Infectious Disease Research

The LaBaer lab is currently working on several projects that track the immune response to various infectious organisms.
 

Mycobacterium Tuberculosis

Investigators: Xiaobo Yu, Ph.D., Mitch Magee, Ph.D., Garrick Wallstrom, Ph.D. and Jennifer Viloria

Collaborators: Robert N. Husson, M.D. (Harvard Medical School), Jacqueline M. Achkar, M.D., M.S. (Albert Einstein College of Medicine), Tuofu Zhu (University of Washington School of Medicine), Sanjeeva Srivastava, Ph.D. (IIT Bombay)
 

Disease Background

Who gets it?
What are the symptoms?
How do we detect it now?
How is it currently treated?
What are the current challenges?
  • 1/3 of the world’s population is infected with Mycobacterium tuberculosis(Mtb).
  • Co-infection of HIV decreases the ability to detect TB infection
  • TB infection is often the cause of death for people infected with HIV.
  • TB treatment is complicated and time-consuming and relies on accurately diagnosing the infection. 
  • Current diagnostics tests have low sensitivity and specificity.
    • Skin test often results in False-positives
    • Sputum test has a low sensitivity: 50% in immunocompetent persons and even less in HIV+/TB+ patients
  • TB treatment is complicated and time-consuming and relies on accurately diagnosing the infection. 
  • In order to interrupt this cycle of infection-transmission, we have to improve the current diagnostic capabilities, especially for difficult to diagnose patients like those co-infected HIV.

Our Approach

Mycobacterium tuberculosis (Mtb) infects approximately one-third of the world’s population, and an estimated 9.4 million people develop active tuberculosis (TB) each year.  The poor detection rate of TB cases, especially in developing countries, promotes this level of ongoing transmission. In resource-poor regions, direct microscopic examination of sputum smears for acid fast bacilli (AFB) provides the prime diagnostic tool. However, the AFB smear test exhibits poor sensitivity at approximately ~50% in immunocompetent persons, and is even less sensitive in HIV+ TB+ patients. In order to interrupt this cycle of infection-transmission, we need improvements in rapid diagnostic capabilities for difficult to diagnose patients like those co-infected HIV.

We are assessing the detection of antibody biomarkers produced in response to active TB to add as an adjunct to the current diagnostic armamentarium. To accomplish this, we are analyzing sera from well-defined patient and control groups, and screen these sera on a unique and novel protein array platform, which allows for display of the complete proteome of Mtb on a microarray. The screen of a complete representative proteome using patient and control sera will allow us to delineate a smaller set of target proteins which we will validate using an acquired set of independent samples obtained in cross-sectional studies of TB suspects.  We predict that the validation study will yield a select set of ~30 candidate proteins that could serve to detect antibody biomarkers in TB patients.  The antigen-antibody interaction provides the foundation for many diagnostics and our goal is to find and delineate the most sensitive and specific Mtb proteins that react with patient antibodies. The successful completion of this program will provide new and improved strategies for detecting TB rapidly and sensitively.


Use of protein microarrays to screen human serum searching for immune responses against P. aeruginosa and V. cholerae

Investigator: Wagner Montor, Ph.D.

Collaborators: Steve Lory (Harvard Medical School), John Mekalanos (Harvard Medical School), Ed Ryan (MGH)

The NAPPA methodology for protein microarrays developed in our lab 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.

 


Panviral Proteome Project

Investigators: Xiaobo Yu, Ph.D., Andrea Throop, Ph.D., Xiaofang Bain and Joshua Labaer, M.D., Ph.D.

Collaborators: Jim Pipas, Ph.D. (University of Pittsburg)

Viruses have co-evolved with every organism that exists or has ever existed on Earth. Throughout the history of life, viruses have sampled every possible way to invade their hosts and evade the organismal and cellular defense systems that guard against infection. Approximately 3,000 viral species are documented, encoding about 93,000 proteins, most of which have not been characterized. In fact, only a very small minority of the known viruses has been studied in detail. Importantly, all viral genomes encode genes that act to alter the cellular environment in a way that favors infection or to antagonize and/or manipulate host antiviral defenses. Cell signaling pathways are vulnerable to intervention at multiple points and, as a group viruses have evolved to target the key regulatory steps of all pathways that impinge on their propagation. The result can cause disease both through the direct consequences of infection and indirectly by affecting host responses that lead to autoimmune, cancers and other chronic diseases.

