Harlow LaboratoryNAPPA - BioDefense Organisms
The Application of NAPPA Technology to Study Immune Responses During Anthrax Infection and the US-Licensed Anthrax Vaccine
Sean Rollins, Ph.D.
LaBaer Laboratory
Collaborators:
Ed Ryan (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.
Use of protein microarrays to screen human serum searching for immune responses against P. aeruginosa and V. cholerae
Wagner Montor, Ph.D.
LaBaer Laboratory
Collaborators:
Steve Lory (HMS)
John Mekalanos (HMS)
Ed Ryan (MGH)
The NAPPA methodology for protein microarrays developed at HIP is also being used to screen human serum in order to find antibodies against specific pathogens. Pseudomonas aeruginosa is responsible for potentially life threatening infections in individuals with compromised defense mechanisms and those with cystic fibrosis. Although a number of immunogenic proteins are known, no effective vaccine has been approved yet. We have used protein microarrays to execute a proteome-wide study of all in silico predicted outer membrane and exported P. aeruginosa proteins identifying 50 that trigger an adaptive immune response in cystic fibrosis and acutely-infected patients, 12 of which were recognized by numerous patients and show the best potential to be used for diagnostics and vaccine development. We are now expanding this approach to the entire proteome of Vibrio cholerae, an organism that continues to be responsible for pandemic infections in many parts of the world. We have access to serum from infected individuals in Bangladesh acquired on day 2, day 7 and day 21 post infection. This will allow us to use the patients’ serum as their own controls.
Figure 1: Representative protein microarray (NAPPA) results of V. cholerae proteins. ORFs (n = 346) were transferred and arrayed as plasmid DNA onto protein microarrays, and expression on the array was tested. (A and B) The DNA-to-protein relationships. (Upper) Picogreen detection of DNA. (Lower) The corresponding GST protein. (A) Controls spotted onto the array; on the left and right are 8 feature for plasmids that do not encode protein, and in the center are 12 feature of purified GST protein. (B) A comparison of 32 V. cholerae ORFs; all ORFs display DNA, but variation in ORF-specific protein expression/capture. (C) Examples for the entire set of controls and ORFs tested on NAPPA; left array, DNA detection by picogreen staining; right array, protein expression/capture by anti-GST antibody.
Innate immunity studies in cholera
Wagner Montor, Ph.D.
LaBaer Laboratory
Collaborators:
John Mekalanos (HMS)
Peter Yoon (HMS)
Ann Thanawastien (HMS)
Together with the Mekalanos lab, we have developed a high throughput method for screening for proteins that trigger toll like receptor response in cells. We have screened the entire V. cholerae proteome and identified approximately 13 proteins that trigger an innate immune response. One of these has been characterized in detail as acting through TLR4 that is independent of the LPS response.
Figure 1: V. cholerae proteins expressed in vitro are active in biological assays. A. Six different proteins were produced alone or together as a pool. After in vitro protein production, 10 ul of each RRL mixture and its serial dilutions were added to treat A549 reporter cells for 4 h. Luciferase-based NF-kB activation was shown in the bottom. Squares shown in red indicate positive NF-kB activation. FlaD, Flagellin D; FlaC, Flagellin C; NAR, nitrate reductase; OmpA, outer membrane A; FNR, anaerobic transcriptional activator; TRFac, transcription factor. B. Western blot analysis of in vitro-synthesized proteins.
Innate immunity studies in tularemia
Andreas Rolfs, Ph.D.
Zhenwei Shi (Research Assistant)
Collaborator: Prof. D.L. Kasper, Department of Microbiology and Molecular Genetics (HMS)
Innate immunity studies were initiated for the human pathogen F. tularensis to further understand the specific immune biology of this bacterium. Using the ORF clone collection for F. tularensis previously published (Murthy, et al.) we expressed the entire proteome for this organism in vitro after transfer into an appropriate vector, and used these proteins to stimulating a toll like receptor (TLR) response in immunological active cells. For further characterization were those proteins chosen that exhibited in independent experiments stimulation similar to that of a known TLR agonist.
Murthy T, Rolfs A, Hu Y, Shi Z, Raphael J, Moreira D, Kelley F, McCarron S, Jepson D, Taycher E, Zuo D, Mohr SE, Fernandez M, Brizuela L, Labaer J. (2007) A full-genomic sequence-verified protein-coding gene collection for Francisella tularensis. PLoS ONE; 2(6):e577. PMID: 17593976
T-cell responses in tularemia
TVS Murthy, Ph.D. (former HIP member)
Marcin Pacek, Ph.D. (former HIP member)
Andreas Rolfs, Ph.D.
Zhenwei Shi (Research Assistant)
Collaborator:
Dennis Kasper and Lee-Ann Blalock; Department of Microbiology and Molecular Genetics (HMS)
Together with the Kasper group, an assay was developed to use purified proteins as antigen source for ELISPOT assays. HIP used its complete F. tularensis clone collection to produce proteins in E.coli in high throughput for a genome wide screen for proteins that induce a T-cell response. Potential hits have been identified and are currently tested in animal vaccine studies.