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U.S. Department of Health and Human Services

Vaccines, Blood & Biologics

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Biomarkers of Vaccine Safety and Efficacy for Diphtheria Vaccines

Principal Investigator: Michael P. Schmitt, PhD
Office / Division / Lab: OVRR / DBPAP / LRSP

General Overview

The recent diphtheria epidemics in Eastern Europe and waning immunity to diphtheria among many adults in the US demonstrate the importance of maintaining adequate vaccination and developing better vaccines.

Ideally, the next generation of diphtheria vaccines would provide longer lasting immunity against the bacterium that causes this disease, Corynebacterium diphtheriae, eradicate it from people who were previously vaccinated, and reduce the adverse events associated with diphtheria vaccination. In order to develop safer and more effective diphtheria vaccines, scientists will have to learn more about C. diphtheriae and how it causes diseases.

About 25% of all licensed vaccines distributed in the US contain diphtheria toxoid (toxins made by bacteria that are weakened so they can be used as the main component in vaccines). Techniques for making this type of diphtheria vaccine are constantly evolving. These changes frequently include modifications in the way the bacteria are cultivated and in the various tests used to measure the stability, purity, or potency of the final vaccine.

This continuous evolution in vaccine production and testing techniques requires FDA to maintain regulatory and review expertise in this field. Our laboratory is meeting this need with an active C. diphtheriae research program that provides us with experience and expertise in the growth characteristics and media requirements of this microorganism. This expertise is available to manufacturers seeking guidance on how to grow these bacteria so they consistently produce safe and effective toxoid for use in vaccines.

Moreover, my laboratory work has provided the FDA with insight into the use of C. diphtheriae plasmids in vaccine production. (Plasmids are DNA molecules in bacteria that are not connected to chromosomes but still transmit important traits, such as the ability to cause disease and resistance to antibiotics.) Another essential part of our research is developing genetic tools that will facilitate the discovery of vaccine candidates and enhance our understanding of how C. diphtheriae causes diseases. This work will contribute to the development of second-generation diphtheria vaccines that contain toxoids with increased ability to trigger immune responses that limit the ability of the bacteria to colonize individuals (i.e., populate the respiratory tract in preparation for causing disease).

Our recent research identified a variety of proteins on the surface of C. diphtheriae, including two proteins that bind to heme molecules (molecules containing an atom of iron; heme is associated with the hemoglobin protein which is an essential component of red blood cells). Such heme-binding proteins could serve as components of future vaccines. Many of these factors are regulated by iron and bacteria produce them simultaneously with diphtheria toxins. Heme and hemoglobin binding proteins in other bacterial pathogens are important for determining how readily bacteria can invade and cause disease. These proteins are currently being evaluated in clinical studies to determine their effectiveness as vaccines.

Scientific Overview

Corynebacterium diphtheriae is a gram-positive bacterium and the cause of the severe respiratory disease diphtheria. Diphtheria toxin (DT), the primary virulence determinant for this pathogen, has been extensively investigated; however, factors involved in the colonization and survival of this organism on the mucosal surfaces of the human host are only beginning to be identified. A better understanding of the fundamental mechanisms of pathogenesis of C. diphtheriae will facilitate the development of future vaccines that are directed not only against the toxin, but also to other virulence determinants. A second generation of diphtheria vaccines may assist in the eradication of the carrier-state of the bacterium and provide more effective and longer lasting immunity against C. diphtheriae infection.

The research in my laboratory is focused on 1) characterizing a heme-iron transport system in C. diphtheriae and 2) analyzing a heme-dependent two-component regulatory system. C. diphtheriae uses host compounds such as heme and hemoglobin as essential iron sources, and my laboratory has identified and characterized systems associated with the uptake and use of heme-iron.

Heme uptake in C. diphtheriae involves an ABC-type heme transporter as well as a putative surface-anchored protein designated HtaA. Our current research shows that HtaA and a related protein, HtaB, are anchored to the cell surface and are capable of binding heme. Deletion analysis of htaA and the hmuTUV genes indicates that the products of these genes are involved in heme transport in C. diphtheriae, and further suggests that HtaA functions as a cell surface heme receptor in C. diphtheriae. Heme that has entered the cytosol of C. diphtheriae is proposed to be degraded by the heme oxygenase enzyme, HmuO, releasing iron.

The ChrA-ChrS two-component signal transduction system in C. diphtheriae regulates expression of the hmuO gene by a heme-dependent manner; recent studies by our group have shown that this system also controls the expression of two additional operons in C. diphtheriae through a similar regulatory mechanism. We are currently studying the ChrS-ChrA system involved in defining the essential regions and residues in the ChrS sensor-kinase component that are required for signal transduction.


