Expression and immune recognition of Brugia malayi VAL-1, a homologue of vespid venom allergens and Ancylostoma secreted proteins☆
Introduction
The infective third-stage larvae of many parasitic nematodes undergo the most critical transition of the life cycle, from a free-living or arthropod-borne existence to the definitive, often mammalian, host [1], [2]. Products from these stages are of particular interest as key mediators of the invasion process and as potential immunogens for anti-parasite vaccines [3], [4], [5]. When similar proteins are associated with larval stages of many different nematode species, a common pathway or essential component of the infection process may be inferred. One such instance is the group of proteins first described as Ancylostoma caninum secreted protein or ASP [6] with similarity to a wider family including, for example, hymenopteran venom allergens. Significantly, these products are released when resting larvae are stimulated to commence invasion. The venom allergen antigen homologue VAH/ASP family contains two distinct forms, typified in A. caninum [7]: Ac-ASP-1 is a long-form 42 kDa protein while Ac-ASP-2 is a short-form 20–22 kDa product. Sequence analysis reveals that Ac-ASP-1 contains a tandem nonidentical repeat of an ancestral unit represented only once in Ac-ASP-2 [7].
Members of this gene family have now been reported from a range of nematode species. In the sheep intestinal nematode Haemonchus contortus, both the large-form Hc40 [8] and short-form Hc24 [9], [10] have been identified. Multiple homologues have been discovered in the two human hookworm species Ancylostoma duodenale and Necator americanus [11], [12], and also in Meloidogyne incognita [13], Onchocerca volvulus [14] and Toxocara canis [15]. Additional gene members in Ascaris and Strongyloides have also been indicated by hybridisation experiments with Ac-asp-2 probes [7]. Further, a homologue from the filarial nematode Dirofilaria immitis was selected by reactivity with immune dog serum (Tripp C, Wisnewski N, unpublished deposition) and a related gene in Brugia malayi reported from expressed sequence tag (EST) analysis [4]. Significantly, up to 17 homologous genes have been identified in the genome of Caenorhabditis elegans [7], [12]. All but one of these are single-unit forms (e.g. C. elegans C39E9.1), the exception being F11C7.3 which displays a paired but nonidentical repeat structure [8].
More distant relatives of the VAH/ASP family can be found across a wide evolutionary spectrum. Within A. caninum, a related gene encodes a neutrophil inhibitory factor (NIF), which displays an antagonistic binding to the integrin receptor αMβ2 (CD11b/CD18, Mac-1) on mammalian cells [16]. While the aforementioned insect venom allergens have no known function, they are similar to helothermine, a lizard salivary toxin, which blocks the ryanodine receptor [17]. In mammals, similar proteins have been described as the testis-specific proteins TPX-1, sperm-coating glycoprotein (SCG) and cysteine-rich secretory protein (CRISP) [18], as well as P25TI, a novel trypsin inhibitory protein upregulated in some tumour lines [19]. The larger gene family also includes an interesting set of plant defence proteins, all characterised by a conserved signature of key residues [7]. From these similarities, it is difficult to deduce a general function for nematode homologues, but it is attractive to postulate inhibition or activation of host receptor-dependent processes. For example, the Ov-ASP-1 and Ov-ASP-2 proteins from Onchocerca volvulus have been shown to promote corneal angiogenesis when injected into mice [14], a role in keeping with the formation of vascularised nodules in onchocerciasis.
Because vaccines against nematode parasites focus on the infective larval stage, heightened expression of vah/asp genes in larvae make these products an attractive candidate vaccine antigen [20]. Promising results have so far been obtained in two different species. Thus, four out of five sheep vaccinated with a fraction enriched with H. contortus Hc-24 were protected against establishment of adult parasites [21], while ASP-1-immunised mice show up to 80% reduction in infection [22], [23].
The Filarial Genome Project has provided a major resource and impetus for gene discovery from B. malayi and other filarial nematodes [4], [24], [25], [26], [27], [28]. EST sequences deposited for B. malayi include a number corresponding to a homologue of ASP [4], [25], [28]. We describe here the characterisation of this homologue, and its expression as a recombinant probe for immunological reactivity of patients infected with this major human tropical pathogen.
Section snippets
Database searching
The ungapped TBLASTN algorithm was used with the A. caninum ASP-1 protein sequence matched against all deposits in the NCBI dbEST nucleotide database translated into all six frames [29]. Multiple ESTs with significant similarity were found, permitting a putative full-length sequence to be assembled. From this, a 5′ gene-specific primer was designed to amplify the gene by PCR from an L3 cDNA library, 5′-AGT ACT TAC TAG ACG ACA TCT TAC TGT T-3′ (roman typeface nt −68 to −54, 5′ of the start
Search of dbEST for ASP homologues
The A. caninum ASP-1 sequence was used to screen the NCBI dbEST database containing the Filarial Genome Project EST sequences from multiple stages of B. malayi. Of approximately 18 700 sequences, 82 showed high levels of similarity and 72 of these were from the infective L3 stage larval cDNA library. This represents an abundance of 2.22% (72/3249 L3 ESTs). Similar levels of abundance have been observed for the related nematode O. volvulus [14]. Three more cDNAs were noted from the Mf stage,
Conclusions
The VAL family has become one of the most intensively studied sets of genes from nematode parasites, showing strong association with larval invasion of the mammalian host. We report here that these products do not go unnoticed by the host immune system and suggest that an appropriate response to these antigens may prove to be protective. It will now be important to establish the biological function of the VAL proteins in the host–parasite interaction, so that we can begin to appreciate a fuller
Acknowledgements
We thank Mark Blaxter for providing an analysis of the frequency of ESTs corresponding to the val-1 gene and for the constructive comments on the manuscript. We are also grateful for Yvonne Harcus, Judith Allen and Laetitia Le Goff for their invaluable assistance with the vaccination trials. The studies described here were funded by the Wellcome Trust and the European Union (INCO-DC1C18-CT95-0014 and INCO-DC1C18-CT95-0245).
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Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank™ data base with accession number AF334661.