BACKGROUND
Hookworm Infection: The Basics
Epidemiology
In humans, the intestinal nematodes Ancylostoma duodenale and Necator americanus cause hookworm infection. Current estimates of global hookworm prevalence range from 600-740 million cases (1, 2). Additionally, an estimated 3.2 billion people are at risk for acquiring hookworm infection (2). As seen on the map below (Figure 1), hookworm infection is highest in sub-Saharan Africa, regions of Southeast Asia, India, Latin America, China, and the Middle East (3).
Hookworm causes an estimated 65,000 deaths annually. However, the majority of the global burden of disease is due to associated morbidity and not mortality.
Life Cycle
Hookworm infection occurs when third-stage larvae (L3), known as filariform, 
come in contact with and penetrate human skin. Because infective larvae mature in the soil, they tend to invade via the hands or feet. Once inside the human, the larvae migrate through the veins until they reach the lungs. Larvae are then coughed up, swallowed, and travel to the small intestine where they develop into the adult stage. Adults use their teeth or cutting plates to attach to the intestinal lumen, causing blood loss in the infected individual (see Figure 2). Adult hookworms typically live inside their human host for one to two years, and each female produce thousands of eggs per day. The eggs exit the human in the stool and then hatch into the non-infective rhabditiform larvae. Over the course of two to three weeks, rhabditiform larvae molt three times and develop into the infective L3 or filariform larvae. These larvae can then penetrate human skin and repeat the cycle (4-6). Please refer to Figure 3 for an overview of the hookworm life cycle.
Clinical Manifestations
When the filariform larvae first penetrate the skin, an allergic reaction, known as ground itch, may occur. Hookworm infection may also result in pneumonitis when larvae migrate through the lungs. Abdominal pain may occur as adult worms first attach to the intestine (7). However, the main clinical manifestations of hookworm infection are due to prolonged intestinal blood loss. Specifically, if the infected individual’s diet is insufficient in protein and iron, anemia may develop. In general, young children and women of reproductive age have low iron stores and thus, are more susceptible to chronic blood loss and anemia from hookworm infection (5, 6). Severe anemia may result in heart failure, tissue swelling, growth retardation, or mental impairment (7).
Diagnosis & Treatment
Identification of hookworm eggs in stool samples is a common means of diagnosing infection. Once diagnosed, hookworm is usually treated with the drug albendazole. However, mebendazole and pyrantel pamoate also successfully treat hookworm infections. If the person suffers from anemia, iron supplementation may also be appropriate.
Current Prevention & Control Measures
One method of hookworm control is through school-based deworming programs. In areas of high prevalence, it is cost-effective to simply give all school-aged children a single dose of albendazole, rather than attempting to diagnose individual cases.
The benefits of such a school-wide campaign are two-fold. Firstly, children who are infected with hookworm will receive proper treatment, leading to significant overall health improvements. Moreover, in theory, such mass chemotherapy campaigns have a positive effect on the community as a whole, since treatment prevents further transmission. Specifically, when an individual is treated for hookworm, the patient no longer excretes hookworm eggs, so there are no larvae to develop into their filariforms, which can then infect other people. Community-wide treatment can produce significant reductions in hookworm incidence (6).
Though school-based deworming campaigns are effective in ensuring that all vulnerable children receive treatment, there are several limitations to this control method. Firstly, since deworming does not improve host immunity to hookworm, re-infection can occur soon after these campaigns. Re-infection is especially common in regions where hookworm is endemic, since high amounts of infectious larvae exist in the soil even after deworming. Additionally, though hookworm is more common in children, high hookworm intensity may also occur in adults. Therefore, treating solely school-aged children through deworming programs is not effective in completely eliminating hookworm in the larger community (3).
Vaccination is an alternative approach to hookworm control. A vaccine has several advantages over current control methods. Firstly, unlike deworming methods, the basic premise of a vaccine involves building host immunity and thus, eliminating the potential for re-infection. Additionally, using a vaccine as opposed to mass drug treatment circumvents the possibility of hookworm developing drug resistance (3).
Previous Efforts to Develop a Vaccine
Proof of concept that protective immunity against hookworm disease is feasible came from the development of an X-ray irradiated canine vaccine against the dog hookworm Ancylostoma caninum (8). This vaccine contained attenuated third-stage (L3) A. caninum larvae, which retained the ability to secrete proteins. These secreted proteins elicited protective Th2 immunity—a primarily antibody response to extracellular pathogens—to hookworm disease. The vaccine was licensed in 1973 but discontinued in 1975 (9).
Despite its commercial failure, development and subsequent discontinuation of the canine vaccine informed research on vaccines against human hookworm disease. The dog vaccine’s creators established three “characteristics of attenuation” that could be used to assess efficacy of vaccine candidates: 1) reduced larval infectivity determined by adult worm burden (number of worms in the intestine); 2) reduced pathogenicity of adult worms determined by host hemoglobin levels; and 3) reduced female fecundity determined by egg per gram feces (9, 10, 11). In addition, their research proved that a hookworm vaccine did not need to provide sterilizing immunity, immunity preventing infection completely. Instead, it must decrease worm burden enough to eliminate hookworm disease (anemia-associated morbidity and sequelae) (11).