We propose to use the panviral proteome as a unique tool for probing cell biology and disease. We will generate a plasmid library that encodes ~6,000 viral proteins and use it to discover and characterize the host cell pathways they regulate and the cellular systems they control and evaluate the association of these proteins with human disease. Computational methods combined with novel high-throughput biological assays will be used to identify viral proteins that alter or antagonize cellular systems governing proliferation, cell death or survival, metabolism and inflammation. The cellular targets of these proteins will be identified in subsequent genetic and proteomic studies. The goals of these studies are to discover novel viral antigens, uncover novel associations between specific diseases and viruses, and to identify previously unsuspected viruses that elicit a human immune response.

These studies will have a major impact on multiple areas of basic science, especially genomics, cell signaling, immunology, and virology as well on an array of diseases of infectious and noninfectious etiology. This effect is because many of the systems that viruses target during the course of infection are also perturbed in diseases of noninfectious etiology. Viral proteins that target cell proliferation, cell death, metabolic state, migration, differentiation, or inflammatory state will provide a powerful toolbox for investigators studying basic cell biology as well as for probing diseases in which these systems are affected. Finally, the characterization of the panviral proteome will have a direct impact on our understanding of the many acute and chronic diseases that are caused by viruses, and those of a suspected infectious etiology.  


Immunity studies in cholera

Investigator: Wagner Montor, Ph.D.

Collaborators: John Mekalanos (Harvard Medical School), Peter Yoon (Harvard Medical School), Ann Thanawastien (Harvard Medical School)

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.

 


The Application of NAPPA Technology to Study Immune Responses During Anthrax Infection and the US-Licensed Anthrax Vaccine

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.

 


Publications

Sheikh, A., Khanam, F., Sayeed, M. A., Rahman, T., Pacek, M., Hu, Y., Rollins, A., Bhuiyan, M. S., Rollins, S., Kalsy, A., Arifuzzaman, M., Leung, D. T., Sarracino, D. A., Krastins, B., Charles, R. C., Larocque, R. C., Cravioto, A., Calderwood, S. B., Brooks, W. A., Harris, J. B., LaBaer, J., Qadri, F., and Ryan, E. T. (2011) Interferon-gamma and Proliferation Responses to Salmonella enterica Serotype Typhi Proteins in Patients with S. Typhi Bacteremia in Dhaka, Bangladesh. PLoS Negl Trop Dis 5, (6), e1193. Abstract

Montor WR, Huang J, Hu Y, Hainsworth E, Lynch S, Kronish JW, Ordonez CL, Logvinenko T, Lory S, LaBaer J. (2009) Genome-wide study of Pseudomonas aeruginosa outer membrane protein immunogenicity using self-assembling protein microarrays. Infection and Immunity Nov;77(11):4877-86. Epub 2009 Sep 8.

Thanawastien A, Montor WR, Labaer J, Mekalanos JJ, Yoon SS. (2009) Vibrio cholerae proteome-wide screen for immunostimulatory proteins identifies phosphatidylserine decarboxylase as a novel Toll-like receptor 4 agonist. PLoS Pathogens Aug;5(8):e1000556. Epub 2009 Aug 21.

Rolfs A, Montor WR, Yoon SS, Hu Y, Bhullar B, Kelley F, McCarron S, Jepson DA, Shen B, Taycher E, Mohr SE, Zuo D, Williamson J, Mekalanos J, Labaer J. (2008) Production and sequence validation of a complete full length ORF collection for the pathogenic bacterium Vibrio cholerae. Proc Natl Acad Sci U S A. 105(11):4364-9. Epub 2008 Mar 12. PMID: 18337508

Slagowski NL, Kramer RW, Morrison MF, LaBaer J, Lesser CF. (2008) A functional genomic yeast screen to identify pathogenic bacterial proteins. PLoS Pathog. 4(1):e9. PMID: 18208325

Murthy T, Rolfs A, Hu Y, Shi Z, Raphael J, Moreira D, Kelley F, McCarron S, Jepson D, Taycher E, Zuo D, Mohr S E, Fernandez M, Brizuela L, LaBaer J. (2007) A full-genomic sequence-verified protein-coding gene collection for Francisella tularensis. PLoS ONE;2:e577 PMID: 17593976

Labaer J, Qiu Q, Anumanthan A, Mar W, Zuo D, Murthy TV, Taycher H, Halleck A, Hainsworth E, Lory S, Brizuela L. The Pseudomonas aeruginosa PA01 gene collection. Genome Res. 2004 Oct;14(10B):2190-200. PMID: 15489342