J Bacteriol 2015 Feb 1;197(3):553-62
Utilization of host iron sources by Corynebacterium diphtheriae: multiple hemoglobin-binding proteins are essential for the use of iron from the hemoglobin-haptoglobin complex.
Allen CE, Schmitt MP

J Bacteriol 2013 Aug;195(16):3774-83
Pilus Gene Pool Variation and the Virulence of Corynebacterium diphtheriae Clinical Isolates during Infection of a Nematode.
Broadway MM, Rogers EA, Chang C, Huang IH, Dwivedi P, Yildirim S, Schmitt MP, Das A, Ton-That H

J Bacteriol 2013 Jun;195(12):2852-63
Analysis of novel iron-regulated, surface-anchored hemin-binding proteins in Corynebacterium diphtheriae.
Allen CE, Burgos JM, Schmitt MP

J Bacteriol 2012 Jun;194(12):3199-215
Pangenomic Study of Corynebacterium diphtheriae That Provides Insights into the Genomic Diversity of Pathogenic Isolates from Cases of Classical Diphtheria, Endocarditis, and Pneumonia.
Trost E, Blom J, de Castro Soares S, Huang IH, Al-Dilaimi A, Schroder J, Jaenicke S, Dorella FA, Rocha FS, Miyoshi A, Azevedo V, Schneider MP, Silva A, Camello TC, Sabbadini PS, Santos CS, Santos LS, Hirata R Jr, Mattos-Guaraldi AL, Efstratiou A, Schmitt MP, Ton-That H, Tauch A

J Bacteriol 2012 Apr;194(7):1717-29
The ChrA response regulator in Corynebacterium diphtheriae controls hemin-regulated gene expression through binding to the hmuO and hrtAB promoter regions.
Burgos JM, Schmitt MP

Procedia Vaccinol 2011;5:47-59
Alternative methods and strategies to reduce, refine, and replace animal use for human vaccine post-licensing safety testing: state of the science and future directions
Isbrucker R, Levis R, Casey W, McFarland R, Schmitt M, Arciniega J, Descamps J, Finn T, Hendriksen C, Horiuchi Y, Keller J, Kojima H, Sesardic D, Stickings P, Johnson NW, Allen D

J Bacteriol 2011 Oct;193(19):5374-85
Novel hemin binding domains in the Corynebacterium diphtheriae HtaA protein interact with hemoglobin and are critical for heme iron utilization by HtaA.
Allen CE, Schmitt MP

J Bacteriol 2010 Sep;192(18):4606-17
The ABC transporter, HrtAB, confers resistance to hemin toxicity and is regulated in a hemin-dependent manner by the ChrAS two-component system in Corynebacterium diphtheriae.
Bibb LA, Schmitt MP

J Bacteriol 2009 Apr;191(8):2638-48
HtaA is an iron-regulated hemin binding protein involved in the utilization of heme-iron in Corynebacterium diphtheriae.
Allen CE, Schmitt MP

J Bacteriol 2009 Mar;191(5):1595-603
Regulation and Activity of a Zinc Uptake Regulator, Zur in Corynebacterium diphtheriae.
Smith KF, Bibb LA, Schmitt MP, Oram DM

Vaccine 2008 Apr 7;26(16):1913-21
WHO Working Group on revision of the Manual of Laboratory Methods for Testing DTP Vaccines-Report of two meetings held on 20-21 July 2006 and 28-30 March 2007, Geneva, Switzerland.
Corbel MJ, Das RG, Lei D, Xing DK, Horiuchi Y, Dobbelaer R, WHO Working Group

J Bacteriol 2007 May;189(9):3650-4
Comparative Analysis of hmuO Function and Expression in Corynebacterium Species.
Kunkle CA, Schmitt MP

Infect Immun 2007 May;75(5):2421-31
The ChrA-ChrS and HrrA-HrrS Signal Transduction Systems are Required for Activation of the hmuO Promoter and Repression of the hemA Promoter in Corynebacterium diphtheriae.
Bibb LA, Kunkle CA, Schmitt MP

Infect Immun 2005 Nov;73(11):7406-12
Analysis of a Heme-Dependent Signal Transduction System in Corynebacterium diphtheriae: Deletion of the chrAS Genes Results in Heme Sensitivity and Diminished Heme-Dependent Activation of the hmuO Promoter.
Bibb LA, King ND, Kunkle CA, Schmitt MP

J Bacteriol 2005 Jan;187(2):422-33
Analysis of a DtxR-Regulated Iron Transport and Siderophore Biosynthesis Gene Cluster in Corynebacterium diphtheriae.
Kunkle CA, Schmitt MP



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