Sara Lustigman

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.

A research agenda for helminth diseases of humans: The problem of helminthiases

A disproportionate burden of helminthiases in human populations occurs in marginalised, low-income, and resource-constrained regions of the world, with over 1 billion people in developing areas of sub-Saharan Africa, Asia, and the Americas infected with one or more helminth species. The morbidity caused by such infections imposes a substantial burden of disease, contributing to a vicious circle of infection, poverty, decreased productivity, and inadequate socioeconomic development. Furthermore, helminth infection accentuates the morbidity of malaria and HIV/AIDS, and impairs vaccine efficacy. Polyparasitism is the norm in these populations, and infections tend to be persistent. Hence, there is a great need to reduce morbidity caused by helminth infections. However, major deficiencies exist in diagnostics and interventions, including vector control, drugs, and vaccines. Overcoming these deficiencies is hampered by major gaps in knowledge of helminth biology and transmission dynamics, platforms from which to help develop such tools. The Disease Reference Group on Helminths Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR), was given the mandate to review helminthiases research and identify research priorities and gaps. In this review, we provide an overview of the forces driving the persistence of helminthiases as a public health problem despite the many control initiatives that have been put in place; identify the main obstacles that impede progress towards their control and elimination; and discuss recent advances, opportunities, and challenges for the understanding of the biology, epidemiology, and control of these infections. The helminth infections that will be discussed include: onchocerciasis, lymphatic filariasis, soil-transmitted helminthiases, schistosomiasis, food-borne trematodiases, and taeniasis/cysticercosis.

Antibodies against a secreted protein from hookworm larvae reduce the intensity of hookworm infection in humans and vaccinated laboratory animals

The development of a vaccine would provide an important new tool for the control of human hookworm infection. On the basis of successful vaccination of laboratory animals with living irradiated, third‐stage hookworm larvae (L3), we examined the antibody responses of individuals from hookworm endemic areas of Brazil and China against the most abundant L3 secreted antigens, the ancylostoma secreted proteins, ASP‐1 and ASP‐2. Logistic regression was used to investigate the effects of antibody isotype responses to ASPs on the risk of an individual harboring heavy hookworm infection. A significant protective association was observed between increasing anti‐ASP‐2 IgE levels and the risk of heavy hookworm infection. To confirm that ASP‐2 is a protective antigen, laboratory dogs were immunized with recombinant ASP‐2 formulated with the GlaxoSmithKline Adjuvant, AS03. Sera obtained from the immunized dogs exhibited high geometric mean antibody titers, immunoprecipitated native ASP‐2 from L3 extracts and localized the site of ASP‐2 expression to the glandular esophagus and body channels exiting to the cuticle. The sera also exhibited an increased ability to inhibit migration of L3 through tissue in vitro relative to sera from AS03‐injected controls. Upon L3 challenge, the ASP‐2 vaccinated dogs exhibited significant reductions in fecal egg counts and intestinal hookworm burden. These findings provide strong support for the development of an effective recombinant vaccine against hookworm infection in humans.

A Research Agenda for Helminth Diseases of Humans: Intervention for Control and Elimination

Recognising the burden helminth infections impose on human populations, and particularly the poor, major intervention programmes have been launched to control onchocerciasis, lymphatic filariasis, soil-transmitted helminthiases, schistosomiasis, and cysticercosis. The Disease Reference Group on Helminth Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR), was given the mandate to review helminthiases research and identify research priorities and gaps. A summary of current helminth control initiatives is presented and available tools are described. Most of these programmes are highly dependent on mass drug administration (MDA) of anthelmintic drugs (donated or available at low cost) and require annual or biannual treatment of large numbers of at-risk populations, over prolonged periods of time. The continuation of prolonged MDA with a limited number of anthelmintics greatly increases the probability that drug resistance will develop, which would raise serious problems for continuation of control and the achievement of elimination. Most initiatives have focussed on a single type of helminth infection, but recognition of co-endemicity and polyparasitism is leading to more integration of control. An understanding of the implications of control integration for implementation, treatment coverage, combination of pharmaceuticals, and monitoring is needed. To achieve the goals of morbidity reduction or elimination of infection, novel tools need to be developed, including more efficacious drugs, vaccines, and/or antivectorial agents, new diagnostics for infection and assessment of drug efficacy, and markers for possible anthelmintic resistance. In addition, there is a need for the development of new formulations of some existing anthelmintics (e.g., paediatric formulations). To achieve ultimate elimination of helminth parasites, treatments for the above mentioned helminthiases, and for taeniasis and food-borne trematodiases, will need to be integrated with monitoring, education, sanitation, access to health services, and where appropriate, vector control or reduction of the parasite reservoir in alternative hosts. Based on an analysis of current knowledge gaps and identification of priorities, a research and development agenda for intervention tools considered necessary for control and elimination of human helminthiases is presented, and the challenges to be confronted are discussed.

