New technologies for the control of human hookworm infection

Review

TRENDS in Parasitology

Vol.22 No.7 July 2006

New technologies for the control of human hookworm infection Peter J. Hotez1,2, Jeff Bethony1, Maria Elena Bottazzi1, Simon Brooker3, David Diemert2 and Alex Loukas4 1

Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University, 2300 I Street, NW Washington, DC 20037, USA 2 Sabin Vaccine Institute, 1899 F Street, NW Washington, DC 20006, USA 3 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK 4 Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Brisbane, QLD 4029, Australia

Since the 1990s, the major approach to hookworm control has been morbidity reduction in school-aged children by periodic deworming with benzimidazoles. Now, efforts are underway to determine the feasibility of integrating deworming with control programs that target other neglected tropical diseases. However, the sustainability of benzimidazole deworming for hookworm is of concern because of the variable efficacy of mebendazole, high rates of post-treatment reinfection and possible development of drug resistance. This requires parallel efforts to develop new and complementary hookworm control tools, such as new anthelmintic drugs (e.g. tribendimidine) and a recombinant hookworm vaccine. It is hoped that, ultimately, anthelmintic vaccination will be linked to deworming as part of an expanded control package. The global burden of hookworm Despite the availability of low-cost, safe and single-dose benzimidazole anthelmintics, human hookworm infection (‘hookworm’) remains an important public health threat wherever rural poverty occurs in the tropics and subtropics [1,2]. The recent emergence of new and appropriate technologies for hookworm control (Table 1), including new health products (e.g. drugs and vaccines) and computer-based tools (e.g. mathematical modeling, disease burden assessment and cost-effectiveness studies), could lead to substantial reductions in the global prevalence and intensity of hookworm in the coming decade. This brief review describes these new technologies and their potential to have an impact on the global control of hookworm. Hookworm is one of the most common infections of humans, occurring in w600 million people [3]. The lower global prevalence of hookworm compared with previous estimates of 740 million cases in 2003 [4] partly reflects the hookworm reductions in the populous eastern provinces of China, which coincide with the recent economic prosperity of that region [5]. New estimates, based on a 2005 report of a nationwide survey of intestinal Corresponding author: Hotez, P.J. ([email protected]). Available online 18 May 2006

parasites, indicate that the prevalence of hookworm in China has fallen from 17% in the early 1990s [6] to just below 6% [7]. The highest prevalence of hookworm now occurs in sub-Saharan Africa, followed (in descending order) by Southeast Asia, the Indian subcontinent, the Americas, China and the Middle East. Although prevalence data give us some insight into the health impact of hookworm, the global burden is better reflected by estimates of disability-adjusted life years (DALYs). Since DALYs were first used to measure hookworm disease burden in 1990, the global estimates have varied widely, from as low as 1.5 million to as high as 22.1 million DALYs (reviewed by Bethony et al. [3]). This disparity reflects the subjectivity of the different disability weights assigned to the major chronic morbidities commonly attributed to hookworm disease [1,3]. Together, the World Health Organization and the Ellison Institute for World Health at Harvard University have embarked on an initiative to reassess the disease burdens attributed to the major neglected tropical diseases, including hookworm. Through full consideration of the chronic disabilities of hookworm, including the high percentage of iron-deficiency anemia attributable to hookworm in endemic countries [8], it is expected that higher DALY estimates, and therefore heightened public health interest, will emerge. Current approaches to hookworm control The historical record over the past century teaches us that poverty reduction and urbanization are the most effective means for reducing the prevalence and intensity of hookworm [5]. Economic prosperity and the abandonment of agrarian life styles contributed greatly to the control of hookworm and other diseases of poverty (e.g. typhoid fever, malaria) in the Southern USA during the first-half of the 20th century and in Japan and South Korea in the decades immediately following World War II [5]. Therefore, it is reasonable to predict that steady economic improvements and urbanization in Asia and the Americas will also translate into substantial reductions in hookworm transmission, with sub-Saharan Africa lagging behind. Surprisingly, a detailed understanding of the

www.sciencedirect.com 1471-4922/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2006.05.004

