Fusion of Na-ASP-2 with human immunoglobulin Fcc abrogates histamine release from basophils sensitized with anti-Na-ASP-2 IgE B. ZHAN, 1,2 H. SANTIAGO, 3 B. KEEGAN, 1,2 P. GILLESPIE, 1,2 J. XUE, 4 J. BETHONY, 5 L. M. DE OLIVEIRA, 5 D. JIANG, 5 D. DIEMERT, 5 S.-H. XIAO, 4 K. JONES, 1,2 X. FENG, 6 P. J. HOTEZ 1,2 & M. E. BOTTAZZI 1,2 1 Section of Pediatric Tropical Medicine, Departments of Pediatrics and Molecular Virology and Microbiology, and National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA, 2 Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development, Texas Children’s Hospital, Houston, TX, USA, 3 Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD, USA, 4 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, China, 5 Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington, DC, USA, 6 Department Parasitology, Jilin Medical College, Jilin, China SUMMARY Na-ASP-2 is a major protein secreted by infective third- stage larvae (L3) of the human hookworm Necator americ- anus upon host entry. It was chosen as a lead vaccine candidate for its ability to elicit protective immune responses. However, clinical development of this antigen as a recombi- nant vaccine was halted because it caused allergic reactions among some of human volunteers previously infected with N. americanus. To prevent IgE-mediated allergic reactions induced by Na-ASP-2 but keep its immunogenicity as a vac- cine antigen, we designed and tested a genetically engineered fusion protein, Fcc ⁄ Na-ASP-2, composed of full-length Na-ASP-2 and truncated human IgG Fcc1 that targets the negative signalling receptor FccRIIb expressed on pro-allergic cells. The chimeric recombinant Fcc ⁄ Na-ASP-2 protein was expressed in Pichia pastoris and shared the similar antige- nicity as native Na-ASP-2. Compared to Na-ASP-2, the chimeric fusion protein efficiently reduced the release of histamine in human basophils sensitized with anti-Na-ASP-2 IgE obtained from individuals living in a hookworm-endemic area. In dogs infected with canine hookworm, Fcc ⁄ Na-ASP-2 resulted in significantly reduced immediate-type skin reactivity when injected intradermally compared with Na-ASP-2. Hamsters vaccinated with Fcc ⁄ Na-ASP-2 formulated with Alhydrogel Ò produced specific IgG that recognized Na-ASP-2 and elicited similar protection level against N. americanus L3 challenge as native Na-ASP-2. Keywords allergy, FccRIIb, FceRI, hookworm, IgE, Na-ASP-2, Necator americanus , vaccine INTRODUCTION Human hookworm infection is a major neglected tropical disease of humans, with most of the cases caused by Neca- tor americanus (1). Humans become infected when third- stage infective hookworm larvae (L3) penetrate host skin and subsequently migrate to the intestines, where they develop into adult blood-feeding worms. During the skin invasion, L3 secrete excretory ⁄ secretory products (ES) to facilitate the establishment of parasitism in the host (2–5). The Ancylostoma-secreted proteins (ASPs), including ASP- 1 and ASP-2, are the most abundant proteins secreted by the hookworm L3 upon entry into the host (4,5). Several lines of evidence support hookworm ASP-2 as a candidate vaccine antigen that induces host-protective immunity, based on larval challenge and other animal studies con- ducted in dogs (6), mice (7), rats (8) and hamsters (9–11). Specifically, vaccination with recombinant ASP-2 demon- strated significant reduction of adult worm burden and fecundity based on the reduced egg count in faeces follow- ing larval challenge (6,9–11) and recruitment of host immune cells to the challenged larvae (7,8). It was also found that ASP-2 is the major component recognized by the protective immune sera from dogs vaccinated with irradiated hookworm larvae (12). Furthermore, anti-ASP- 2 sera from ASP-2-vaccinated dogs inhibited the migration of larvae through dog skin in vitro, suggesting a possible mechanism underlying protective immunity (6). Epidemio- logical study showed that anti-ASP-2 antibodies in the Correspondence: Bin Zhan, Department of Pediatrics and Tropical Medicine, Baylor College of Medicine, Texas Children Hospital, 1102 Bates St., Ste.550, Houston, TX 77030, USA (e-mail: bzhan@bcm.edu). Disclosures: All authors disclose no potential conflicts of interest in any company or institution that might benefit from the publication. Received: 20 February 2012 Accepted for publication: 25 May 2012 Parasite Immunology, 2012, 34, 404–411 DOI: 10.1111/j.1365-3024.2012.01371.x 404 Ó 2012 Blackwell Publishing Ltd