A Research Agenda for Helminth Diseases of Humans: Diagnostics for Control and Elimination Programmes

Diagnostic tools appropriate for undertaking interventions to control helminth infections are key to their success. Many diagnostic tests for helminth infection have unsatisfactory performance characteristics and are not well suited for use in the parasite control programmes that are being increasingly implemented. Although the application of modern laboratory research techniques to improve diagnostics for helminth infection has resulted in some technical advances, uptake has not been uniform. Frequently, pilot or proof of concept studies of promising diagnostic technologies have not been followed by much needed product development, and in many settings diagnosis continues to rely on insensitive and unsatisfactory parasitological or serodiagnostic techniques. In contrast, PCR-based xenomonitoring of arthropod vectors, and use of parasite recombinant proteins as reagents for serodiagnostic tests, have resulted in critical advances in the control of specific helminth parasites. The Disease Reference Group on Helminths Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR) was given the mandate to review helminthiases research and identify research priorities and gaps. In this review, the diagnostic technologies relevant to control of helminth infections, either available or in development, are reviewed. Critical gaps are identified and opportunities to improve needed technologies are discussed.

Identification of Potential Vaccine and Drug Target Candidates by Expressed Sequence Tag Analysis and Immunoscreening of Onchocerca volvulus Larval cDNA Libraries

ABSTRACT The search for appropriate vaccine candidates and drug targets against onchocerciasis has so far been confronted with several limitations due to the unavailability of biological material, appropriate molecular resources, and knowledge of the parasite biology. To identify targets for vaccine or chemotherapy development we have undertaken two approaches. First, cDNA expression libraries were constructed from life cycle stages that are critical for establishment of Onchocerca volvulus infection, the third-stage larvae (L3) and the molting L3. A gene discovery effort was then initiated by random expressed sequence tag analysis of 5,506 cDNA clones. Cluster analyses showed that many of the transcripts were up-regulated and/or stage specific in either one or both of the cDNA libraries when compared to the microfilariae, L2, and both adult stages of the parasite. Homology searches against the GenBank database facilitated the identification of several genes of interest, such as proteinases, proteinase inhibitors, antioxidant or detoxification enzymes, and neurotransmitter receptors, as well as structural and housekeeping genes. Other O. volvulus genes showed homology only to predicted genes from the free-living nematode Caenorhabditis elegans or were entirely novel. Some of the novel proteins contain potential secretory leaders. Secondly, by immunoscreening the molting L3 cDNA library with a pool of human sera from putatively immune individuals, we identified six novel immunogenic proteins that otherwise would not have been identified as potential vaccinogens using the gene discovery effort. This study lays a solid foundation for a better understanding of the biology of O. volvulus as well as for the identification of novel targets for filaricidal agents and/or vaccines against onchocerciasis based on immunological and rational hypothesis-driven research.

Cloning of a Cysteine Protease Required for the Molting of Onchocerca volvulus Third Stage Larvae

We have investigated the involvement of a cysteine protease in the development of Onchocerca volvulus fourth stage larvae (L4) by testing the effect of cysteine protease inhibitors on the survival of third stage larvae (L3), and the molting of L3 to L4 in vitro. When larvae were cultured in the presence of specific inhibitors, the peptidyl monofluoromethylketones, viability of either L3 or L4 was not affected. However, the inhibitors reduced the number of L3 that molted to L4 in vitro in a time- and dose-dependent manner. Molting was completely inhibited in the presence of 50-250 μM inhibitor. Ultrastructural examination of L3 that did not molt in the presence of inhibitors indicated that new L4 cuticle was synthesized, but there was no separation between the L3 and the L4 cuticles. The endogenous cysteine protease was detected in molting larvae after binding to labeled inhibitors, and by antibodies directed against a recombinant O. volvulus L3 cysteine protease that was cloned and expressed. The enzyme was detected in cuticle regions where the separation between the cuticles occurs in molting larvae. These studies suggest that molting and successful development of L4 depends on the expression and release of a cysteine protease.

