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Title: Development of a vaccine against the human hookworm, Necator americanus
Author: Girod, Nadine
ISNI:       0000 0001 3500 5563
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2001
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Necator americanus infections cause iron-deficiency anaemia contributing, after years of infection, to a deficit in both physical and intellectual development, particularly in children. Despite the wide range of anthelminthic treatments available, the prevalence of N. americanus infection remains a major public health concern. Therefore, the development of a vaccine, providing life-long protection, represents a useful means of controlling hookworm infection. In this thesis, an attempt was made to identify candidate vaccine molecules and establish which immune responses can confer protection. Since N. americanus infect their host by skin penetration, the migratory behaviour of infective larvae through human, hamster and mouse ex vivo skins was studied by incubation with proteinase inhibitors. Skin penetration was shown to involve serine, aspartyl and métallo proteinases. Pepstatin A, an aspartyl proteinase inhibitor, was the most potent inhibitor, significantly inhibiting by up to 50 % larval penetration through human and hamster skins. The aspartyl proteinase(s) was also important in the penetration of gamma-irradiated (40 krad) larvae through ex vivo skin. However the migration of irradiated larvae, through ex vivo skin, was delayed compared to that of live larvae, despite the production of more excretory/secretory proteins containing proteolytic activity. A successful vaccination protocol was then established, using gamma-irradiated larvae as the vaccine. Indeed, two immunisations with 300 gamma-irradiated larvae (40 krad) were shown to induce a high level of proteetion against N. americanus infection in BALB/c mice, based on worm recovery in the lungs and intestines. Inflammatory foci and mast cell proliferation were present in the skin of vaccinated animals. In addition, analysis of cytokine mRNA in the skin by RT-PCR indicated that Th2 type cytokines were dominant in the vaccinated group. Reduced pathology was also observed in the lungs of vaccinated animals. Levels of IL4 and y-IFN cytokines, determined in ConA stimulated lymphocytes, from the spleen, axillary and mesenteric lymph nodes, using ELISA, indicated a higher IL4/y-IFN ratio in the vaccinated groups, especially in the skin draining lymph nodes. On the other hand, a primary infection suppressed a Th2 response to induce a Thl response. It was therefore clear that irradiated larvae promote a protective Th2 response against subsequent infection, initiated by the entrance of the parasite into the skin. Based on the previous findings, excretory/secretory products from irradiated larvae and larval aspartyl proteinases were used as candidate vaccines. Mice were vaccinated with excretory/secretory products fi*om irradiated larvae in alum, a Th2 adjuvant. This vaccination resulted in a significant reduction (50.8 %) in worm burden in the intestine of vaecinated animals. However, a comparative study showed that alum induced higher levels of protection than Freund's adjuvant, a Thl adjuvant. In an attempt to improve antigen presentation, mice were vaccinated intradermally with mature dendritic cells pulsed with worm cell lysate. Finally, since the aspartyl protease appeared crucial for infection, a recombinant aspartyl proteinase was then used as a vaccine; unfortunately this was not successful. It is therefore likely that a successful vaeeine against N. americanus will contain several defined antigens targeted against different stages of the parasite's life cycle, together with an adjuvant capable of inducing a Th2 response.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Parasite; Immune response; Skin