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

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.

Global metabolomic analysis of a mammalian host infected with Bacillus anthracis.

Whereas DNA provides the information to design life and proteins provide the materials to construct it, the metabolome can be viewed as the physiology that powers it. As such, metabolomics, the field charged with the study of the dynamic small-molecule fluctuations that occur in response to changing biology, is now being used to study the basis of disease. Here, we describe a comprehensive metabolomic analysis of a systemic bacterial infection using Bacillus anthracis, the etiological agent of anthrax disease, as the model pathogen. An organ and blood analysis identified approximately 400 metabolites, including several key classes of lipids involved in inflammation, as being suppressed by B. anthracis. Metabolite changes were detected as early as 1 day postinfection, well before the onset of disease or the spread of bacteria to organs, which testifies to the sensitivity of this methodology. Functional studies using pharmacologic inhibition of host phospholipases support the idea of a role of these key enzymes and lipid mediators in host survival during anthrax disease. Finally, the results are integrated to provide a comprehensive picture of how B. anthracis alters host physiology. Collectively, the results of this study provide a blueprint for using metabolomics as a platform to identify and study novel host-pathogen interactions that shape the outcome of an infection.

Reduced ethanol consumption and preference in cocaine- and amphetamine-regulated transcript (CART) knockout mice.

Cocaine- and amphetamine-regulated transcript (CART) is a neuropeptide implicated in addiction to drugs of abuse. Several studies have characterized the role of CART in addiction to psychostimulants, but few have examined the role of CART in alcohol use disorders including alcoholism. The current study utilized a CART knockout (KO) mouse model to investigate the role of CART in ethanol appetitive behaviors. A two-bottle choice, unlimited-access paradigm was used to compare ethanol appetitive behaviors between CART wild type (WT) and KO mice. The mice were presented with an ethanol solution (3%-21%) and water, each concentration for 4 days, and their consumption was measured daily. Consumption of quinine (bitter) and saccharin (sweet) solutions was measured following the ethanol preference tests. In addition, ethanol metabolism rates and ethanol sensitivity were compared between genotypes. CART KO mice consumed and preferred ethanol less than their WT counterparts in both sexes. This genotype effect could not be attributed to differences in bitter or sweet taste perception or ethanol metabolism rates. There was also no difference in ethanol sensitivity in male mice; however, CART KO female mice showed a greater ethanol sensitivity than the WT females. Taken together, these data demonstrate a role for CART in ethanol appetitive behaviors and as a possible therapeutic drug target for alcoholism and abstinence enhancement.

Immunotherapeutic strategies to prevent and treat human herpesvirus 6 reactivation after allogeneic stem cell transplantation.

Human herpesvirus (HHV) 6 causes substantial morbidity and mortality in the immunocompromised host and has no approved therapy. Adoptive transfer of virus specific T cells has proven safe and apparently effective as prophylaxis and treatment of other virus infections in immunocompromised patients; however, extension to subjects with HHV6 has been hindered by the paucity of information on targets of cellular immunity. We now characterize the cellular immune response from 20 donors against 5 major HHV6B antigens predicted to be immunogenic and define a hierarchy of immunodominance of antigens based on the frequency of responding donors and the magnitude of the T-cell response. We identified specific epitopes within these antigens and expanded the HHV6 reactive T cells using a GMP-compliant protocol. The expanded population comprised both CD4(+) and CD8(+) T cells that were able to produce multiple effector cytokines and kill both peptide-loaded and HHV6B wild-type virus-infected target cells. Thus, we conclude that adoptive T-cell immunotherapy for HHV6 is a practical objective and that the peptide and epitope tools we describe will allow such cells to be prepared, administered, and monitored in human subjects.