Comparative analysis of glycosylated and nonglycosylated filarial homologues of the 20-kilodalton retinol binding protein from Onchocerca volvulus (Ov20)

Abstract

Ov20 is a structurally novel 20-kDa retinol binding protein secreted by Onchocerca volvulus. Immunological and biological investigation of this protein has been hampered by the inability to maintain O. volvulus in a laboratory setting. In an effort to find a system more amenable to laboratory investigation, we have cloned, sequenced, and expressed cDNA encoding homologues of Ov20 from two closely related filarial species, Brugia malayi (Bm20) and Acanthocheilonema viteae (Av20). Sequence comparisons have highlighted differences in glycosylation of the homologues. We present here an analysis of mouse immune responses to Ov20, Bm20, and Av20. The results suggest a strong genetic restriction in response to native Bm20 that is overcome when recombinant, nonnative material is used. Reactivity of human filarial sera to the three recombinant proteins confirmed previous specificity studies with Ov20 but highlighted important differences in the reactivity patterns of the O. volvulus and B. malayi homologues that may be due to differences in glycosylation patterns. Ov20 is a dominant antigen in infected individuals, while Bm20 is not. The availability of the B. malayi homologue enabled us to use defined murine reagents and inbred strains for genetic analysis of responsiveness in a way that is not possible for Ov20. However, the close sequence similarity between Ov20 and Av20 suggests that the A. viteae model may be more suited to the investigation of the biological functions of Ov20.

FIG. 1

Alignment of the predicted amino acid sequences of the retinol binding proteins Bm20, Ov20, and Av20. N-linked glycosylation sites are underlined, and areas of homology among the three recombinants are indicated (∗). The fragment representing Ov11 is highlighted.

FIG. 2

Reactivity of BALB/c, BALB.k, C57BL/6, and CBA sera from mice implanted intraperitoneally with live adult B. malayi. Plates were coated with MBP, Bm20, and B. malayi extract (Bma). The y axis represents optical density at 405 nm. Dotted bars, control unimplanted animals; solid bars, parasite-exposed animals. Standard deviations represent two to four animals in control groups and three to five animals in implanted group.

FIG. 3

Reactivity of sera from C57BL/6 mice implanted intraperitoneally with B. malayi. Plates were coated with recombinant Bm20, Ov20, and Av20. Standard deviations represent four to six animals in each group. C57cont and C57imp, control and implanted C57BL/6 mouse sera; O.D., optical density.

FIG. 4

Reactivity of sera from mice immunized with recombinant Bm20, Ov20, and Ov11. Plates were coated with recombinant Bm20, Av20, and Ov20. Standard deviations represent four to six animals in each group. C57αBm20, BALBαBm20, and B10αBm20, sera from C57BL/6, BALB/c, and B10.D2 mice immunized with Bm20; αOv20, sera from mice immunized with Ov20; αOv11, sera from mice immunized with Ov11; BALBnms and B10nms, normal serum controls from BALB/c and B10.D2 mice; O. D., optical density.

FIG. 5

Reactivity of human filarial (W. bancrofti and B. malayi) and onchocerciasis sera to the recombinant proteins Bm20, Ov20, and Av20. Standard deviations represent 35 samples in the onchocerciasis (Oncho) serum group, 18 samples in the Brugia serum group, 22 samples in the Wuchereria (Wuch) serum group and 7 to 8 samples in the control (uninfected) group. O. D., optical density.