Hookworm Na-ASP-2 - putative functions, allergenicity and implications for vaccine development

Necator americanus is one of several hookworms that infect humans. The other notable species are Ancylostoma duodenale and Ancylostoma ceylanicum. Hookworms infect approximately 540-730 million people worldwide with more than a billion at risk however there are currently no vaccines to limit the global burden of disease. Activation associated secreted protein 2 (ASP-2) is one of the most highly up-regulated genes upon the transition of hookworm larvae from the free-living to the infective stage. Vaccine trials in numerous animal models of hookworm disease showed that ASP-2 was efficacious and worthy of development as a human hookworm vaccine. Na-ASP-2 was safe and immunogenic in a phase 1a human clinical trial in hookwormnaive volunteers in the U.S. However in a phase 1b trial in a hookworm-endemic area of Brazil, a subset of the trial patients exhibited immediate systemic hypersensitivity reactions, due to circulating IgE antibodies against the vaccine as a result of prior hookworm exposure, hence the vaccine was shelved. These studies highlighted the fact that there was insufficient knowledge about the biology of ASP-2 and its interactions with the human host. My hypothesis was twofold: 1) De-allergenisation (reduce IgE reactivity) of Na-ASP-2 to get a better vaccine; 2) ASP-2 has specific molecular and/or cellular binding partners within the human host to exert its effector functions. In this thesis, I mapped the IgG and IgE epitopes of Na-ASP-2 to both recombinant fragments of the protein as well as overlapping synthetic 13mer peptides. I then used this information to design and express site-directed mutants where single amino acids in major IgE epitopes were substituted to Alanine in an attempt to de-allergenise the protein. Four distinct IgE mutants were expressed in recombinant form and screened for reduced IgE reactivity using sera from phase 1b trial patients. Single point mutations resulted in reduced IgE binding, albeit non-significant and future efforts should focus on a single protein that incorporates all four mutations. The crystal structure of Na-ASP-2 from N. americanus reveals a putative equatorial binding groove containing a conserved tandem Histidine motif located in the centre of the groove. This putative binding groove was thought to provide a yet to be determined catalytic activity, prompting speculation that it interacts with proteins from its human host. In this thesis I employed two distinct approaches to elucidate putative host-derived binding partners for ASP-2. Firstly, by panning a random 12-mer peptide phage library, 16 peptides were identified after three successive rounds of panning. Most of the identified peptides were enriched for Glutamine and Histidine residues, and 3 peptides possessed a HXXQH motif, and a fourth peptide contained the highly similar HXXXH motif. BLASTp searches of public databases with an emphasis on the human proteome identified many distinct proteins containing varying degrees of similarity to different regions within the peptides; however, no peptide had a perfect match across its entire sequence length (12 residues) to known proteins, implying that binding of Na-ASP-2 to its native ligand is dependent on shorter peptides, possibly those containing an HXXQH motif. One such protein was the SK3 small conductance calcium-activated potassium channel, a transmembrane protein that contained a HNHQH motif within its extracellular domain. Binding of the synthetic SK3 peptide to recombinant Na-ASP-2 was confirmed by differential scanning fluorimetry. Potential binding modes of the peptide to Na-ASP-2 were studied by molecular dynamics simulations which clearly identify a preferred topology of the Na-ASP-2:SK3 peptide complex. To further explore potential binding partners of Na-ASP-2, the recombinant hookworm protein was used to probe a human proteome microarray, whereupon it bound selectively to the B cell Igα receptor CD79a. This is the first description of a host-pathogen protein:protein interaction identified using proteome microarrays. Using flow cytometry I confirmed the association that Na-ASP-2 bound to human B lymphocytes ex vivo. To investigate the biological effects of ASP-2 on human cells, B cell RNA was extracted and submitted to Next Generation RNA Sequencing, and revealed downregulated transcription of approximately 1000 B cell mRNAs while only approximately 100 mRNAs were upregulated, compared with control-treated cells. The expression of a range of molecules was affected by Na-ASP-2, including factors involved in leukocyte transendothelial migration pathways and the B cell signaling receptor pathway. Of note was the downregulated transcription of lyn and pi3k, molecules that are known to interact with CD79A and control B cell receptor signaling processes. Together, these results highlight a previously unknown interaction between a hookworm-secreted protein and B cells, which has implications for helminth-driven immunomodulation and vaccine development. Further, the novel use of human protein microarrays to identify host–pathogen interactions, coupled with ex vivo binding studies and subsequent analyses of global gene expression in human host cells, demonstrates a new pipeline by which to explore the molecular basis of infectious diseases.