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  1. Biologics Research Projects

Identification of Targets for Development of Vaccines and Biological Therapies Against Gastrointestinal Pathogens

Paul E. Carlson, Jr., PhD
Office of Vaccines Research and Review
Division of Bacterial, Parasitic and Allergenic Products
Lab of Mucosal Pathogens and Cellular Immunology

Paul.Carlson@fda.hhs.gov


Biosketch

Paul Carlson, Ph. D., is a principal investigator in the Laboratory of Mucosal Pathogens and Cellular Immunology, Division of Bacterial, Parasitic, and Allergenic Products, Office of Vaccines Research and Review, CBER, FDA. Dr. Carlson received his PhD from the University of Pittsburgh; he performed postdoctoral research at the University of Michigan in the laboratory of Phil Hanna. His research at FDA has focused on infections caused by the enteric pathogens Clostridium difficile and Vancomycin resistant Enterococcus (VRE) species, specifically,  1) mechanisms of C. difficile pathogenesis; 2) development of genetic tools to study C. difficile; 3) host response to C. difficile; 4) the role of the host microbiota in C. difficile colonization resistance; 5) the interactions between the host immune system and the microbiome; 6) bacteriophage therapy against VRE.

Dr. Carlson is co-chair of the FDA microbiome working group, the Joint Agency Microbiome (JAM) working group, and a member of the Microbiome Interagency Working Group (MIWG). His regulatory responsibilities include product (Chemistry, Manufacturing, and Control) review for fecal microbiota transplantation (FMT), defined live biotherapeutic products, and bacteriophage therapies.


General Overview

Our laboratory studies bacterial pathogens that live in the host gastrointestinal tract. Since these pathogens represent serious public health threats, we are working to identify new targets for therapeutics to fight these infections. These targeted treatments include vaccines, Fecal Microbiota for Transplantation (FMT), live biotherapeutic products, and bacteriophage therapy.

Clostridium difficile (commonly called C-diff, or Cd) is a leading cause of antibiotic- associated diarrhea and hospital-acquired infection worldwide. Cd causes nearly 500,000 infections per year in the United States alone. Antibiotic therapy is the primary risk factor for Cd infection (CDI), as these drugs disrupt the normal host microbiota, allowing Cd colonization. Our laboratory is investigating the mechanisms by which this bacterium causes disease. We seek to understand how Cd responds to the host environment during infection in order to identify specific mechanisms by which Cd causes disease. A greater understanding of how Cd survives, grows, and causes disease in the host can help direct the development of new therapies against this pathogen.

While initial cases of CDI are usually treated with antibiotics, many patients (20%-35%) develop recurrent disease despite repeated courses of such therapy. Currently, the most effective treatment for recurrent CDI is FMT. FMT is a process in which stool from a healthy donor is transplanted into the infected individual. The bacterial species present in the feces of the healthy donor compete with Cd for resources in the host intestine, leading to clearance of Cd in >85% of cases. Although this treatment method is effective, its safe implementation is a challenge for both physicians and FDA regulators. Our laboratory is working to identify potential alternatives to FMT, specifically in the development of targeted probiotics, or live biotherapeutic products (LBPs).

Our laboratory is studying safety, efficacy, and potency measurements in FMT products. These studies include 1) assessing donor screening methods; 2) understanding how manufacturing procedures can alter the microbial composition of an FMT product; 3) identifying potential biomarkers of FMT potency. Data on putative biomarkers can also be used to develop rationally designed LBTs that can outcompete Cd in animal models. In addition, we are developing methods for characterizing complex, multi-strain LBTs.   

We are also studying the potential of bacteriophage therapy to reduce natural colonization levels of Vancomycin Resistant Enterococcus (VRE) infections. Enterococcus species are natural members of the human microbiome that can cause disease in immunocompromised patients.  The acquisition of vancomycin resistance in these organisms, which were already resistant to many other antibiotics, has limited treatment options for these infections. We are investigating whether bacteriophage therapy can be used as a treatment to decolonize patients and health care workers to reduce the spread of these infections. These studies will also help answer critical regulatory questions regarding the use of phage therapy to treat bacterial infections.


Scientific Overview

We study bacterial pathogenesis using both large-scale analyses (microarray technology, RNA sequencing, etc.) and basic bacterial genetics techniques. We first seek to understand the global responses of a pathogen to the host environment or the response of the host to infection with a given bacterium. Our hypothesis is that genes identified as being turned on or off in response to a given environment could be important for survival of the bacterium in the host. We use findings from these studies, to identify specific targets for further investigation, using bacterial genetics to understand the role of individual genes during infection. We then delete these genes and examine the resulting strain for altered phenotypes. Examples of conditions currently being studied in the laboratory include nutrient starvation (i.e., iron starvation) and oxygen resistance.

Our laboratory also studies the response of host cells to Cd infection. Using a custom-designed environmental control chamber, we create conditions for growing both host cells and anaerobic Cd bacilli.  This chamber allows us to examine the response of host cells to live Cd in vitro. We are currently studying the responses of both host epithelial cells and cells of the innate immune system. Assessment of these responses is done both globally (RNA sequencing) and at the targeted protein level (ELISA).