Review

328

TRENDS in Parasitology

Vol.22 No.7 July 2006

Table 1. Milestones in the development and implementation of new technologies for hookworm Date

Benzimidazole anthelmintics

1960s

Thiabendazole, first benzimidazole licensed for humans [57]

1970s

(i) Albendazole developed for veterinary use [58];

1980s

1990s

New anthelmintic drugs (tribendimidine) None

Anthelmintic vaccines

Other technologies

Preclinical development of attenuated Ancylostoma caninum larval vaccines [41] Commercialization of attenuated A. caninum larval vaccines [41]

None

Preclinical studies with purified and recombinant proteins [61,62] (i) Discovery of ASP-2 from A. caninum [45];

Theoretical framework of Anderson and May [10]

(ii) Discovery of APR-1 from A. caninum [63] None

(ii) First disease burden assessments in DALYs [64] None

None

None

None

Preclinical testing of ASP-2 [47–50]

None

None

Process development of Na-ASP-2 from Necator americanus [51] (i) cGMP manufacture of Na-ASP-2 [51]; (ii) Preclinical testing of APR-1 [55] Phase I clinical testing of Na-ASP-2

None

(i) Linking Na-ASP-2 vaccination with deworming in clinical trials in Brazil; (ii) Process development of Na-APR-1

Reassessment of hookworm disease burden (DALYs)

None

(ii) Mebendazole efficacy against hookworm demonstrated, approved for humans [59]; (iii) Benzimidazole resistance described [38,60] Albendazole approved for humans [58]

Synthesis of tribendimidine [40]

Large-scale trials of single-dose benzimidazoles (albendazole and mebendazole) [12,58]

Randomized, controlled trials of tribendimidine in humans [40]

2000

None

2001

2003

Adoption of World Health Assembly Resolution 54.19 Initiation of large-scale deworming of school-aged children [24] None

2004

None

None

2005

Proposed integration of deworming with other neglected tropical disease control [26] Proof of concept for integrated neglected tropical disease control [26,27]

None

2002

2006 (anticipated)

Clinical observation for the efficacy of tribendimidine against hookworm in humans [40] None

Equivalency trials in Africa (tribendimidine versus albendazole) [40]

specific mechanisms by which urbanization and poverty reduction reduce hookworm transmission remains elusive. Improvement in each individual component ordinarily attributed to poverty (e.g. sanitation, health education, footwear and underlying nutritional status) often has minimal impact on transmission [1]. For example, a recent study in Salvador, Brazil, found that improved drainage and sewerage had some impact on the prevalence, but not on the intensity, of hookworm [9]. Simply waiting for poverty reduction to occur in areas of intense hookworm transmission is not an acceptable option. Building on a mathematical framework developed by Anderson and May [10], a strategy emerged in the early 1990s to focus global anthelmintic chemotherapy (‘deworming’) efforts on school-aged children [11,12]. Among the reasons for focusing on school-aged children is that this population typically harbors a higher worm burden of soil-transmitted helminths than any other group [10–12], and, as a result, suffers from impairments in physical growth, cognitive and motor development, www.sciencedirect.com

None

(i) Mathematical modeling of hookworm transmission [34];

None

None

immune function and school performance [1,13–19]. Regular and periodic treatment with a benzimidazole anthelmintic has been shown significantly to improve childhood growth [14], physical fitness [15], cognition [16], school attendance [17] and host iron status [18,19]. In 2001, the 54th World Health Assembly passed a resolution (54.19) urging its member states to control the morbidity of hookworm and other soil-transmitted helminth infections by large-scale deworming (typically with one of the benzimidazoles, mebendazole or albendazole) of at least 75% of school-aged children at risk for infection (World Health Organization Partners for Parasite Control: www. who.int/wormcontrol). A major step forward in international coordination was achieved when a framework to Focus Resources on Effective School Health (FRESH) was launched at the World Education Forum in Dakar in April 2000 (www.freshschools.org and www.schoolsandhealth. org). This FRESH framework provides a common approach of agreed good practice for the effective implementation of deworming and other health services

