled O. volvulus Ov-fba-1 DNA probe. (B) Northern blot analysis of the total RNA from O. volvulus (lane 1) and human leukocytes (lane 2) with a labeled Ov-fba-1 DNA probe.

FIG. 3.

(A) Immunoblot analysis using polyclonal murine antisera to purified recombinant (lanes 1 and 2) Ov-FBA-1 or control antisera from adjuvant-immunized mice (lanes 3 and 4). Lanes 1 and 3, O. volvulus adult antigen; lanes 2 and 4, purified recombinant aldolase. (B) Immunblot analysis of antibody response to Ov-FBA-1 among human subjects. Lanes 1 to 4, control unexposed subjects; lanes 5 to 8, O. volvulus-infected subjects; lanes 9 to 12, PI subjects.

FIG. 4.

(A) Immunohistochemical localization of Ov-FBA-1. Paraffin sections of an adult female O. volvulus were incubated with antibodies prepared against Ov-FBA-1 and binding detected with horseradish peroxidase-conjugated secondary antibodies. (a and b) Transverse section showing the presence of early morula-stage microfilariae inside the uterus of the female worm. (c and d) Similar transverse sections showing late morula-stage microfilariae. Positive staining was localized to the musculature of the body wall, the ovarian/uterine wall, the esophageal wall and in the late-morula-stage, developing microfilariae. Panels a and c are adjacent serial sections of panels b and d and represent controls with sera from adjuvant-immunized animals.

FIG. 5.

Ultrastructural localization by immunoelectron microscopy of the parasite protein recognized by rabbit antibodies against recombinant Ov-FBA-1. Thin sections of O. volvulus larvae during molting of L3 to L4 in vitro were incubated first with antibodies raised against recombinant aldolase and then with protein A coupled to 15-nm gold particles for indirect antigen localization (bar = 0.5 mm). Note the regions where the cuticles of L3 (arrowheads) and L4 (arrows) separate (A), the channels (small arrowheads) connecting the esophagus (Eo) to the cuticle (B), and the basal lamina (open arrowheads) surrounding the esophagus and the body cavity (C).

FIG. 6.

Ov-FBA-1 vaccination studies. Male mice (3 to 6 weeks old; six animals per group) were vaccinated with Ov-FBA-1 or with adjuvant alone and implanted with diffusion chambers containing 25 O. volvulus L3. Chambers were recovered 21 days later, and larval survival was assessed.