Progress toward the Development of a NEAT Protein Vaccine for Anthrax Disease

Abstract

Bacillus anthracis is a sporulating Gram-positive bacterium that is the causative agent of anthrax and a potential weapon of bioterrorism. The U.S.-licensed anthrax vaccine is made from an incompletely characterized culture supernatant of a nonencapsulated, toxigenic strain (anthrax vaccine absorbed [AVA]) whose primary protective component is thought to be protective antigen (PA). AVA is effective in protecting animals and elicits toxin-neutralizing antibodies in humans, but enthusiasm is dampened by its undefined composition, multishot regimen, recommended boosters, and potential for adverse reactions. Improving next-generation anthrax vaccines is important to safeguard citizens and the military. Here, we report that vaccination with recombinant forms of a conserved domain (near-iron transporter [NEAT]), common in Gram-positive pathogens, elicits protection in a murine model of B. anthracis infection. Protection was observed with both Freund's and alum adjuvants, given subcutaneously and intramuscularly, respectively, with a mixed composite of NEATs. Protection correlated with an antibody response against the NEAT domains and a decrease in the numbers of bacteria in major organs. Anti-NEAT antibodies promote opsonophagocytosis of bacilli by alveolar macrophages. To guide the development of inactive and safe NEAT antigens, we also report the crystal structure of one of the NEAT domains (Hal) and identify critical residues mediating its heme-binding and acquisition activity. These results indicate that we should consider NEAT proteins in the development of an improved antianthrax vaccine.

NMR experiments redefine the hemoglobin binding properties of bacterial NEAr‐iron Transporter domains

“…Their ability to bind heme is well accepted because they exhibit characteristic UV–vis spectra and their primary sequences harbor the conserved iron coordinating tyrosine residue. In addition, for three of the domains their atomic structures in complex with heme have been determined ( Ba IsdX1 N , Ba IsdX2 N5 , and Ba Hal N ) . However, it is unclear how the nine dual domains might also engage Hb as their primary sequences lack the Hb‐interacting (F/Y)YH(Y/F) aromatic motif and their binding to Hb has only been demonstrated in SPR or ELISA experiments using immobilized Hb obtained from commercial sources.…”

“…(c) in blue] . Evidence for their ability to bind heme is strong and is based on the presence of a Soret band in their UV–vis spectra, and in some instances has been validated by X‐ray crystallography . In addition, their primary sequences contain all of the residues within the conserved heme‐binding S/YXXXY sequence element; Hal N is the lone exception as it lacks the second, nonmetal coordinating tyrosine residue [Fig.…”

Heme catabolism in the causative agent of anthrax

“…Nine‐week‐old A/J mice were obtained from Jackson Laboratories and housed for at least 1 week in BCM’s Center for Comparative Medicine. Wild‐type and mutant bacillus spores were generated as previously described (Balderas et al , ). On the day before the infection, spores were heated to 65°C for 10 min to kill any vegetative cells and serial diluted on LB agar in triplicate to determine the number of colony forming units (cfus) per ml of spore mixtures.…”

Confronting the threat of bioterrorism: realities, challenges, and defensive strategies

“…In this study, 4418 women received the vaccine in the first trimester and 423 in the second and third trimeseter. More effective anthrax vaccines that require fewer doses are constantly being tested, 104,105 such as the NEAT protein anthrax vaccine, 106 a dual purpose influenza vaccine that protects against anthrax, 107 and a combined anthrax-plague vaccine. 108 Apart from the 17D yellow fever live attenuated vaccine and the Junin virus vaccine, no vaccines for haemorrhagic fevers have been licensed.…”