Development of Immunotherapy Strategies: Targeting Gram-positive and Gram-negative Pathogenic Bacteria

The rapid surge in drug-resistant bacterial infections has now become one of the primary public health crises of the 21st century. In a world without effective antibiotics, modern surgical and medical procedures will become too dangerous or impossible due to the threat of untreatable bacterial infections. As discussed in Chapter 1, the emergence of antibiotic resistant bacteria threatens to render a majority of the current antimicrobial therapeutics ineffective. Every year in the United States alone, over two million people are afflicted with bacterial infections that are resistant to FDA-approved antibiotics. According to the CDC, over 20,000 of those patients died as a result of drug-resistant Gram-positive bacterial infections, such as Streptococcus pneumoniae (S. pneumoniae), Enterococcus faecium (E. faecium), and Staphylococcus aureus (S. aureus). Equally alarming is the emergence of multidrug Gram-negative pathogenic bacteria, including strains that are resistant to all currently available antibiotics. As the number of efficacious antibiotics continues to rapidly dwindle without replenishment, the possibility of entering a post-antibiotic era can become a reality. Discovery and development of drug leads against the most serious pathogenic bacteria is desperately needed to reinvigorate the antibiotic pipeline and reverse this alarming trend. This thesis will discuss the design of two immunotherapy strategies that target bacterial cells for destruction via surface modeling conjugates that specifically home to bacterial cell surfaces. The Pires lab has pioneered the field of bacterial immunotherapy for the eradication of Gram-positive bacteria. In Chapter 2, we will highlight previous reports of facile bacterial surface modulation strategies that act to stimulate or attenuate the host immune system. We have extended our techniques of bacterial surface remodeling with the goal of reactivating the host immune system to seek out and directly clear pathogenic bacteria. In Chapter 3, we set out to leverage the surface-homing properties of vancomycin to specifically tag the surface of Gram-positive Staphylococcus aureus with immune cell attractants. Vancomycin was conjugated to a small molecule hapten, known to effectively recruit endogenous antibodies. In combination with sortase A-mediated surface remodeling, which are house-keeping enzymes that catalyze selective and covalent modification of bacterial cell walls, we successfully demonstrated the tagging and recruitment of endogenous anti-DNP antibodies to the surface of S. aureus. We also showed, for the first time, in vivo selective targeting of S. aureus in live C. elegans, a widely used model host to understand bacterial pathogenesis and host-pathogen interactions. Together, our results pave the way for a narrow-spectrum strategy for the destruction of bacterial infections caused by S. aureus (drug-sensitive and -resistant) through bacterial immunotherapy.Chapter 4 will discuss our goal is to eradicate Gram-negative superbugs by targeting problematic pathogenic bacteria for destruction by the host immune system. In this second major strategy, we report the design and development of a series of polymyxin B conjugates (a last resort antibiotic against Gram-negative pathogens), which are, to our knowledge, the first class of synthetic molecules that remodel Gram-negative bacterial cell surfaces with immune cell attractants. Given the inherent antimicrobial activity of polymyxin B, we designed agents to display dual activities against bacteria (membrane-disruption and immune activation). By leveraging the power of the immune system in clearing pathogens, this new class of molecules was shown to uniquely target Gram-negative bacteria and, additionally, potentiate existing FDA-approved antibiotics. Additionally, in this study, the recruitment of antibodies from pooled human serum is shown, thus validating the biological relevance of this immunotherapy. We hope to establish this approach as a potential treatment option and further refine this methodology to address the clinical challenge of Gram-negative bacterial pathogens. The last chapter of this thesis focuses on the development of a facile assay to monitor the activity and inhibition of two isoforms of the Peptidylarginine deiminases (PAD) family: PAD2 and PAD4. PADs are post-translational modifiers that catalyze the calcium-dependent conversion of arginine residues to unnatural citrulline residues in a protein substrate. The full extent of the role PADs play in normal physiology and diseased states is not yet fully understood. PADs have important roles in the formation of Neutrophil Extracellular Traps (NETs), which was a recently discovered response of the immune system against bacterial pathogens. NETs are biomolecules that encase invading pathogens, which immobilize them to assist in their clearance by the human immune system. We report on a new, fluorescence-based assay, which is readily performed under ambient conditions and is compatible with high-throughput screening platforms. Furthermore, through a collaboration with Penn State Hershey Medical Center, we utilized the assay in a high-throughput screen for potential PAD4 inhibitors

