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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 21, 2005 as doi:10.1096/fj.05-3936fje. |
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* Department of Microbiology and Tropical Medicine, The George Washington University, Washington DC, USA;
Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Brisbane, and Australian Centre for International Tropical Health and Nutrition, The University of Queensland, Australia;
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK;
Lindsley F. Kimball Research Institute, New York Blood Center;
|| Centro de Pesquisas René Rachou, Fundação Instituto Oswaldo Cruz (FIOCRUZ), Belo Horizonte, Minas Gerais, Brazil; and
|| Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, China
1 Correspondence: Department of Microbiology and Tropical Medicine, The George Washington University, Ross Hall, Room 736, 2300 Eye St. NW, Washington DC 20037, USA. E-mail: jeff{at}cpqrr.fiocruz.br; or photez{at}gwu.edu
SPECIFIC AIMS
We examined the antibody responses of individuals from hookworm endemic areas of Brazil and China against the most abundant antigens secreted by third-stage infective hookworm larvae (L3), the Ancylostoma Secreted Proteins, ASP-1 and ASP-2. These results suggested that host antibodies against ASP-2 could protect again heavy infection and led us to further test whether ASP-2 could serve as a vaccine antigen in a permissive, naturally occurring animal model (canine). Canine anti-ASP-2 antibodies were examined for their ability to inhibit hookworm larval migration through tissue in vitro and to reduce host hookworm burdens and fecal egg counts in vivo.
PRINCIPAL FINDINGS
1. Cloning and expression of ASPs
During the 1960s and 1970s, canines were successfully vaccinated with irradiated L3 of the canine hookworm, Ancylostoma caninum. Based on the importance of L3 secreted products in mediating protection obtained with this vaccine, we cloned the genes encoding the most abundant L3 secreted antigens: ASP-1 and ASP-2. ASP-1 and ASP-2 were expressed in eukaryotic vectors, the yeast Pichia pastoris, and Sf9 insect cells, respectively. Recombinant ASP-1 is a 44 kDa protein and ASP-2 is a 24 kDa protein with one putative N-linked glycosylation site. The recombinant proteins were secreted by host cells and purified by nickel affinity chromatography.
2. Human immunoepidemiology studies with ASP-1 and ASP-2
We examined the antibody responses of individuals from hookworm endemic areas of Brazil (Minas Gerais State) and China (Hainan Province) against ASP-1 and ASP-2. The mean age for these two populations was 36 and 33 years of age, respectively, with the prevalence of hookworm infection 54% in Brazilian endemic area and 59% in the Chinese endemic area. The mean quantitative fecal egg counts were 1645 eggs per gram of feces (EPG) for Brazil and 1723 EPG for China. Because > 20% of the study sample had EPGs over 3999, both sites qualify as high intensity hookworm transmission areas by WHO standards. Necator americanus was identified as the predominant hookworm in both regions.
Logistic regression was used to investigate the association of antibody levels to ASP-1 and ASP-2 on the risk of an individual harboring a heavy hookworm infection. After controlling for age, sex, and study area, a negative association (odds ratio [OR]=0.38; 95% confidence interval [CI]=0.15–0.94; P<0.0001) was observed between the risk of heavy hookworm infection and increasing IgE anti-ASP-2 levels (i.e., increasing IgE anti-ASP-2 levels reduced the risk of heavy hookworm infection by 62%) (Table 1
). In contrast, a significant positive association was observed on the risk of heavy hookworm infection and increasing IgG4 anti-ASP-2 levels (OR=4.967; 95% CI=1.87–13.18; P<0.0001). In a separate analysis, a significant positive association (OR=2.95; 95% CI=1.122-5.66; P<0.0001) was observed for the ratio of IgG4/IgE anti-ASP-2. These findings suggest that heavy infection is influenced by a balance between the protective effects of IgE and the risk effects of IgG4 against ASP-2. Additional analyses did not show significant relationships between age, sex, area (Brazil or China), or antibody responses to crude hookworm antigen extracts or recombinant ASP-1 (data not shown). These results suggest that host antibodies against ASP-2 could influence worm burdens and led us to test whether ASP-2 could serve as a vaccine antigen in a permissive, naturally occurring animal model.
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3. Canine vaccination with ASP-2: in vitro effects of anti-ASP-2 antibody
To confirm that ASP-2 was a protective antigen when used as a vaccine, a randomized, controlled vaccine trial was conducted using helminth-naive, purpose-bred beagle pups, who received either an intramuscular injection with 4 doses of recombinant Ac-ASP-2 formulated with the GlaxoSmithKline Adjuvant System 03 (AS03) or 4 doses of the adjuvant AS03 alone (control). Sera obtained from the immunized dogs prior to challenge exhibited high anti-ASP-2 IgG1 and IgG2 titers (geometric mean antibody titers [GMAT] of 13,500 and 63,850, respectively), but only moderate IgE titers (GMAT of 1204). IgG from canines vaccinated with ASP-2 immunoprecipitated the native protein from L3 extracts (data not shown), indicating that dogs vaccinated with recombinant ASP-2 recognized the native ASP-2 protein in crude L3 antigen extract. ASP-2 immunolocalized to the glandular esophagus of A. caninum L3 in addition to the basal lamina of the body cavity or the channels that connect the glandular esophagus to the L3 surface. ASP-2 was also detected on the L3 cuticle and epicuticle (Fig. 1
). In triplicate experiments, pooled sera recovered from the ASP-2 vaccinated dogs inhibited tissue penetration by 60% percent compared with a pool of control sera (P=<0.0001).
