(Received for publication, June 30, 1995; and in revised form, September 11, 1995)
From the
Protective immunity against human onchocerciasis may best be reflected by the existence of individuals who in spite of exposure to the filarial nematode Onchocerca volvulus do not develop disease (putatively immune). We observed preferential recognition of an O. volvulus antigen of approximately 90 kDa by sera from putatively immune individuals compared with sera from diseased individuals. Screening of an adult worm cDNA library with one serum recognizing this antigen almost exclusively led to the identification of a full length clone of 2043 base pairs designated E1. The open reading frame of 462 amino acid residues shows similarity to human brain ankyrin. E1 appears to represent a small transcript of the O. volvulus ankyrin gene. The nonfusion protein obtained by expression of the complete E1 cDNA exhibits an apparent molecular mass of 90 kDa on SDS-polyacrylamide gel electrophoresis. An antiserum against the recombinant protein reacts with the 90-kDa antigen in O. volvulus extract. In O. volvulus, E1 was localized in the neuronal cell bodies, the nerve ring, and the extracellular clefts of the basal labyrinth. These results identify an ankyrin-related O. volvulus protein as an immunogen to putatively immune individuals, suggesting that neuronal proteins may be important targets for immunity against O. volvulus in vivo.
Human onchocerciasis, a chronic disease caused by infection with the filarial nematode Onchocerca volvulus, affects millions of people in Africa and Latin America(1) . The debilitating dermal and ocular complications led to major efforts toward disease control including the search for a vaccine.
Evidence for protective immunity
against O. volvulus is based on observations that in endemic
areas a group of exposed individuals exists that in contrast to the
majority of the exposed population did not develop
onchocerciasis(2, 3) . These putatively immune
individuals (PI) ()appear to mount a more effective immune
response against O. volvulus(2) than diseased
individuals(4) , which may result in successful elimination of
the parasite. Preferential antibody recognition of onchocercal antigens
by PI sera compared with sera from diseased individuals has been
reported(5) ; however such antigens, which may be potentially
protective, remain poorly defined. Recently, one such antigen from the
filarial parasite Wuchereria bancrofti(6) was cloned
and shown to encode for a chitinase-like protein(7) . Here we
report the cloning and characterization of an O. volvulus antigen identified using sera from individuals putatively immune
to O. volvulus, which is associated with the nervous system of
the worm.
Figure 1: A, immunoblot of O. volvulus antigen with serum pools from GEN (lane 1), SOW (lane 2), and PI (lane 3). B, immunoblot of O. volvulus antigen with serum from a PI. The arrows indicate position of the 90-kDa band.
Figure 2:
A, Northern blot of adult O. volvulus RNA hybridized with a P-labeled E1 probe. B,
Southern blot analysis of restriction enzyme-digested O. volvulus genomic DNA. Size-separated DNA digested with HindIII (lane 1) and EcoRI (lane 2) was hybridized
with the complete E1 cDNA probe. C, polymerase chain reaction
amplification of E1 from O. volvulus L3 cDNA with E1-specific
primers. Lane 2 contained polymerase chain reaction-amplified
E1 cDNA of O. volvulus L3. Lane 1 contained no
DNA.
Expression of the mRNA of E1 in the infective stage (L3) was detected by reverse transcriptase-polymerase chain reaction amplification of O. volvulus L3 cDNA with E1-derived primers. A signal corresponding to the expected size was observed (Fig. 2C).
Figure 3: Nucleotide and derived amino acid sequence of E1. The deduced amino acid sequence is shown in single letter code below the nucleotide sequence; both are numbered on the right. The in-frame stop codon preceding the probable initiation codon and the termination codon are marked by asterisks. The putative polyadenylation signals are underlined. The potential leucine zipper motif is boxed, and the putative N-linked glycosylation site is boxed and shaded.
Figure 4: Alignment of the predicted protein sequence of E1 and human brain ankyrin 2 (BRANK2 from (20) ). Identical amino acids are boxed. Gaps introduced to maximize homology are indicated by dashes.
Figure 5: A, SDS-PAGE of recombinant E1 protein and Coomassie Brilliant Blue staining. B, immunoblot of E1 protein with the PI serum used for screening of the cDNA expression library. Lanes 1 in A and B contain vector without insert; lanes 2 in both panels contain vector plus E1 insert. The arrows indicate the positions of the recombinant E1 protein. C, immunoblot of the recombinant E1 protein (lane 1) and O. volvulus antigen (lane 2) with a rabbit antibody against E1 recombinant protein.
The recombinant protein is recognized by the PI serum used for screening of the cDNA library, which reacts almost exclusively with the 90-kDa antigen (Fig. 5B). Furthermore, the rabbit antibody against the recombinant protein recognizes a 90-kDa antigen in whole O. volvulus protein extract (Fig. 5C). This strongly suggests that E1 is identical to the 90-kDa O. volvulus antigen recognized by PI individuals. In immunoblots the recombinant E1 antigen was recognized by all of 10 PI sera, by 4 of 6 SOW sera, and 1 of 5 GEN sera tested (not shown).