Review

TRENDS in Parasitology

within the context of school health programs. However, school-aged children are not the only at-risk populations that might benefit from periodic deworming. In many regions, both preschool children and pregnant women also suffer from high-intensity hookworm infections and anemia [8,20–22], and, consequently, deworming is now also recommended as part of maternal health packages [23], although actual implementation is, in practice, variable. Frequent and periodic deworming with a single dose of either albendazole (400 mg) or mebendazole (500 mg) has now become the major approach to control the morbidity of hookworm and other soil-transmitted helminth infections. The frequency of deworming depends on the communitylevel prevalence or intensity, with moderate prevalence and low intensity infections requiring targeted treatment of school-aged children once a year, and high prevalence or high intensity infections requiring targeted treatment of school-aged children 2–3 times per year, in addition to treatment of preschool children and women of reproductive age in mother-and-child health programs when appropriate [24]. Short-term prospects: a ‘pro-poor’ package In much of the developing world, individuals infected with hookworm are also at high risk for other neglected tropical diseases, such as schistosomiasis, lymphatic filariasis, onchocerciasis and trachoma. The geographical and epidemiological overlap of these diseases is particularly extensive in sub-Saharan Africa and Brazil, where the incidence of polyparasitism is high [25,26]. Currently, the control programs for the major neglected tropical diseases are organized into vertical public–private partnerships (PPPs) that independently target at least one and up to four of these neglected tropical diseases. The PPPs operate in parallel, using either donated drugs or drugs obtained at reduced cost, but there are theoretical benefits of coordinating their activities to integrate the control of seven neglected tropical diseases: hookworm, ascariasis, trichuriasis, schistosomiasis, lymphatic filariasis, onchocerciasis and trachoma [26]. However, proof of concept for success in integrating neglected tropical disease control efforts will depend on several factors, including issues of compliance, drug interactions, emerging drug resistance, and harmonization of the timing of interventions and the populations targeted (e.g. children versus adults) for integration. In parallel with control program integration is a nascent effort to link neglected tropical diseases control with that of the ‘big three’ diseases: HIV/AIDS, tuberculosis and malaria [27]. This notion arose with the realization that these diseases are occurring predominantly in polyparasitized individuals. In sub-Saharan Africa for instance, the geographical overlap between hookworm and Plasmodium falciparum malaria is extensive, and the magnitude of anemia in patients with coinfection is greater than that experienced with individuals infected with either parasite alone (Brooker, S. et al., unpublished). Moreover, there is a suggestion that helminth infections might increase host susceptibility to malaria and HIV/AIDS (reviewed by Druilhe et al. [28], www.sciencedirect.com

Vol.22 No.7 July 2006

329

Bundy et al. [29], Borkow et al. [30] and Gallagher et al. [31]), although other evidence has failed to identify associations between worms and other diseases [32,33]. However, with the current widespread implementation of deworming at scale, there is an important opportunity, and indeed a scientific imperative, to investigate these associations further. If substantiated, deworming could become an important tool for helping to mitigate the disease burden of the better-known killer infectious diseases. Long-term prospects: incorporating new hookworm control tools There is a risk that the exclusive reliance on benzimidazoles for the control of soil-transmitted helminth infections will have less of an impact on hookworm than on ascariasis and trichuriasis. Unlike ascariasis and trichuriasis, in which the highest intensity infections occur almost exclusively in school-aged children, high hookworm intensities can also occur in adults [1,11,34]. Therefore, school-based deworming programs are not expected to reduce hookworm transmission significantly, whereas they might have some effect on ascariasis and trichuriasis transmission [34]. In addition, single-dose mebendazole often fails to remove adult hookworms effectively from the host gastrointestinal tract [35], and the rates of hookworm reinfection are high in areas of intense transmission [36]. Finally, the efficacy of benzimidazole anthelmintics against hookworm can diminish with increased and frequent use [37], leading to concerns that anthelmintic drug resistance might emerge in hookworms as it has for nematode parasites of ruminants [38]. The concerns about the heavy dependence on benzimidazoles for the control of hookworm have stimulated interest in the development of alternative or complementary control tools. Because hookworm is a neglected tropical disease with low commercial market potential, the pharmacopeia for this infection has not changed substantially over the past three decades. Of the few anthelmintic agents in the drug discovery pipeline (reviewed by Utzinger and Keiser [39]), the most promising is tribendimidine, which was first synthesized in China in the 1980s [40]. In humans, tribendimidine has efficacy superior to that of albendazole for the treatment of hookworm when used as a single dose of 400 mg [40]. Ultimately, it might be possible to forestall the emergence of drug resistance by rotating the benzimidazoles with tribendimidine or another anthelmintic drug. However, even a drug more efficacious than albendazole would not be expected to prevent the occurrence of posttreatment hookworm reinfection. This would require an effective vaccine. Proof of concept that it is possible to develop an anthelmintic vaccine against hookworm is based on the successful development of an irradiationattenuated larval vaccine for Ancylostoma caninum infection in dogs [41], which in turn was based on earlier work showing that antigens secreted by infective larvae elicit host immunity [42]. These early efforts stimulated a decade-long search to identify the major antigens secreted by infective hookworm larvae following host entry (reviewed by Hotez et al. [43]), which resulted in the