Current Efforts: the Human Hookworm Vaccine Initiative (HHVI)
Founded in 2000, the Human Hookworm Vaccine Initiative (HHVI) is an arm of the Sabin Vaccine Institute in Washington, D.C., funded by the Bill and Melinda Gates Foundation to develop a recombinant protein vaccine against human hookworm disease (12, 13). Because it is unadvisable to use live attenuated parasites for human vaccination, HHVI relied on information about secreted hookworm proteins in dogs to identify and characterize possible antigens for their recombinant vaccines. Researchers incorporated the “characteristics of attenuation” when devising a scoring system to evaluate potential recombinant antigens. Criteria include (9):
1) pathology—reductions in worm burden and host blood loss in animal models
2) transmission—reduction in fecal egg count
3) development feasibility—previously known protein function and structure
4) immunoepidemiology—correlation between immune responses and infection intensities in naturally infected populations
5) existence of protective homologues—presence in other pathogens
Using these criteria, the HHVI isolated and tested over 20 vaccine candidates. They are simultaneously developing separate larval and adult hookworm antigens, Necator americanus (Na)-ASP-2 and Na-APR-1 proteins, respectively. The ultimate goal is to combine these two vaccines into a single bivalent formulation.
Larval Antigen: the Na-ASP-2 vaccine
Targeting the infectious larval stage, the Na-ASP-2 vaccine contains recombinant Ancylostoma secreted protein 2 (ASP-2), cloned from a Chinese strain of N. americanus and expressed in yeast Pichia pastoris, plus Alhydrogel® adjuvant. Secreted by both N. americanus and A. duodenale, ASP-2 is a larvae-specific, cysteine-rich member of the pathogenesis related protein (PRP) superfamily. Host antibodies for Na-ASP-2, known as anti-Na-ASP-2 antibodies, inhibit tissue penetration and migration of L3, so fewer parasites reach the intestines to feed, mate, and lay eggs (13, 14). Thus, vaccination reduces worm burden and transmission without directly targeting adult stages.
Laboratory and epidemiologic evidence suggests that this larval antigen elicits protective immunity against disease. In China and Brazil, individuals positive for anti-ASP-2 antibodies exhibit more mild hookworm infection (15). Furthermore, Na-ASP-2 plus Alhydrogel® inoculation is immunogenic and provides lasting protection in rats. A three vaccine schedule produced significant increases in antibody levels with a marked Th2-type cytokine profile. That antibody titers were maintained for up to 3 months post-vaccination and boosted by additional inoculation indicates durable immunity exists in rats (14).
A Phase I clinical trial of the Na-ASP-2 vaccine among healthy human volunteers in the US was completed in May 2006; results soon to be published (16). A second trial among previously infected adults in Brazil will begin later this year. HHVI is also working to transfer Na-ASP-2 development and manufacturing to Instituto Butantan, a biomedical center affiliated with the State Secretary of Health, São Paulo, Brazil (17). Efforts will culminate in Phase II studies of efficacy among hookworm-infected, school-aged children in Brazil (16).
Adult Hookworm Antigen: the Na-APR-1 vaccine and other potential candidates
Currently in an earlier stage of development than the larval vaccine, Na-APR-1 antigen is the lead candidate for vaccination against adult hookworms. Wild-type Apr-1 encodes a cathepsin D-like aspartic protease, expressed in the gastrointestinal tract of adult hookworms and necessary for proteolytic cleavage of human hemoglobin (termed a hemoglobinase). When ingested by the parasite, antibodies for Na-APR-1 may bind to and neutralize the hemoglobinase such that adult worms are unable to digest blood meals and “starve” (18).
Promising preclinical trials used A. caninum (Ac)-APR-1, an orthologue of Na-APR-1, to vaccinate beagles. Intramuscular injection induced antibody and cellular responses; decreased fecal egg count; and reduced adult worm burden after subsequent challenge with infective L3. Most importantly, vaccinated dogs had significantly higher hemoglobin concentrations compared to controls, and four out of five immunized beagles did not develop anemia (9).
Given these findings, the HHVI has begun pilot manufacturing and process development of the Na-APR-1 vaccine with the hopes of submitting an Investigation New Drug (IND) application in 2009 (13). The adult vaccine will undergo Phase I clinical testing. While Na-APR-1 remains the chief vaccine candidate, researchers are investigating other adult antigens, including GSP-1 and CP-2 (16).
The Bivalent Human Hookworm Vaccine (HHV)
After completion of Phase II trails for the larval and Phase I trials for the adult antigen, the vaccines will be combined with at least one adjuvant to create an injectable, bivalent Human Hookworm Vaccine (HHV) (13). As with the canine vaccine, HHV will not prevent infections but control hookworm disease and interrupt transmission to limit morbidity and incidence (9). It will disrupt the hookworm lifecycle at multiple developmental stages to ensure more successful disease prevention. The larval antigen elicits antibodies that inhibit migration and decrease the number of feeding adults in the intestines (9). Fewer adults result in less blood loss, which prevents the development of anemia and limits female fecundity. The adult antigen elicits antibodies against a parasitic digestive enzyme to limit blood feeding. Because each adult consumes less blood, risk of anemia decreases. The antigen also reduces fecal egg count by depriving worms of food stores. Clinical trials of the bivalent formulation are anticipated to begin no earlier than 2011 (13).