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4. Canine vaccination with ASP-2: in vivo effects of anti-ASP-2 antibody
Two weeks following the final immunization, vaccinated dogs and control dogs were infected with hookworm by percutaneous administration of 500 A. caninum L3. Relative to controls, the vaccinated dogs exhibited reductions in fecal egg counts (69%, 10,613±5,306 vs. 33362±1,681; P=0.05). A reduction of 26% (204±12 vs. 151±21; P=0.03) in adult worm burdens recovered from the host intestine was also observed.
CONCLUSIONS AND SIGNIFICANCE
The studies here report on 3 independent lines of evidence indicating that ASP-2 is a lead vaccine candidate for human hookworm infection: 1) increasing IgE antibodies against ASP-2 protected against heavy hookworm infection in humans; 2) anti-ASP-2 antibodies immunoprecipitated native ASP-2 from larval extracts and inhibited hookworm larval invasion through tissue in vitro; and 3) vaccination with ASP-2 resulted in lower worm burdens and fecal egg counts in dogs. Previously, we have shown that vaccination with ASP-2 reduces hookworm burden in hamsters (a permissive but not naturally occurring host) challenged with the hookworm Ancylostoma ceylanicum. Other investigators have found that ASP-2-like molecules are protective antigens for onchocerciasis in laboratory mice and for ruminant haemonchosis caused by the trichostrongyle nematode, Haemonchus contortus. Our study also shows that it is important for an infected person to mount the appropriate antibody response (IgE and not IgG4) to ASP-2 in order to exhibit a reduction in the intensity of hookworm infection. Our canine vaccine trials show that vaccination with ASP-2 results in a strong and protective IgG response. The possibility remains that the protection from a vaccine is mediated by a different process than the protection that occurs during a chronic hookworm infection.
The mechanism by which antibodies against ASP-2 reduce host hookworm burdens and fecal egg counts is not known. Evidence to date indicates that anti-ASP-2 antibodies interact primarily with the tissue invading L3 stage. So far, ASP-2 protein has not been detected in other hookworm life-history stages and asp-2 mRNA was found only in L3 and not adult hookworms.
From the immunolocalization studies reported here, L3 produce ASP-2 in their glandular esophagus prior to secretion. Secretion probably occurs by at least 2 different mechanisms. First, ASP-2 was found in the lumen of the L3 esophagus suggesting that it could be secreted from its oral opening during entry into the host. Second, the protein was also detected in channels that connect the esophagus to the cuticle, and to the parasite surface itself. Therefore, anti-ASP-2 antibody could interfere with larval migration either by an interaction with its target antigen on the parasite surface or oral opening. This also includes the possibility that anti-ASP-2 antibody interacts with the parasite surface through an association with effector leukocytes (antibody dependent cellular cytotoxicity). Alternatively, anti-ASP-2 antibody may neutralize some critical biochemical function of the target protein. Based on the recent report of the crystal structure of ASP-2, it has been proposed that it could function as an immunomodulator by mimicking chemokines. Our observation reported here that anti-ASP-2 antibodies inhibit L3 invasion through tissue in vitro further supports the hypothesis that the immune response resulting from ASP-2 vaccination interferes with the early stages of parasite invasion in the host. Therefore, ASP-2 vaccination would decrease the number of L3 that reach the gastrointestinal tract, leading to reduced adult hookworm burdens and host blood loss (Fig. 2
). Moreover, anti-ASP-2 antibodies likely have an attenuating effect on worms that do reach maturity, given the large reduction in fecundity of worms from vaccinated dogs.
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In public health terms, the impact of a hookworm vaccine using ASP-2 would be the reduction of moderate-heavy hookworm infection and the morbidity associated with the hookworm -induced blood loss and iron deficiency anemia (IDA) that accompanies this level of infection. Similar reductions in worm burdens using anthelminthic drugs were proposed at the World Health Assembly 2001 Resolution 54.19, with the goal of attaining a minimum target of regular administration of chemotherapy (anthelminthic deworming) of 75% of all school-age children at risk of morbidity by 2010. The development of new vaccine technologies does not necessarily require a choice between drugs vs. vaccines to control infection. These are different types of tools that can be applied at the same time to control hookworm infection. A hookworm vaccine could be integrated into current soil-transmitted helminth chemotherapy control programs, with chemotherapy given first to treat existing infections followed by a vaccine administered to prevent or significantly delay reinfection. Studies are in progress to develop and test human hookworm vaccines comprised of an ASP-2 antigen.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3936fje; doi: 10.1096/fj.05-3936fj
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