330

Review

TRENDS in Parasitology

discovery of two Ancylostoma-secreted proteins (ASPs), ASP-1 and ASP-2, belonging to the pathogenesis-related protein superfamily [44–46]. ASP-2 was subsequently shown to be an immunodominant antigen associated with protection from the attenuated canine larval vaccine [47]. Recombinant ASP-2 vaccines partially protected both dogs and hamsters challenged with either A. caninum [48] or Ancylostoma ceylanicum [49,50], respectively. In addition, studies in Brazil and China showed an association between individuals producing IgE against ASP-2 and a reduced risk of acquiring heavy hookworm burdens [48]. On the basis of these studies, Na-ASP-2 from Necator americanus was selected as a lead candidate for further vaccine development. The recombinant protein was expressed in yeast, and antibodies against the Na-ASP-2 hookworm vaccine (Na-ASP-2 adsorbed to Alhydrogelw) recognize the native antigen and also inhibit hookworm larval tissue migration in vitro [51]. The vaccine has recently completed process development and pilot ‘current Good Manufacturing Practices’ manufacture [51,52]. Through the sponsorship of the Human Hookworm Vaccine Initiative, a Washington, DC-based PPP (www.sabin.org/hookworm.htm), an investigational new drug application for the Na-ASP-2 hookworm vaccine was submitted to the US Food and Drug Administration, and Phase I testing in healthy adult volunteers has commenced. Pending regulatory approval in Brazil, plans are underway to test the vaccine in adults living in a hookworm-endemic area, followed by age de-escalation studies to school-aged children. Because the vaccine targets only the infective larvae, it will be necessary to deworm study participants before vaccination. Linking vaccination with deworming affords opportunities to integrate vaccine development with school-based programs, and provide a model that could guide future international recommendations for hookworm vaccine use. Future directions Because the Na-ASP-2 hookworm vaccine might be only partially effective in reducing worm burdens and host blood loss [52], the Human Hookworm Vaccine Initiative plans to develop and add a second antigen to the vaccine – one derived from the adult blood-feeding stage. The antigen showing the greatest promise is an aspartic protease (Na-APR-1) required by the parasite to initiate the ordered digestion of host hemoglobin [53,54]. Dogs vaccinated with recombinant APR-1 exhibited diminished host blood loss, worm burdens and fecal egg counts. It was also shown that the gut of hookworms recovered from the vaccinated dogs was damaged after ingesting host antibody with the blood meal [55]. Studies are in progress to take Na-APR-1 through process development and manufacture, followed by coformulation of Na-ASP-2 and Na-APR-1 into a bivalent human hookworm vaccine (HHV). By diminishing reliance on anthelmintic drugs, it is anticipated that widespread use of the HHV will prolong the interval required between periodic drug treatments and reduce the likelihood or frequency of anthelmintic www.sciencedirect.com

