A new generation of vaccines for the neglected tropical diseases (NTDs) have now advanced into clinical development, with the Na-GST-1/Alhydrogel Hookworm Vaccine already being tested in Phase 1 studies in healthy adults. The current manuscript focuses on the often overlooked critical aspects of NTD vaccine product development, more specifically, vaccine stability testing programs. A key measure of vaccine stability testing is "relative potency" or the immunogenicity of the vaccine during storage. As with most NTD vaccines, the Na-GST-1/Alhydrogel Hookworm Vaccine was not developed by attenuation or inactivation of the pathogen (Necator americanus), so conventional methods for measuring relative potency are not relevant for this investigational product. Herein, we describe a novel relative potency testing program and report for the first time on the clinical lot of this NTD vaccine during its first 60 months of storage at 2–8°C. We also describe the development of a complementary functional assay that measures the ability of IgG from animals or humans immunized with Na-GST-1/Alhydrogel to neutralize this important hookworm enzyme. While 90% inhibition of the catalytic activity of Na-GST-1 was achieved in animals immunized with Na-GST-1/Alhydrogel, lower levels of inhibition were observed in immunized humans. Moreover, anti-Na-GST-1 antibodies from volunteers in non-hookworm endemic areas were better able to inhibit catalytic activity than anti-Na-GST-1 antibodies from volunteers resident in hookworm endemic areas. The results described herein provide the critical tools for the product development of NTD vaccines.

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CRISPR‐Cas comprise adaptive immune systems against foreign nucleic acids in prokaryotes. However, little is known about how the expression of CRISPR‐Cas genes is regulated in response to viral infection. Type‐III CRISPR interference results in the production of cyclic oligoadenylate (cOA) second messengers, which are known to bind various CRISPR Associated Rossmann Fold (CARF) ‐containing nuclease receptors to regulate their functions. Despite conservation of the CARF domain across Csa3 transcription factors, the basis for cOA binding specificity was unclear. In this study, we extend the known receptor repertoire of cOAs to include transcriptional factors by demonstrating specific binding of cA4 to Saccharolobus solfataricus Csa3 (Csa3Sso) (KD of 5.8 ± 0.03 μM). We determined a 2.0 Å resolution X‐ray crystal structure of cA4‐bound Csa3Sso, which reveals the binding of the Csa3Sso CARF domain to an elongated conformation of cA4. Binding affinity analyses of Csa3Sso mutants targeting the observed Csa3Sso•cA4 structural interface identified Csa3Sso residues that are required for ligand binding and specificity. We determined an essential role of a conserved glutamate (Glu122) for the low affinity interaction with cA4; a mutation of this residue to Ala and Gln resulted in a ~145 and ~125‐fold increase in cA4 binding affinity by Csa3Sso. Our complementary SAXS analyses detected a cA4‐induced conformational change in Csa3Sso, involving an asymmetric quaternary rearrangement of the C‐terminal winged helix‐turn‐helix (wHTH) domains supporting an allosteric mode of Csa3 regulation by cA4. Overall, our results support cA4 and Csa3‐mediated cross‐talk between type‐III and type‐I CRISPR systems that co‐exist in several prokaryotes.

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