Peptide-Based Subunit Vaccine against Hookworm Infection

et al. (2012) Peptide-Based Subunit Vaccine against Hookworm Infection. PLoS ONE 7(10): e46870. doi:10.1371/journal.pone.0046870 Peptide-Based Subunit Vaccine against Hookworm Infection Mariusz Skwarczynski 0 Annette M. Dougall 0 Makan Khoshnejad 0 Saranya Chandrudu 0 Mark S. Pearson 0 Alex Loukas 0 Istvan Toth 0 David Joseph Diemert, The George Washington University Medical Center, United States of America 0 1 The University of Queensland, School of Chemistry and Molecular Biosciences , St. Lucia, Queensland , Australia , 2 Centre for Biodiscovery and Molecular Development of Therapeutics, Queensland Tropical Health Alliance, James Cook University , Cairns, Queensland , Australia , 3 The University of Queensland, School of Pharmacy , Wooloongabba, Queensland , Australia Hookworms infect more people than HIV and malaria combined, predominantly in third world countries. Treatment of infection with chemotherapy can have limited efficacy and re-infections after treatment are common. Heavy infection often leads to debilitating diseases. All these factors suggest an urgent need for development of vaccine. In an attempt to develop a vaccine targeting the major human hookworm, Necator americanus, a B-cell peptide epitope was chosen from the apical enzyme in the hemoglobin digestion cascade, the aspartic protease Na-APR-1. The A291Y alpha helical epitope is known to induce neutralizing antibodies that inhibit the enzymatic activity of Na-APR-1, thus reducing the capacity for hookworms to digest hemoglobin and obtain nutrients. A291Y was engineered such that it was flanked on both termini by a coil-promoting sequence to maintain native conformation, and subsequently incorporated into a Lipid Core Peptide (LCP) self-adjuvanting system. While A291Y alone or the chimeric epitope with or without Freund's adjuvants induced negligible IgG responses, the LCP construct incorporating the chimeric peptide induced a strong IgG response in mice. Antibodies produced were able to bind to and completely inhibit the enzymatic activity of Na-APR-1. The results presented show that the new chimeric LCP construct can induce effective enzyme-neutralising antibodies in mice, without the help of any additional toxic adjuvants. This approach offers promise for the development of vaccines against helminth parasites of humans and their livestock and companion animals. - Funding: This work was supported by the National Health and Medical Research Council of Australia (NHMRC Programme Grant 496600), http://www.nhmrc.gov. au/. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors also thank Thalia Guerin for her correction of this manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Hookworm infection causes one of the worlds most debilitating neglected tropical diseases. The human hookworm (Necator americanus) infects over 700 million people worldwide, predominantly in indigent rural and tropical regions [1]. Chronic infection results in long-term pathological consequences primarily due to ongoing intestinal blood loss resulting from the feeding activities of these hematophagous parasites. Heavy infection leads to irondeficiency anemia and can manifest as impaired neurological and intellectual functioning in children, reduced work capacity in adults, and severe adverse outcomes in pregnancy [2]. Those most vulnerable to the harmful effects of hookworm include children and pregnant women, who are unable to tolerate the chronic blood loss and iron deficiency anemia due to their lower iron reserves [3]. These factors have significant influence on current and future productivity and economic well being of infected populations. Benzimidazole drugs are commonly used for the treatment and eliminate adult parasites. However, chemotherapy has limited efficacy and reinfection after treatment is common [4,5]. The problems with drug effectiveness and the looming threat of drug resistance suggest that alternatives to mass drug administration are urgently needed. Development of a vaccine that would prevent the acquisition of moderate or heavy intensity hookworm infection would be a major advance in reducing the morbidity caused by this parasite [6,7]. Currently, there is no human hookworm vaccine on the market or in advanced clinical trials. Hookworms obtain their nourishment primarily by ingesting blood and digesting the hemoglobin and serum proteins released from lysed erythrocytes. Na-APR-1 is a cathepsin D aspartic protease derived from the gut of adult N. americanus where it initiates the hemoglobin digestive cascade [8,9]. Therefore, blocking the catalytic activity of Na-APR-1 via the induction of neutralizing antibodies should result in starving and ultimately killing of the parasite. Indeed, it was demonstrated that APR-1 could be used as an efficacious hookworm vaccine antigen against A. caninum in dogs. Vaccination with the recombinant N. americanus or A. caninum enzymes induced antibodies that bound to the gut of the parasite and neutralized the enzymatic activity of the protease in vitro. When vaccinated dogs were then challenged with hookworm larvae they had significantly diminished adult parasite burdens and a reduction in blood loss was observed [8,10]. However, production of APR-1 at a commercial scale has proven to be challenging due to protein aggregation and low manufacturing yield obtained from eukaryotic expression systems. Previously, the immunogenic peptide epitope from N. americanus APR-1 (A291Y, AGPKAQVEAIQKY) was shown to induce the production of neutralizing antibodies in vivo. These antibodies were able to inhibit enzymatic activity of APR-1 against synthetic peptide and natural protein substrates [11]. Peptide antigens are not immunogenic by themselves and require appropriate delivery systems and strong, often toxic, adjuvants to stimulate desired immune responses [12]. For example, in the above-mentioned study the use of toxic complete Freunds adjuvant composed of inactivated and dried mycobacteria was necessary to stimulate immune responses against the A291Y epitope. To avoid this problem, lipidation of peptides emerged as a promising strategy for delivery of peptide subunit vaccines. The self-adjuvanting Lipid Core Peptide (LCP) delivery system has a demonstrated ability to induce strong immune responses against the incorporated peptide-epitopes without the help of external adjuvants, and is considered to be a capable platform for development of human vaccines [13,14]. In this paper, we have designed, synthesized and characterized a short series of A291Y conjugates. We have used the coilpromoting sequence from the yeast GCN4 protein to induce native helical A291Y epitope conformation [11,13,15,16]. Subsequently, chimeric and parent A291Y epitopes were incorporated into the LCP delivery system. The various (...truncated)