Figure 6:
Immunohistochemical localization of the E1
protein in sections of live onchocercal worms. A, in a cross
section of an adult O. volvulus female, intense labeling of
the basal labyrinth of the hypodermis and the uterine epithelium is
seen. i, intestine. B, distinct staining pattern of
the basal labyrinth of the lateral hypodermal chord (arrow) is
shown in a longitudinal section of O. gibsoni. C,
anterior end of adult O. volvulus with distinct staining of
the nerve ring around the esophagus. A neuronal cell body is indicated
by the arrow. D, neuronal cell body (arrow)
in the inner part of the hypodermal chord, intensively stained using
immune serum (1:3200). E, basal labyrinth of a median chord
with the pouch of the nerve axon (arrow). F and G, basal labyrinth (arrows) in the outer zone of the
epithelium of the uterus (F) and intestine (G)
labeled. The basal lamina remains unstained. H, schematic
representation of the body wall of an adult female O. volvulus as seen by electron microscopy(24) . bly, basal
labyrinth; cu, cuticle; hy, hypodermis; i,
intestine; mu, muscle; u, uterus. A and C-H, O. volvulus, alkaline
phosphatase-anti-alkaline phosphatase. A, F, and G, with toluidin. A, 280; C,
670; B and E-G
800; A-C and E-G, affinity purified rabbit antibody to E1
protein.
Here we report the cloning and characterization of an O. volvulus antigen selected based on its immunogenicity for putatively immune individuals. Its predicted amino acid sequence indicates that it is related to ankyrins, a group of membrane-associated proteins with diverse functions such as involvement in polarization of membrane proteins, attachment of voltage-gated ion channels to the cytoskeleton, and axon guidance (for reviews see Refs. 25 and 26). E1 is most closely related to human brain ankyrin (BRANK2); however, it lacks the membrane-binding and most of the spectrin-binding domains, which are typical for all ankyrin sequences described to date. Although the region of similarity between E1 and human and mouse ankyrins is evolutionary conserved (26, 27) and is apparently also similar in the free living nematode C. elegans(26, 27) , no similarity in the remaining 239 amino acids of E1 to human or mouse ankyrins is observed. This is consistent with the reported high divergence between species in this part of the regulatory domain(26) . An interesting but yet unresolved phenomenon shared by E1, human, and brain ankyrins is the observed discrepancy between the size of the recombinant proteins predicted by the cDNA sequences and by their migration on SDS-PAGE, with differences between 30-41%, which are attributable to regions in or adjacent to the regulatory domains(21, 23) .
E1 appears to be a small transcript of the O. volvulus ankyrin gene. Similar small transcripts of mammalian ankyrin genes lacking the membrane-binding and large parts of the spectrin-binding domains have been observed in a number of rat and mouse tissues including brain(22, 27, 28) . The current hypothesis is that these small ankyrins may be involved in functions other than that of membrane-cytoskeleton linkers(22) .
We found that in O. volvulus the E1 protein is localized to the nerve ring, the neuronal cell bodies, and the basal labyrinth within the extracellular clefts of the hypodermis, where nerve axons are located in O. volvulus(30) as well as in C. elegans(31) . In mammals, brain ankyrin is not only found in the axonal membrane (32) but also in intercellular connections in brain, where it colocalizes with neuronal cell adhesion molecules(33) , which bind to ankyrin(34) . The leucine zipper motif in the E1 sequence may be important in binding to other proteins(35) . A leucine zipper motif has also been found in the 43-kDa protein (36) suggested to arise from the small ankyrin transcripts observed in mouse skeletal muscle(28) . This protein is involved in the immobilization of the acetylcholine receptor at the postsynaptic membrane of the neuromuscular junction (32) . The observed localization of E1 in the neuronal cell bodies as well as in the extracellular clefts adjacent to the basal lamina raises the question of whether E1 is related to proteins involved in transynaptic signaling(37) .
Both the sequence similarity to brain ankyrins and the localization within O. volvulus indicate that E1 is associated with the nervous system of the worm. In C. elegans, ankyrin is important in the development and function of the nervous system. The ankyrin gene of C. elegans is one of the axonal guidance genes whereby mutants of this unc-44 gene are characterized by the development of abnormally short chemosensory cilia, abnormal neurons, and defects in axon guidance and fasciculation(38, 39) . In nematodes some of these neuronal structures are not only essential for development but are also directly exposed to the environment. Thus the chemosensory cilia represent neuronal endings accessible through the amphid openings in the head(40, 41) , which are necessary for the uptake and processing of environmental signals(40, 42) . Truncated cilia apparently rend neurons insensitive to exogenous signals(42) . For parasitic nematodes like O. volvulus, such defects could be critical, because the developmental changes undergone upon encounter with their hosts appear to depend on the evaluation of environmental signals(42) . Interestingly, in O. volvulus these cilia are only directly exposed to the environment in the stages present in the human host(41) .
It thus is conceivable that antibodies directed against proteins present in the nervous system of the worm such as E1 may have access to the parasite's neuronal structures. That antibody binding can interfere with neuronal function and development has been demonstrated in vitro and in vivo. In insect embryos antibodies raised against a single neuronal protein involved in growth cone guidance, fasciclin, can stall growth of axons(43) . It will be of interest to examine if antibody to E1 has similar effects, particularly because ankyrin appears functionally related in nematodes(29, 39) . Antibody binding could lead to an impairment of parasite neuronal functions and, if larval stages are affected, possibly to a developmental arrest.
Further experiments will include analysis of the E1 antibody effects on the parasite in vitro as well as of epitopes possibly relevant to protective immunity. In vivo studies may be feasible in cattle, natural mammalian hosts for Onchocerca species similar to O. volvulus(44) , because antibody to E1 identified a similar protein in these parasites.
The present results raise the question of whether antibodies directed against the parasite's neuronal structures could provide protection for the host by inhibition of essential parasite functions. Neuronal proteins have not yet been associated with protection against helminths. Nevertheless, the nervous system may represent an Achilles' heel of the parasite accessible for immune-mediated intervention. The present results suggest that this could be exploited in efforts to develop a vaccine against O. volvulus.
This paper is dedicated to the memory of Bruce M. Greene, MD.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X84359[GenBank].
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