Investigator: Garrick Wallstrom, Ph.D.
Collaborators: Sean Rollins, Ph.D. and Ed Ryan, Ph.D. (MGH), Conrad Quinn (CDC)
Bacillus anthracis spores have long been recognized as a potential biological weapon. Several events have demonstrated the potential for significant illness, mortality and societal disruption after an aerosol release of B. anthracis spores. The current FDA approved anthrax vaccine (AVA) was licensed 38 years ago and is comprised of a six-dose immunization schedule with annual boosters. There is limited human efficacy data pertaining to AVA vaccination, since anthrax is primarily a veterinary disease with limited human incidence. Additionally, AVA has many reported side effects including localized swelling and pain at the injection site; head, joint and muscle aches; malaise; nausea and fever. The primary immunogen of AVA is Protective Antigen. Protective Antigen alone is protective but the addition of cell culture filtrate provides a greater level of protection. We are applying NAPPA technology to identify which B. anthracis proteins are responsible for this enhanced level of protection. By reducing the total number of proteins used to achieve this level of protection, potentially many of the observed side effects attributed to AVA vaccination could be reduced. Furthermore, supplementation of a higher dose of newly identified immunogens could provide additional protection and longevity to the immune response, possibly reducing the number of vaccine inoculations.
We have produced a sequence verified collection of B. anthracis protein coding genes that is 96% complete. We have transferred all of these genes into a plasmid vector that is compatible with NAPPA and are producing all of these proteins on arrays. These slides are being immuno-screened with sera from 1 human inhalation anthrax patient, 7 human cutaneous anthrax patients and 4 human AVA vaccines. An additional set of sera, from 9 rhesus macaques that have been vaccinated with dilutions of AVA and inhalationally challenged with fully virulent Ames strain spores, have also been immuno-screened. Patients and macaques are demonstrating a specific immune response to Protective Antigen control spots. We are in the process of accessing immunogenicity for the screened experimental B. anthracis proteins. We expect to have the nearly full B. anthracis ORFeome immuno-screened with these sera within three months and will perform validation immunogenicity experiments, thereafter. Identified antigens will be strong candidates for protective immunization studies using animal models of anthrax.
NAPPA Self-assembling Bacillus anthracis protein microarrays: The pictured protein microarrays contain 1728 spots representing 752 Bacillus anthracis proteins printed in duplicate.
(A) Self assembly of Bacillus anthracis proteins: Spots that light up represent proteins that have self-assembled efficiently. Red indicates highly efficient protein assembly, green and blue represents reduced efficiency and black represents no protein expression or efficiency below the limit of detection. Most gaps in the grid arise from intentionally printing negative control spots that should not produce protein.
(B) Detecting immune responses using self-assembling protein microarrays: The pictured microarray slide was screened with sera from a macaque monkey that was vaccinated with AVA and subsequently challenged with aerosolized Bacillus anthracis spores. Colored spots indicate antibodies binding to the newly synthesized proteins. Red indicates robust antibody binding, green and blue indicate reduced antibody binding and black is below the limit of detection. The arrows indicate a positive immune response to an established anthrax antigen.