Vol.22 No.7 July 2006

drug resistance, while at the same time preventing hookworm disease. To ensure the sustainability of the HHV, additional efforts are in progress to transfer the vaccine development technology to manufacturers located in innovative developing countries where hookworm is endemic [56]. This, together with efforts to create an international consensus for use of the HHV in schools, comprises the core of a global access plan [52]. The coming decade promises to be an exciting one in the history of hookworm control as new and appropriate technologies are folded into FRESH and other school-based health systems. Acknowledgements The authors are supported by the Human Hookworm Vaccine Initiative of the Sabin Vaccine Institute and the Bill and Melinda Gates Foundation. In addition, J.B. is supported by an International Research Scientist Development Award from the NIH-Fogarty International Center (K01TW00009), A.L. is supported by an R.D. Wright Career Development Award from the National Health and Medical Research Council of Australia and S.B. is supported by a Wellcome Trust Advanced Research Fellowship (073656).

References 1 Brooker, S. et al. (2004) Human hookworm infection in the 21st century. Adv. Parasitol. 58, 197–288 2 Bungiro, R. and Cappello, M. (2004) Hookworm infection: new developments and prospects for control. Curr. Opin. Infect. Dis. 17, 421–426 3 Bethony, J. et al. (2006) Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet 367, 1521–1532 4 de Silva, N.R. et al. (2003) Soil-transmitted helminth infections: updating the global picture. Trends Parasitol. 19, 547–551 5 Hotez, P.J. (2002) China’s hookworms. China Q. 172, 1029–1041 6 Xu, L-Q. et al. (1995) Soil-transmitted helminthiases: nationwide survey in China. Bull. World Health Organ. 73, 507–513 7 National Institute of Parasitic Diseases, China CDC (2005) Report on the National Survey of Current Situation of Major Human Parasitic Diseases in China. Ministry of Health, P.R. China. 8 Stoltzfus, R.J. et al. (1997) Hookworm control as a strategy to prevent iron deficiency. Nutr. Rev. 55, 223–232 9 Moraes, L.R. et al. (2004) Impact of drainage and sewerage on intestinal nematode infections in poor urban areas in Salvador, Brazil. Trans. R. Soc. Trop. Med. Hyg. 98, 197–204 10 Anderson, R.M. and May, R.M. (1982) Population dynamics of human helminth infections: control by chemotherapy. Nature 297, 557–563 11 Bundy, D.A.P. (1988) Population ecology of intestinal helminth infections in human communities. Philos. Trans. R. Soc. Lond. B Biol. Sci. 321, 405–420 12 Bundy, D.A. et al. (1990) Control of geohelminths by delivery of targeted chemotherapy through schools. Trans. R. Soc. Trop. Med. Hyg. 84, 115–120 13 Partnership for Child Development (1997) Better health, nutrition and education for the school-aged child. Trans. R. Soc. Trop. Med. Hyg. 91, 1–2 14 Stephenson, L.S. et al. (1989) Treatment with a single dose of albendazole improves growth of Kenyan schoolchildren with hookworm, Trichuris trichiura, and Ascaris lumbricoides infections. Am. J. Trop. Med. Hyg. 41, 78–87 15 Stephenson, L.S. et al. (1990) Improvements in physical fitness of Kenyan schoolboys infected with hookworm, Trichuris trichiura and Ascaris lumbricoides following a single dose of albendazole. Trans. R. Soc. Trop. Med. Hyg. 84, 277–282 16 Sakti, H. et al. (1999) Evidence for an association between hookworm infection and cognitive function in Indonesia school children. Trop. Med. Int. Health 4, 322–334 17 World Bank (2003) School deworming at a glance. http://siteresources. worldbank.org/INTPHAAG/Resources/AAGDewormingEng110603. pdf