Our microbiome studies use mouse models of Cd infection, including knockout mice lacking specific components of the immune system, and germ-free mice, which do not contain a microbiome of their own. These studies assess changes to the microbiome using DNA sequencing technologies to determine which organisms are present in feces following a variety of treatments, including antibiotic regiments and FMT, as well as the effect that stool processing has on overall viability in an FMT. Specifically, we are optimizing methods for sequencing genes only from living organisms in order to understand the effects of manufacturing conditions (oxygen exposure, lyophilization, and freeze/thaw cycles) on the viability of the microbes in an FMT product.  Additionally, we found that mice deficient in Mucosal Associated Invariant T (MAIT) cells, which recognize bacterial metabolites and require the presence of a microbiome for natural development (germ free mice do not have MAIT cells) have a unique microbiota that provides resistance to CDI, even following antibiotic treatment.  Our lab showed that this resistance to CDI is microbiome-dependent and transferrable by FMT. We are now working with this unique set of microbes to identify specific organisms or metabolites that provide protection against Cd colonization, which can be used as biomarkers for potency of FMT products and for developing defined Live Biotherapeutic Products to target CDI.

We use a variety of methods to study VRE decolonization using phages. The lab characterizes phage that have been isolated from raw sewage by examining their host range across a wide range of VRE isolates. For complete characterization, we will also sequence the genomes of all phage used in our studies. These genome sequences will provide a baseline comparison for studies of phage evolution and adaptation.  Finally, several promising candidate phages have been tested for their ability to reduce VRE colonization and transmission in a mouse model of disease.  Interestingly, we found that the composition of the microbiota at the time of treatment appears to play a significant role in the overall efficacy of the phage cocktails in our mouse models.  We are currently working to understand the role of the microbiota during bacteriophage therapy for the elimination of VRE from the gut.  We are also assessing the potential for these phage cocktails as treatments for VRE infections, using various mouse models of disease.


Publications

  1. Metabolites 2020 Mar 26;10(4):127
    Bile acid profile and its changes in response to cefoperazone treatment in MR1 deficient mice.
    Sun J, Cao Z, Smith AD, Carlson PE Jr, Coryell M, Chen H, Beger RD
  2. Cell Host Microbe 2020 Feb 12;27(2):173-5
    Regulatory considerations for fecal microbiota transplantation products.
    Carlson PE Jr
  3. PLoS One 2019 Sep 27;14(9):e0223025
    Microbiota of MR1 deficient mice confer resistance against Clostridium difficile infection.
    Smith AD, Foss ED, Zhang I, Hastie JL, Giordano NP, Gasparyan L, VinhNguyen LP, Schubert AM, Prasad D, McMichael HL, Sun J, Beger RD, Simonyan V, Cowley SC, Carlson PE Jr
  4. Infect Immun 2019 May 21;87(6):e00085-19
    Bacteriophage resistance alters antibiotic mediated intestinal expansion of enterococci.
    Chatterjee A, Johnson CN, Luong P, Hullahalli K, McBride SW, Schubert AM, Palmer KL, Carlson Jr. PE, Duerkop BA
  5. J Bacteriol 2018 Sep 24;200(20):e00175-18
    A novel Bvg-repressed promoter causes vrg-like transcription of fim3 but does not result in the production of serotype 3 Fimbriae in the Bvg(-) mode Bordetella pertussis.
    Chen Q, Lee G, Craig C, Ng V, Carlson PE Jr, Hinton DM, Stibitz S
  6. mSphere 2018 Sep 5;3(5):e00335-18
    Germinant synergy facilitates Clostridium difficile spore germination under physiological conditions.
    Kochan TJ, Shoshiev MS, Hastie JL, Somers MJ, Plotnick YM, Gutierrez-Munoz DF, Foss ED, Schubert AM, Smith AD, Zimmerman SK, Carlson PE Jr, Hanna PC
  7. Infect Immun 2018 Jul 23;86(8):e00326-18
    Cysteine desulfurase IscS2 plays a role in oxygen resistance in Clostridium difficile.
    Giordano N, Hastie JL, Smith AD, Foss ED, Gutierrez-Munoz DF, Carlson PE Jr
  8. J Bacteriol 2018 Jul 25;200(16):e00218-18
    Updates to Clostridium difficile spore germination.
    Kochan TJ, Foley MH, Shoshiev MS, Somers MJ, Carlson PE, Hanna PC
  9. Pathog Dis 2018 Mar 1;76(2):fty010
    Transcriptomic profiling of Clostridium difficile grown under microaerophillic conditions.
    Giordano N, Hastie JL, Carlson PE Jr
  10. Pathog Dis 2018 Mar 1;76(2):fty009
    Transcriptional response of Clostridium difficile to low iron conditions.
    Hastie JL, Hanna PC, Carlson PE Jr
  11. Microbiol Spectr 2017 Sep;5(5):BAD-0017-2017
    U.S. regulatory considerations for development of live biotherapeutic products as drugs.
    Dreher-Lesnick SM, Stibitz S, Carlson PE Jr
  12. PLoS Pathog 2017 Jul 13;13(7):e1006443
    Intestinal calcium and bile salts facilitate germination of Clostridium difficile spores.
    Kochan TJ, Somers MJ, Kaiser AM, Shoshiev MS, Hagan AK, Hastie JL, Giordano NP, Smith AD, Schubert AM, Carlson PE Jr, Hanna PC