Angiogenic activity of Onchocerca volvulus recombinant proteins similar to vespid venom antigen 5

Although the mechanisms underlying the host inflammatory response in ocular onchocerciasis have been examined, the role of particular parasite proteins in this process remains largely unexplored. Recently, it was found that one of the most abundant expressed sequence tags in Onchocerca volvulus infective larvae encoded a protein with similarities to a component of vespid venom. This clone was designated O. volvulus Activation associated Secreted Protein -1 (Ov-asp-1). We report the characterization of three members of a family of proteins, designated the Ov-ASP family, of which Ov-ASP-1 is a member. Sequence based and phylogenetic analyses suggest that these proteins form a filarial specific protein family related to both the vespid venom antigen 5 and the vertebrate CRISP/Tpx family of proteins. The three members of the Ov-ASP family exhibit distinct patterns of expression in the life cycle of O. volvulus. Genomic Southern blot analyses indicate that several genes encoding sequences related to the Ov-asp family are present in the genome of O. volvulus. Recombinant proteins expressed from full length cDNAs encoding two members of the Ov-asp family were found to induce an angiogenic response after injection into corneas of naive mice, and vessel formation was associated with only minor inflammatory cell infiltration. These data suggest that Ov-ASP proteins may directly induce an angiogenic response and may therefore contribute to corneal neovascularization in onchocercal keratitis.

The mature-parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum associates with the erythrocyte membrane skeletal protein, band 4.1

Several proteins synthesized by mature asexual stages of Plasmodium falciparum interact with the erythrocyte membrane skeleton. One of these is the mature-parasite-infected erythrocyte surface antigen (MESA; also called PfEMP2), a phosphoprotein of 250-300 kDa, which is found on the internal face of the erythrocyte membrane. When MESA is precipitated with anti-MESA antibodies, another phosphoprotein of 80 kDa is co-precipitated. This 80-kDa phosphoprotein was identified by peptide mapping as the erythrocyte membrane component band 4.1. Thus, MESA is apparently anchored at the erythrocyte membrane through an association with band 4.1. Band 4.1 is more intensely phosphorylated in infected erythrocytes and is increased in relative molecular mass in erythrocytes infected by isolates of P. falciparum that cytoadhere.

Ancylostoma caninum MTP-1, an astacin-like metalloprotease secreted by infective hookworm larvae, is involved in tissue migration

ABSTRACT Infective larvae (L3) of nematodes secrete macromolecules that are critical to infection and establishment of the parasite in the host. The dog hookworm Ancylostoma caninum secretes an astacin-like metalloprotease, Ac-MTP-1, upon activation in vitro with host serum. Recombinant Ac-MTP-1 was expressed in the baculovirus/insect cell system as a secreted protein and was purified from culture medium by two separate methods, cation-exchange fast-performance liquid chromatography and gelatin-affinity chromatography. Recombinant MTP-1 was catalytically active and digested a range of native and denatured connective tissue substrates, including gelatin, collagen, laminin, and fibronectin. A dog was immunized with recombinant Ac-MTP-1 formulated with AS03 adjuvant, and the antiserum was used to immunolocalize the anatomic sites of expression within A. caninum L3 to secretory granules in the glandular esophagus and the channels that connect the esophagus to the L3 surface and to the cuticle. Antiserum inhibited the ability of recombinant MTP-1 to digest collagen by 85% and inhibited larval migration through tissue in vitro by 70 to 75%, in contrast to just 5 to 10% inhibition obtained with preimmunization serum. The metalloprotease inhibitors EDTA and 1,10-phenanthroline also reduced the penetration of L3 through skin in vitro by 43 to 61%. The data strongly suggest that Ac-MTP-1 is critical for the invasion process of hookworm larvae, and moreover, that antibodies against the enzyme can neutralize its function and inhibit migration.