[Show abstract][Hide abstract] ABSTRACT: Hookworm infection is one of most important parasitic infection of humans, occurring in 740 million people. Here we report the protective vaccination of dogs with Ac-16, an immunodominant surface antigen from the hookworm Ancylostoma caninum. We show that immunization with Ac-16 formulated with AS03 elicited specific humoral and cellular immune responses and provided partial protection against hookworm infection and morbidity as evidenced by a significant reduction of hookworm egg counts (64% reduction; P = 0.0078) and worm-induced blood loss (P < 0.05). Moreover, specific anti-Ac-16 antibodies recognized the native protein on the surface of third-stage larvae and blocked their migration through tissue in vitro. Our data support the use of Ac-16 as a potential candidate for vaccination against hookworm infection.
[Show abstract][Hide abstract] ABSTRACT: While X-irradiated live parasites are not an acceptable proposition for human vaccination, they offer a ready experimental system to explore mechanisms by which immunity against hookworm infection may be induced in humans. As such, we sought to further elucidate the details of this highly protective immune response induced by the irradiated vaccine in canids, with special emphasis on the cellular aspects of the response. Vaccination with irradiated L3 induced high production of antibodies and strong PBMCs proliferation to crude L3 antigen preparation. Elevated IL-4 production was also observed in vaccinated dogs, especially in relation to IFN-gamma production (IL-4/IFN-gamma ratio). Serum from vaccinated animals inhibited penetration of L3 through canine skin in vitro by 60%. Finally, vaccinated animals had a strong antibody response to ASP-2, a promising vaccine antigen that is an excretory-secretory product of L3. These results add further support the idea that the Th2 immune response is required to generate protective immunity against hookworm larvae and that ES molecules released during this developmental stage are likely targets of this response.
[Show abstract][Hide abstract] ABSTRACT: 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.
The FASEB Journal 11/2005; 19(12):1743-5. DOI:10.1096/fj.05-3936fje · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the cloning and expression of Ac-GST-1, a novel glutathione S-transferase from the adult hookworm Ancylostoma caninum, and its possible role in parasite blood feeding and as a vaccine target. The predicted Ac-GST-1 open reading frame contains 207 amino acids (mass, 24 kDa) and exhibited up to 65% amino acid identity with other nematode GSTs. mRNA encoding Ac-GST-1 was detected in adults, eggs, and larval stages, but the protein was detected only in adult hookworm somatic extracts and excretory/secretory products. Using antiserum to the recombinant protein, Ac-GST-1 was immunolocalized to the parasite hypodermis and muscle tissue and weakly to the intestine. Recombinant Ac-GST-1 was enzymatically active, as determined by conjugation of glutathione to a model substrate, and exhibited a novel high-affinity binding site for hematin. The possible role of Ac-GST-1 in parasite heme detoxification during hemoglobin digestion or heme uptake prompted interest in evaluating it as a potential vaccine antigen. Vaccination of dogs with Ac-GST-1 resulted in a 39.4% reduction in the mean worm burden and 32.3% reduction in egg counts compared to control dogs following larval challenge, although the reductions were not statistically significant. However, hamsters vaccinated with Ac-GST-1 exhibited statistically significant worm reduction (53.7%) following challenge with heterologous Necator americanus larvae. These studies suggest that Ac-GST-1 is a possible drug and vaccine target for hookworm infection.
Infection and Immunity 11/2005; 73(10):6903-11. DOI:10.1128/IAI.73.10.6903-6911.2005 · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The recombinant larval protein Na-ASP-2 from the human helminth parasite Necator americanus is a lead candidate for a human hookworm vaccine. We have characterized the humoral and cellular immune responses elicited by the Na-ASP-2 formulated with the aluminum-based adjuvant Alhydrogel in the Sprague Dawley rat. We demonstrated that Na-ASP-2 vaccine induced a strong antibody response at all doses tested, as evidenced by high specific IgG1, IgG2a and IgM titers. High IgG antibody titers were maintained up to three months post vaccination and were boosted by an additional vaccine dose. Specific cell proliferation and a Th2 cytokine profile were also observed in peripheral blood of all rats immunized with the formulated vaccine. Host IL-6 levels were also significantly elevated. These data provide evidence that the immune response of the Na-ASP-2 vaccine is robust and durable and therefore suitable for the further clinical development of the Na-ASP-2 vaccine.
Human vaccines 05/2005; 1(3):123-8. DOI:10.4161/hv.1.3.1924 · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A cDNA encoding a surface-associated antigen was cloned from an Ancylostoma caninum infective larvae (L(3)) cDNA library by immunoscreening with pooled human immune sera. The sera were obtained from individuals living in an Ancylostoma duodenale hookworm-endemic region of China, who had light intensity infections and high antibody titers against A. caninum L(3). Ancylostoma caninum surface-associated antigen-1 is encoded by an 843 bp mRNA with a predicted open reading frame of 162 amino acids. Recombinant Ancylostoma caninum surface-associated antigen-1 was expressed in Escherichia coli and used to prepare a specific antiserum. A Western blot with anti-Ancylostoma caninum surface-associated antigen-1 specific antiserum showed that native Ancylostoma caninum surface-associated antigen-1 protein is expressed by both L(3) and adult hookworms; RT-PCR confirmed that the mRNA is transcribed in both stages. In adult hookworms, the protein localised to the basal layer of the cuticle and hypodermis of adult worms. Serological analysis determined that recombinant Ancylostoma caninum surface-associated antigen-1 protein is recognised by 61% of human sera from a Necator americanus hookworm endemic area in China, indicating the antigen is immunodominant. Anti-Ancylostoma caninum surface-associated antigen-1 antiserum partially inhibited (46.7%) invasion of hookworm L(3) into dog skin in vitro. Together these results suggest that Ancylostoma caninum surface-associated antigen-1 offers promise as a protective vaccine antigen.
International Journal for Parasitology 09/2004; 34(9):1037-45. DOI:10.1016/j.ijpara.2004.04.015 · 3.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Parasitic helminths induce chronic infections in their hosts although, with most human helminthiases, protective immunity gradually develops with age or exposure of the host. One exception is infection with the human hookworm, Necator americanus, where virtually no protection ensues over time. Such observations suggest these parasites have developed unique mechanisms to evade host immunity, leading us to investigate the role of the excretory/secretory (ES) products of adult N. americanus in manipulating host immune responses. Specifically, we found that a protein(s) from ES products of adult N. americanus bound selectively to mouse and human NK cells. Moreover, incubation of purified NK cells with N. americanus ES products stimulated the production of augmented (4- to 30-fold) levels of IFN-gamma. This augmentation was dependent on the presence of both IL-2 and IL-12 and was endotoxin-independent. This is the first report of a pathogen protein that binds exclusively to NK cells and the first report of a nematode-derived product that induces abundant levels of cytokines from NK cells. Such an interaction could provide a means of cross-regulating deleterious Th2 immune responses in the host, thereby contributing to the long-term survival of N. americanus.
The Journal of Immunology 09/2004; 173(4):2699-704. DOI:10.4049/jimmunol.173.4.2699 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.
International Journal for Parasitology 10/2003; 33(11):1245-58. DOI:10.1016/S0020-7519(03)00158-9 · 3.40 Impact Factor