Review

TRENDS in Parasitology

18 Bhargava, A. et al. (2003) Anthelmintic treatment improves the hemoglobin and serum ferritin concentrations of Tanzanian schoolchildren. Food Nutr. Bull. 24, 332–342 19 Stoltzfus, R.J. et al. (2004) Low dose daily iron supplementation improves iron status and appetite, but not anemia, whereas quarterly anthelminthic treatment improves growth, appetite and anemia in Zanzibari preschool children. J. Nutr. 134, 348–356 20 Brooker, S. et al. (1999) The epidemiology of hookworm infection and its contribution to anaemia among pre-school children on the Kenyan coast. Trans. R. Soc. Trop. Med. Hyg. 93, 240–246 21 Bundy, D.A. et al. (1995) Hookworm infection in pregnancy. Trans. R. Soc. Trop. Med. Hyg. 89, 521–522 22 Christian, P. et al. (2004) Antenatal anthelmintic treatment, birthweight, and infant survival. Lancet 364, 981–983 23 Allen, H.E. et al. (2002) New policies for using anthelmintics in high risk groups. Trends Parasitol. 18, 381–382 24 World Health Organization (2002) Prevention and Control Of Schistosomiasis and Soil-Transmitted Helminthiasis. Report of a WHO expert committee. WHO Technical Report Series 912, p. 35, World Health Organization 25 Raso, G. et al. (2004) Multiple parasite infections and their relationship to self-reported morbidity in a community of rural Cote d’Ivoire. Int. J. Epidemiol. 33, 1092–1102 26 Molyneux, D.H. et al. (2005) “Rapid-impact interventions”: how a policy of integrated control of Africa’s neglected tropical diseases could benefit the poor. PLoS Med 2, e336 27 Hotez, P.J. et al. (2006) Incorporating a rapid impact package for neglected tropical diseases with programs for HIV/AIDS, tuberculosis and malaria, a comprehensive pro-poor health policy and strategy for the developing world. PLoS Med 3, e102 28 Druilhe, P. et al. (2005) Worms can worsen malaria: towards a new means to roll back malaria? Trends Parasitol. 21, 359–362 29 Bundy, D. et al. (2000) Good worms or bad worms: do worm infections affect the epidemiological patterns of other diseases. Parasitol. Today 16, 273–274 30 Borkow, G. and Bentwich, Z. (2004) Chronic immune activation associated with chronic helminthic and human immunodeficiency virus infections: role of hyporesponsiveness and anergy. Clin. Microbiol. Rev. 17, 1012–1030 31 Gallagher, M. et al. (2005) The effects of maternal helminth and malaria infections on mother-to-child HIV transmission. AIDS 19, 1849–1855 32 Shapiro, A.E. et al. (2005) Epidemiology of helminth infections and their relationship to clinical malaria in southwest Uganda. Trans. R. Soc. Trop. Med. Hyg. 99, 18–24 33 Brown, M. et al. (2004) Helminth infection is not associated with faster progression of HIV disease in coinfected adults in Uganda. J. Infect. Dis. 190, 1869–1879 34 Chan, M.S. et al. (1997) Transmission patterns and the epidemiology of hookworm infection. Int. J. Epidemiol. 26, 1392–1400 35 Bennett, A. and Guyatt, H. (2000) Reducing intestinal nematode infection: efficacy of albendazole and mebendazole. Parasitol. Today 16, 71–74 36 Albonico, M. et al. (1995) Rate of reinfection with intestinal nematodes after treatment of children with mebendazole or albendazole in a highly endemic area. Trans. R. Soc. Trop. Med. Hyg. 89, 538–541 37 Albonico, M. et al. (2003) Efficacy of mebendazole and levamisole alone or in combination against intestinal nematode infections after repeated targeted mebendazole treatment in Zanzibar. Bull. World Health Organ. 81, 343–352 38 Albonico, M. et al. (2004) Monitoring drug efficacy and early detection of drug resistance in human soil-transmitted nematodes: a pressing public health agenda for helminth control. Int. J. Parasitol. 34, 1205–1210 39 Utzinger, J. and Keiser, J. (2004) Schistosomiasis and soil-transmitted helminthiasis: common drugs for treatment and control. Expert Opin. Pharmacother. 5, 263–286 40 Xiao, S.H. et al. (2005) Tribendimidine: a promising, safe and broadspectrum anthelmintic agent from China. Acta Trop. 94, 1–14 41 Miller, T.A. (1978) Industrial development and field use of the canine hookworm vaccine. Adv. Parasitol. 16, 333–342

