William F. Gregory

Draft Genome of the Filarial Nematode Parasite Brugia malayi

Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the ∼90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict ∼11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during ∼350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.

Bm-CPI-2, a cystatin homolog secreted by the filarial parasite Brugia malayi, inhibits class II MHC-restricted antigen processing

While interference with the class I MHC pathway by pathogen-encoded gene products, especially those of viruses, has been well documented, few examples of specific interference with the MHC class II pathway have been reported. Potential targets for such interference are the proteases that remove the invariant chain chaperone and generate antigenic peptides. Indeed, recent studies indicate that immature dendritic cells express cystatin C to modulate cysteine protease activity and the expression of class II MHC molecules [1]. Here, we show that Bm-CPI-2, a recently discovered cystatin homolog produced by the filarial nematode parasite Brugia malayi (W. F. Gregory et al., submitted), inhibits multiple cysteine protease activities found in the endosomes/lysosomes of human B lymphocyte lines. CPI-2 blocked the hydrolysis of synthetic substrates favored by two different families of lysosomal cysteine proteases and blocked the in vitro processing of the tetanus toxin antigen by purified lysosome fractions. Moreover, CPI-2 substantially inhibited the presentation of selected T cell epitopes from tetanus toxin by living antigen-presenting cells. Our studies provide the first example of a product from a eukaryotic parasite that can directly interfere with antigen presentation, which, in turn, may suggest how filarial parasites might inactivate the host immune response to a helminth invader.

The Abundant Larval Transcript-1 and -2 Genes of Brugia malayi Encode Stage-Specific Candidate Vaccine Antigens for Filariasis

ABSTRACT Lymphatic filariasis is a major tropical disease caused by the mosquito-borne nematodes Brugia and Wuchereria. About 120 million people are infected and at risk of lymphatic pathology such as acute lymphangitis and elephantiasis. Vaccines against filariasis must generate immunity to the infective mosquito-derived third-stage larva (L3) without accentuating immunopathogenic responses to lymphatic-dwelling adult parasites. We have identified two highly expressed genes, designated abundant larval transcript-1 and -2 (alt-1 and alt-2), from each of which mRNAs account for >1% of L3 cDNAs. ALT-1 and ALT-2 share 79% amino acid identity across 125 residues, including a putative signal sequence and a prominent acidic tract. Expression ofalt-1 and alt-2 is initiated midway through development in the mosquito, peaking in the infective larva and declining sharply following entry into the host. Humans exposed toBrugia malayi show a high frequency of immunoglobulin G1 (IgG1) and IgG3 antibodies to ALT-1 and -2, distinguishing them from adult-stage antigens, which are targeted by the IgG4 isotype. Immunization of susceptible rodents (jirds) with ALT-1 elicited a 76% reduction in parasite survival, the highest reported for a single antigen from any filarial parasite. ALT-1 and the closely related ALT-2 are therefore strong candidates for a future vaccine against human filariasis.

Differentially expressed, abundant trans-spliced cDNAs from larval Brugia malayi

Isolation and cloning of abundant reverse transcriptase-polymerase chain reaction (RT-PCR) products from the filarial nematode Brugia malayi using the conserved nematode spliced leader sequence and poly A as amplification targets has allowed us to identify abundant, stage specific transcripts from infective and post-infective larvae. The predicted protein products of the most prominent full-length transcripts from mosquito-derived L3 parasites are: (i) Bm-ALT-1, a homologue of a Dirofilaria immitis abundant larval protein: (ii) Bm-CPI-1, a cystatin-type cysteine protease inhibitor; (iii) Bm-ALT-3, a novel predicted 6 kDa glycine/tyrosine-rich protein; and (iv) Bm-TPH-1, a homologue of a mammalian translationally-controlled tumour protein. Some transcripts were not full-length but had mis-primed at A-rich stretches of coding sequence: the most abundant of these was Bm-col-3, a which encodes a collagen homologous to Bp-COL-1 of Brugia pahangi. Similar analysis of abundant spliced leader (SL)/oligo-dT products from fourth-stage larvae 9 days post-infection yielded two dominant transcripts: (i) Bm-cdd-1, which encodes a protein with homology to cytidine deaminase, differing at only one amino acid position from its homologue described in Brugia pahangi; and (ii) the same truncated form of Bm-col-3 found in L3 preparations. Expression of the major transcripts was assessed by PCR amplification of cDNA libraries derived from each stage of the life cycle. alt1, alt-3 and cpi-1 were all found to be specific to the L3 stage, while cdd-1 was found only in the L4 cDNA library. Expression of these larval-specific transcripts was not detected in either microfilarial or adult libraries.