Acute myeloid leukemia in SRP54‐mutated congenital neutropenia

Abstract SRP54 mutations have recently been implicated in congenital neutropenia (CN) and the in‐frame deletion, p.Thr117del, is the most common pathogenic mutation reported. The largest study of SRP54‐mutated CN to‐date followed 23 patients for a median of 15 years. No patients developed a hematologic malignancy in that study. Given the known risk of leukemia in other CNs it is crucial to know whether patients with SRP54‐mutated CN have an increased risk of leukemia. We report the first case of leukemia in a patient with SRP54‐mutated CN. A 15‐year‐old male with SRP54‐mutated CN (p.Thr117del) was diagnosed with acute myeloid leukemia with myelodysplasia‐related changes on a screening bone marrow evaluation. Next generation sequencing of the leukemia cells identified CSF3R and RUNX1 mutations. These mutations commonly co‐exist in CN‐associated malignancies and suggest leukemogenesis in SRP54‐mutated CN may occur in a similar manner to other CNs. He was successfully treated with CPX‐351 followed by hematopoietic cell transplant (HCT) and remains in remission at a follow‐up time of 9 months. Although conclusions from this single report must be limited, this has potentially significant implications for both screening and treatment practices for these patients, including the role and timing of HCT

d‑Amino Acid Mediated Recruitment of Endogenous Antibodies to Bacterial Surfaces

The number of antibiotic resistant bacterial strains has been continuously increasing over the last few decades. Nontraditional routes to combat bacteria may offer an attractive alternative to the ongoing problem of drug discovery in this field. Herein, we describe the initial framework toward the development of bacterial d-amino acid antibody recruitment therapy (DART). DART represents a promising antibiotic strategy by exploiting the promiscuity of bacteria to incorporate unnatural d-amino acids and subsequently recruit antibodies to the bacterial surface. The conjugation of 2,4-dinitrophenyl (DNP) to various d-amino acids led to the discovery of a d-amino acid that specifically tags the surface of <i>Bacillus subtilis</i> and <i>Staphylococcus aureus</i> for the recruitment of anti-DNP antibodies (a highly abundant antibody in human serum). This system represents a novel strategy as an antibacterial therapy that targets planktonic Gram-positive bacteria

Improved chemotherapy modeling with RAG-based immune deficient mice.

We have previously characterized an acute myeloid leukemia (AML) chemotherapy model for SCID-based immune deficient mice (NSG and NSGS), consisting of 5 days of cytarabine (AraC) and 3 days of anthracycline (doxorubicin), to simulate the standard 7+3 chemotherapy regimen many AML patients receive. While this model remains tractable, there are several limitations, presumably due to the constitutional Pkrdcscid (SCID, severe combined immune deficiency) mutation which affects DNA repair in all tissues of the mouse. These include the inability to combine preconditioning with subsequent chemotherapy, the inability to repeat chemotherapy cycles, and the increased sensitivity of the host hematopoietic cells to genotoxic stress. Here we attempt to address these drawbacks through the use of alternative strains with RAG-based immune deficiency (NRG and NRGS). We find that RAG-based mice tolerate a busulfan preconditioning regimen in combination with either AML or 4-drug acute lymphoid leukemia (ALL) chemotherapy, expanding the number of samples that can be studied. RAG-based mice also tolerate multiple cycles of therapy, thereby allowing for more aggressive, realistic modeling. Furthermore, standard AML therapy in RAG mice was 3.8-fold more specific for AML cells, relative to SCID mice, demonstrating an improved therapeutic window for genotoxic agents. We conclude that RAG-based mice should be the new standard for preclinical evaluation of therapeutic strategies involving genotoxic agents