www.sciencedirect.com

Vol.22 No.7 July 2006

331

42 Cort, W.W. and Otto, G.F. (1940) Immunity in hookworm disease. Rev. Gastroenterol. 7, 2–14 43 Hotez, P.J. et al. (2003) Progress in the development of a recombinant vaccine for human hookworm disease: the Human Hookworm Vaccine Initiative. Int. J. Parasitol. 33, 1245–1258 44 Hawdon, J.M. et al. (1996) Cloning and characterization of Ancylostoma secreted protein. A novel protein associated with the transition to parasitism by infective hookworm larvae. J. Biol. Chem. 271, 6672–6678 45 Hawdon, J.M. et al. (1999) Ancylostoma secreted protein 2: cloning and characterization of a second member of a family of nematode secreted proteins from Ancylostoma caninum. Mol. Biochem. Parasitol. 99, 149–165 46 Asojo, O.A. et al. (2005) X-ray structure of Na-ASP-2, a pathogenesisrelated-1 protein from the nematode parasite, Necator americanus, and a vaccine antigen for human hookworm infection. J. Mol. Biol. 346, 801–814 47 Fujiwara, R.T. et al. (2005) Vaccination with irradiated Ancylostoma caninum third stage larvae induces a Th2 protective response in dogs. Vaccine 24, 501–509 48 Bethony, J. et al. (2005) Antibodies against a secreted protein from hookworm larvae reduce the intensity of hookworm infection in humans and vaccinated laboratory animals. FASEB J. 19, 1743–1745 49 Goud, G.N. et al. (2004) Cloning, yeast expression, isolation, and vaccine testing of recombinant Ancylostoma secreted protein (ASP)-1 and ASP-2 from Ancylostoma ceylanicum. J. Infect. Dis. 189, 919–929 50 Mendez, S. et al. (2005) Effect of combining the larval antigens Ancylostoma secreted protein 2 (ASP-2) and metalloprotease 1 (MTP1) in protecting hamsters against hookworm infection and disease caused by Ancylostoma ceylanicum. Vaccine 23, 3123–3130 51 Goud, G.N. et al. (2005) Expression of the Necator americanus hookworm larval antigen Na-ASP-2 in Pichia pastoris and purification of the recombinant protein for use in human clinical trials. Vaccine 23, 4754–4764 52 Brooker, S. et al. (2005) Epidemiologic, immunologic and practical considerations in developing and evaluating a human hookworm vaccine. Expert Rev. Vaccines 4, 35–50 53 Williamson, A. et al. (2002) Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity. FASEB J. 16, 1458–1460 54 Williamson, A. et al. (2004) A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms. J. Biol. Chem. 279, 35950–35957 55 Loukas, A. et al. (2005) Vaccination with recombinant aspartic hemoglobinase reduces parasite load and protects against anemia after challenge infection with blood-feeding hookworms. PLoS Med 2, e295 56 Morel, C.M. et al. (2005) Health innovation networks to help developing countries address neglected diseases. Science 309, 401–404 57 Salunkhe, D.S. et al. (1964) Clinical evaluation of a new anthelmintic thiabendazole (2-(4 0 -thiazolyl)-benzimidazole). Am. J. Trop. Med. Hyg. 13, 412–416 58 Horton, J. (2002) Albendazole: a broad spectrum anthelmintic for treatment of individuals and populations. Curr. Opin. Infect. Dis. 15, 599–608 59 Banerjee, D. et al. (1972) A clinical trial of mebendazole (R17,635) in cases of hookworm infection. Indian J. Med. Res. 60, 562–566 60 Kates, K.C. et al. (1973) Experimental development of a cambendazole-resistant strain of Haemonchus contortus in sheep. J. Parasitol. 59, 169–174 61 Hotez, P.J. et al. (1987) Hookworm antigens: the potential for vaccination. Parasitol. Today 3, 247–249 62 Carr, A. and Pritchard, D.I. (1987) Antigen expression during development of the human hookworm, Necator americanus (Nematoda). Parasite Immunol. 9, 219–234 63 Harrop, S.A. et al. (1996) Acasp, a gene encoding a cathepsin D-like aspartic protease from the hookworm Ancylostoma caninum. Biochem. Biophys. Res. Commun. 227, 294–302 64 Murray, C.J. et al. (1994) The global burden of disease in 1990: summary results, sensitivity analysis and future directions. Bull. World Health Organ. 72, 495–509