MIF homologues from a filarial nematode parasite synergize with IL-4 to induce alternative activation of host macrophages

Macrophage migration inhibitory factor (MIF) is a highly conserved cytokine considered to exert wide‐ranging, proinflammatory effects on the immune system. Recently, members of this gene family have been discovered in a number of invertebrate species, including parasitic helminths. However, chronic helminth infections are typically associated with a Th2‐dominated, counter‐inflammatory phenotype, in which alternatively activated macrophages (AAMs) are prominent. To resolve this apparent paradox, we have analyzed the activity of two helminth MIF homologues from the filarial nematode Brugia malayi, in comparison with the canonical MIF from the mouse. We report that murine MIF (mMIF) and Brugia MIF proteins induce broadly similar effects on bone marrow‐derived mouse macrophages, eliciting a measured release of proinflammatory cytokines. In parallel, MIF was found to induce up‐regulation of IL‐4R on macrophages, which when treated in vitro with MIF in combination with IL‐4, expressed markers of alternative activation [arginase, resistin‐like molecule α (RELM‐α) or found in inflammatory zone 1, Ym‐1, murine macrophage mannose receptor] and differentiated into functional AAMs with in vitro‐suppressive ability. Consistent with this finding, repeated in vivo administration of Brugia MIF induced expression of alternative macrophage activation markers. As mMIF did not induce RELM‐α or Ym‐1 in vivo, alternative activation may require components of the adaptive immune response to Brugia MIF, such as the production of IL‐4. Hence, MIF may accentuate macrophage activation according to the polarity of the environment, thus promoting AAM differentiation in the presence of IL‐4‐inducing parasitic helminths.

Cystatins from filarial parasites: Evolution, adaptation and function in the host-parasite relationship

Cystatins, together with stefins and kininogens, are members of the cystatin superfamily of cysteine protease inhibitors (CPI) present across the animal and plant kingdoms. Their role in parasitic organisms may encompass both essential developmental processes and specific interactions with the parasites vector and/or final host. We summarise information gathered on three cystatins from the human filarial nematode Brugia malayi (Bm-CPI-1, -2 and -3), and contrast them those expressed by other parasites and by the free-living nematode Caenorhabditis elegans. Bm-CPI-2 differs from C. elegans cystatin, having acquired the additional function of inhibiting asparaginyl endopeptidase (AEP), in a manner similar to some human cystatins. Thus, we propose that Bm-CPI-2 and orthologues from related filarial parasites represent a new subset of nematode cystatins. Bm-CPI-1 and CPI-3 share only 25% amino acid identity with Bm-CPI-2, and lack an evolutionarily conserved glycine residue in the N-terminal region. These sequences group distantly from the other nematode cystatins, and represent a second novel subset of filarial cystatin-like genes. Expression analyses also show important differences between the CPI-2 and CPI-1/-3 groups. Bm-cpi-2 is expressed at all time points of the parasite life cycle, while Bm-cpi-1 and -3 expression is confined to the late stages of development in the mosquito vector, terminating within 48h of infection of the mammalian host. Hence, we hypothesise that CPI-2 has evolved to block mammalian proteases (including the antigen-processing enzyme AEP) while CPI-1 and -3 function in the milieu of the mosquito vector necessary for transmission of the parasite.

