A highly expressed intestinal cysteine protease of Ancylostoma ceylanicum protects vaccinated hamsters from hookworm infection

Abstract

Background Human hookworms ( Necator americanus , Ancylostoma duodenale , and Ancylostoma ceylanicum ) are intestinal blood-feeding parasites that infect ~500 million people worldwide and are among the leading causes of iron-deficiency anemia in the developing world. Drugs are useful against hookworm infections, but hookworms rapidly reinfect people, and the parasites can develop drug resistance. Therefore, having a hookworm vaccine would be of tremendous benefit. Methodology/Principal findings We investigated the vaccine efficacy in outbred Syrian hamsters of three A . ceylanicum hookworm antigen candidates from two classes of proteins previously identified as promising vaccine candidates. These include two intestinally-enriched, putatively secreted cathepsin B cysteine proteases (AceyCP1, AceyCPL) and one small Kunitz-type protease inhibitor (AceySKPI3). Recombinant proteins were produced in Pichia pastoris , and adsorbed to Alhydrogel. Recombinant AceyCPL (rAceyCPL)/Alhydrogel and rAceySKPI3/Alhydrogel induced high serum immunoglobulin G (IgG) titers in 8/8 vaccinates, but were not protective. rAceyCP1/Alhydrogel induced intermediate serum IgG titers in ~60% of vaccinates in two different trials. rAceyCP1 serum IgG responders had highly significantly decreased hookworm burdens, fecal egg counts and clinical pathology compared to Alhydrogel controls and nonresponders. Protection was highly correlated with rAceyCP1 serum IgG titer. Antisera from rAceyCP1 serum IgG responders, but not nonresponders or rAceyCPL/Alhydrogel vaccinates, significantly reduced adult A . ceylanicum motility in vitro . Furthermore, rAceyCP1 serum IgG responders had canonical Th2-specific recall responses (IL4, IL5, IL13) in splenocytes stimulated ex vivo . Conclusions/Significance These findings indicate that rAceyCP1 is a promising vaccine candidate and validates a genomic/transcriptomic approach to human hookworm vaccine discovery.

Arachidonic Acid Is a Safe and Efficacious Schistosomicide, and an Endoschistosomicide in Natural and Experimental Infections, and Cysteine Peptidase Vaccinated Hosts

“…We proposed that immune cells-activating antibodies to the cysteine peptidase immunogens and ARA, together, mediate demise of juvenile worms. The importance of antibodies in mediating the cysteine peptidase vaccine resistance to infection was demonstrated in the S. mansoni ( 30 ) and the Ancylostoma ceylanicum ( 55 ) models. It has been reported that free ARA is readily incorporated by S. mansoni ( 56 ), and likely activates the parasite voltage-gated channels, and enzymes, namely tegument-associated nSMase with subsequent apical SM hydrolysis, and exposure of the otherwise hidden surface membrane antigens to antibody- mediated effector functions, and parasite demise.…”

Comparison of percutaneous vs oral infection of hamsters with the hookworm Ancylostoma ceylanicum: Parasite development, pathology and primary immune response

“…Studies using the hamster model suggest that oral infections with Ancylostoma ceylanicum [26,41] closely approximate some observations made in human infections [27,31,32,43], which has added to our understanding of hookworm pathogenesis and the host-parasite interaction. Establishment of patent A. ceylanicum infections in outbred Golden Syrian hamsters has made possible detailed characterizations of hookworm disease pathogenesis [27], evaluation of therapeutic interventions [36,[44][45][46][47], vaccine development [27,[48][49][50][51][52][53][54], and characterization of innate/ acquired immune responses [31,34,35,45].…”

“…Establishment of patent A . ceylanicum infections in outbred Golden Syrian hamsters has made possible detailed characterizations of hookworm disease pathogenesis [ 27 ], evaluation of therapeutic interventions [ 36 , 44 – 47 ], vaccine development [ 27 , 48 – 54 ], and characterization of innate/acquired immune responses [ 31 , 34 , 35 , 45 ].…”

Specific Antibodies and Arachidonic Acid Mediate the Protection Induced by the Schistosoma mansoni Cysteine Peptidase-Based Vaccine in Mice

“…(3) The increase in uric acid was limited but significant ( p < 0.05) subsequent to cysteine peptidase catabolic activity and was sufficient to direct the immune responses towards the type 2 axis, increase the antibody response, and elicit an increase in free ARA release, as has been well demonstrated [ 19 , 22 , 23 , 24 , 25 , 28 , 29 , 33 ]. (4) The importance of antibodies in mediating the cysteine peptidase vaccine protection was demonstrated in nude mice- S. mansoni [ 9 ], and recently, hamster- Ancylostoma ceylanicum [ 46 ] models. Specific IgG1, IgG2a, and IgG2b antibodies may interact with secreted-excreted cathepsin peptidases [ 47 , 48 ], which are stagnant in the liver sinusoids, leading to eosinophils, basophils, mast cells, neutrophils, and macrophages arming, activation, and the release of cytotoxic and inflammatory mediators.…”