Expression and immune recognition of Brugia malayi VAL-1, a homologue of vespid venom allergens and Ancylostoma secreted proteins

https://doi.org/10.1016/S0166-6851(01)00374-7Get rights and content

Abstract

Several important nematode parasites have been found to express members of a gene family variously termed as venom allergen antigen homologue (vah) or Ancylostoma secreted protein (asp). In some cases these products are secreted by infective larval stages and have been suggested to be effective vaccine immunogens. We isolated the corresponding gene from the human filarial nematode, Brugia malayi, by first searching the expressed sequence tag (EST) dataset generated by the Filarial Genome Project and then using gene-specific nondegenerate primers matching the selected gene for PCR, from B. malayi cDNA libraries. We report here the full-length gene sequence, which we have designated as Bm-val-1, for vah/asp-like. The corresponding protein (Bm-VAL-1) contains 232 amino acids in a single homology unit, unlike products from some other species in which there is a tandem repeat. A putative signal sequence is present at the 5′ end and there are two potential N-glycosylation sites. Murine antibodies to recombinant Bm-VAL-1 react with a 28 kDa protein in L3 extracts and recombinant Bm-VAL-1 is recognised by murine T cells primed with soluble L3 proteins. Of 82 ESTs corresponding to Bm-val-1, 72 are recorded from the infective larval (L3) stage. However, PCR on the first-strand cDNA from later mammalian stages revealed some expression at most subsequent time points. Over 95% (20/21) of microfilaraemic human filariasis patients are seropositive for antibodies to Bm-VAL-1, with particularly high levels of IgG3 and IgG4 isotypes. The IgG4 subclass may indicate stimulation by adult and/or microfilarial-derived immunogens. The association of Bm-VAL-1 with the infective stage and its recognition by humans exposed to filariasis suggests that further evaluation of this antigen as a vaccine candidate should be performed.

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).

References (45)

  • J. Morrissette et al.

    Primary structure and properties of helothermine, a peptide toxin that blocks ryanodine receptors

    Biophys. J.

    (1995)
  • T. Yamakawa et al.

    cDNA cloning of a novel trypsin inhibitor with similarity to pathogenesis-related proteins, and its frequent expression in human brain cancer cells

    Biochim. Biophys. Acta

    (1998)
  • L. Sen et al.

    Hookworm burden reductions in BALB/c mice vaccinated with recombinant Ancylostoma secreted proteins (ASPs) from Ancylostoma duodenale, Ancylostoma caninum and Necator americanus

    Vaccine

    (2000)
  • S.A. Williams et al.

    The filarial genome project: analysis of the nuclear, mitochondrial and endosymbiont genomes of Brugia malayi

    Int. J. Parasitol.

    (2000)
  • R.M. Maizels et al.

    Immune evasion genes from filarial nematodes

    Int. J. Parasitol.

    (2001)
  • S.F. Altschul et al.

    Basic local alignment search tool

    J. Mol. Biol.

    (1990)
  • N. Gomez-Escobar et al.

    A novel member of the transforming growth factor-β (TGF-β) superfamily from the filarial nematodes Brugia malayi and B. pahangi

    Exp. Parasitol.

    (1998)
  • T. Megraw et al.

    Sequence and expression of Drosophila Antigen 5-related 2, a new member of the CAP gene family

    Gene

    (1998)
  • R.M. Maizels et al.

    T cell activation and the balance of antibody isotypes in human filariasis

    Parasitol. Today

    (1995)
  • A.E. Bianco et al.

    Developmentally regulated expression and secretion of a polymorphic antigen by Onchocerca infective-stage larvae

    Mol. Biochem. Parasitol.

    (1990)
  • M.S. Ibrahim et al.

    Surface-associated antigens of Brugia malayi L2 and L3 parasites during vector-stage development

    Mol. Biochem. Parasitol.

    (1992)
  • W.F. Gregory et al.

    The abundant larval transcript 1/2 genes of Brugia malayi encode stage-specific candidate vaccine antigens for filariasis

    Infect. Immun.

    (2000)
  • Cited by (85)

    View all citing articles on Scopus

    Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank™ data base with accession number AF334661.

    View full text