Immuno-epidemiology of human Schistosoma haematobium infection: preferential IgG3 antibody responsiveness to a recombinant antigen dependent on age and parasite burden

BackgroundSchistosomiasis is a major parasitic disease affecting over 200 million people in the developing world with a further 400 million people at risk of infection. The aim of this study was to identify a single antigen from adult Schistosoma haematobium worms and subsequently use this antigen to study the development of schistosome-acquired immunity in a human population.MethodsThe full-length cDNA sequence of a S. haematobium protein, a putative orthologue of the S. mansoni tegumental antigen Sm13, was obtained from a cDNA library of adult S. haematobium worms and named Sh13 following a small-scale expressed sequence tags (EST) project. The recombinant Sh13 protein expressed in E. coli, was used to investigate immuno-epidemiological patterns in 147 Zimbabweans (7–18 years old) exposed to S. haematobium.ResultsSequence analysis of the full-length cDNA sequence of the S. haematobium protein Sh13, indicated that the protein has an N-terminal signal peptide and encodes an 85-amino acid mature protein with a highly conserved predicted transmembrane domain (86 % identity with the S. mansoni tegumental antigen Sm13). The recombinant Sh13 protein was used in ELISA assays to determine the reactivity of sera from the study participants. Antibody responses against Sh13 were predominantly IgG3 isotype compared to responses against crude worm antigens which were predominantly IgG1 and IgG4. The relationship between anti-Sh13 IgG3 levels and infection intensity varied significantly with host age. The youngest children (7–10 years old) had relatively low levels of both infection and anti-Sh13 IgG3. In older children (11–12 years old) rising infection levels were accompanied by a significant increase in anti-Sh13 IgG3 levels. Subsequently, infection intensity declined significantly in 13–18 year olds but levels of the antibody continued to rise. The changing relationship between infection intensity and anti-Sh13 IgG3 levels with host age is consistent with the profile of a protective immune response predicted from theoretical work.ConclusionWe have identified and characterised a novel S. haematobium antigen Sh13, a putative tegumental protein, and shown that it is recognised predominantly by IgG3 antibodies from people infected with/exposed to S. haematobium parasites. We have also shown that, the anti-Sh13 IgG3 response is maximal in older individuals with the lowest infection intensity, and that the age profile of the relationship between anti-Sh13 IgG3 and infection intensity is consistent with that predicted by theoretical work for a protective response stimulated by and directed against adult worms.

HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33

&NA; Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin‐33 (IL‐33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL‐33‐suppressive 26‐kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL‐33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL‐33 (in the cytokines activated state) and also to nuclear DNA via its N‐terminal CCP module pair (CCP1/2), tethering active IL‐33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy. Graphical Abstract Figure. No caption available. HighlightsHpARI is a suppressor of IL‐33 release and consequent allergic sensitizationHpARI binds active IL‐33 and nuclear DNA, tethering IL‐33 within necrotic cellsHpARI is active against both human and murine IL‐33 &NA; Osbourn et al identified HpARI, a protein secreted by a helminth parasite that is capable of suppressing allergic responses. HpARI binds to IL‐33 (a critical inducer of allergy) and nuclear DNA, preventing the release of IL‐33 from necrotic epithelial cells.

Evaluation of Viremia Frequencies of a Novel Human Pegivirus by Using Bioinformatic Screening and PCR

Bioinformatic screening and PCR-based approaches detected active infection with human hepegivirus-1 in exposed populations.

High prevalence and diversity of bovine astroviruses in the faeces of healthy and diarrhoeic calves in South West Scotland

Abstract Astroviruses (AstV) are single-stranded, positive-sense RNA viruses and one of the major causes of infant diarrhoea worldwide. Diarrhoea is a common and important cause of morbidity and mortality in calves; therefore, we investigated whether the presence of AstV is associated with calf diarrhoea. We identified diverse AstV lineages from faecal samples of both healthy and diarrhoeic calves and healthy adult cattle in South West Scotland. AstV was common in calves (present in 74% (85/115) of samples) but uncommon in adult cattle (present in 15% (3/20) of samples). No association was found between the presence of AstV and calf diarrhoea or the presence of a specific AstV lineage and calf diarrhoea. AstV was strongly associated with the presence of rotavirus Group A (RVA), and a protective effect of age was evident for both AstV and RVA. Co-infections with multiple AstV lineages were detected in several calves and serial infection with different viruses could also be seen by longitudinal sampling of individuals. In summary, our study found genotypically diverse AstV in the faeces of calves in South West Scotland. However, no association was identified between AstV and calf diarrhoea, which suggests the virus does not play a primary role in the aetiology of calf diarrhoea in the group studied.