Development of a recombinant antigen vaccine against infection with the filarial worm Onchocerca volvulus

Abstract

Efforts to control Onchocerca volvulus, the etiologic agent of river blindness, have been limited to vector control and drug treatment to eliminate microfilariae, with no means available to prevent infection. The goal of this study was to develop a vaccine against this infection using recombinant antigens that are expressed in the early larval stages of the parasite. Five recombinant antigens, Ov7, Ov64, OvB8, Ov9M, and Ov73k, were identified by screening adult and larval cDNA libraries with antibodies from immune humans, chimpanzees, or rabbits. When mice were immunized with the five individual recombinant antigens, statistically significant reductions in parasite survival were induced in mice immunized with Ov7, OvB8, or Ov64, when administered in alum but not when injected in Freund's complete adjuvant (FCA). Live larvae recovered from control and immunized mice were analyzed to determine their developmental stages. A decrease in the percentage of larvae molting from the third stage to the fourth stage was observed with mice immunized with Ov7, Ov64, or OvB8 in alum but not with mice immunized with Ov9 and Ov73k or with mice immunized with any of the five antigens in FCA. Mice immunized with a cocktail of the three protective antigens developed protective immunity equal to that seen with mice immunized with individual antigens. This study has identified, for the first time, three recombinant antigens capable of inducing protective immunity to O. volvulus. Furthermore, since the antigens functioned with alum as the adjuvant, this vaccine could potentially be used clinically to prevent river blindness in humans.

FIG. 1

Ultrastructural localization by immunoelectron microscopy of parasite proteins recognized by antibodies from mice that developed protective immunity after immunization with irradiated L3. Thin sections of O. volvulus larvae during molting of L3 to L4 in vitro were incubated first with mouse antibodies and then with rabbit anti-mouse Ig antibodies followed by protein A coupled to 15-nm gold particles for indirect antigen localization (bars, 0.5 nm). Note the labeling in the regions where the cuticles of L3 (arrowheads) and L4 (arrows) separate (A), the channels (small arrowheads) connecting the esophagus to the cuticle (B), and the basal lamina (open arrowheads) surrounding the esophagus and the body cavity (C). Labeling was not observed in sections incubated in normal mouse serum (D).

FIG. 2

Ultrastructural localization by immunoelectron microscopy of the parasite proteins corresponding to Ov7, Ov64, OvB8, Ov73k, and Ov9M recombinant proteins. Thin sections of O. volvulus larvae during molting of L3 to L4 in vitro were incubated first with antibodies raised against GST-Ov7 (A), GST-Ov64 (B), GST-OvB8 (C), GST-Ov73k (D), and GST-Ov9M (E) fusion polypeptides and then with protein A coupled to 15-nm gold particles for indirect antigen localization (bars, 0.5 nm). Note the labeling in the areas of the L4 epicuticle-cuticle (small arrows, panels A and D) and the basal layer of the L3 cuticle (arrowheads, panels A and D) where the separation between cuticles takes place, the cuticle of L3 day 1 (B), channels connecting the esophagus (Eo) and the cuticle (small arrowheads, panels A to D), the basal lamina (open arrowheads) surrounding the esophagus and the body cavity (C and D), and muscles (Mu) (E). Serum raised against GST did not ross-react with any proteins in the larvae (data not shown).

FIG. 3

IgG1, IgE, and IgG2a antibody responses of mice immunized with Ov7, Ov64, OvB8, Ov9M, or Ov73k with either alum or FCA. The mean responses measured for control mice were subtracted from the responses measured for individual immunized mice. Values presented are the means and standard deviations of the measurements from the immunized mice after subtraction of the control values. Asterisks represent significant differences between responses seen for mice immunized with alum and those for mice immunized with FCA.

FIG. 4

IgG1 and IgE antibody responses of mice immunized with Ov7, Ov64, and OvB8, with alum injected individually or as a cocktail of three antigens. The mean responses measured for control mice were subtracted from the responses measured for individual immunized mice. Values presented are the means and standard deviations of the measurements from the immunized mice after subtraction of the control values. Asterisks represent significant differences between responses seen for mice immunized with single antigens and those for mice immunized with three antigens.