Incorporation of D-amino acids into peptidoglycan is a unique metabolic feature of bacteria. Since D-amino acids are not metabolic substrates in most mammalian tissues, this difference can be exploited to detect living bacteria in vivo. Given the prevalence of D-alanine in peptidoglycan muropeptides, as well as its role in several antibiotic mechanisms, we targeted this amino acid for positron emission tomography (PET) radiotracer development. D-[3-11 C]Alanine and the dipeptide D-[3-11 C]alanyl-Dalanine were synthesized via asymmetric alkylation of glycine-derived Schiff-base precursors with [ 11 C]methyl iodide in the presence of a cinchonidinium phase-transfer catalyst. In cell experiments, both tracers showed accumulation by a wide variety of both Grampositive and Gram-negative pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. In a mouse model of acute bacterial myositis, D-[3-11 C]alanine was accumulated by living microorganisms but was not taken up in areas of sterile inflammation.When compared to existing clinical nuclear imaging tools, specifically 2-deoxy-2-[ 18 F]fluoro-D-glucose and a gallium citrate radiotracer, D-alanine showed more bacteria-specific uptake. Decreased D-[3-11 C]alanine uptake was also observed in antibioticsensitive microbes after antimicrobial therapy, when compared to that in resistant organisms. Finally, prominent uptake of D-[3-11 C]alanine uptake was seen in rodent models of discitis-osteomyelitis and P. aeruginosa pneumonia. These data provide strong justification for clinical translation of D-[3-11 C]alanine to address a number of important human infections.
Currently, there exists no accurate, noninvasive clinical imaging method to detect living bacteria in vivo. Our goal is to provide a positron emission tomography (PET) method to image infection by targeting bacteria-specific metabolism. Standard of care methodologies detect morphologic changes, image immunologic response to infection, or employ invasive tissue sampling with associated patient morbidity. These strategies, however, are not specific for living bacteria and are often inadequate to detect bacterial infection during fever workup. As such, there is an unmet clinical need to identify and validate new imaging tools suitable for noninvasive, in vivo (PET) imaging of living bacteria. We have shown that D-[methyl-11 C]methionine (D-[ 11 C]Met) can distinguish active bacterial infection from sterile inflammation in a murine infection model and is sensitive to both Gram-positive and Gram-negative bacteria. Here, we report an automated and >99% enantiomeric excess (ee) synthesis of D-[ 11 C]Met from a linear D-homocysteine precursor, a significant improvement over the previously reported synthesis utilizing a D-homocysteine thiolactone hydrochloride precursor with approximately 75−85% ee. Furthermore, we took additional steps toward applying D-[ 11 C]Met to infected patients. D-[ 11 C]Met was subject to a panel of clinically relevant bacterial strains and demonstrated promising sensitivity to these pathogens. Finally, we performed radiation dosimetry in a normal murine cohort to set the stage for translation to humans in the near future.
Conflict of Interest Statement: JL, SG, LH, TL and JP are or were employed by CellSight Technologies. CellSight Technologies Incorporated is commercializing [ 18 F]F-AraG as a PET tracer for evaluation of immune response in immunotherapy. No other potential conflicts of interest relevant to this article exist.
A major obstacle to achieving long-term antiretroviral (ART) free remission or functional cure of HIV infection is the presence of persistently infected cells that establish a long-lived viral reservoir. HIV largely resides in anatomical regions that are inaccessible to routine sampling, however, and non-invasive methods to understand the longitudinal tissue-wide burden of HIV persistence are urgently needed. Positron emission tomography (PET) imaging is a promising strategy to identify and characterize the tissue-wide burden of HIV. Here, we assess the efficacy of using immunoPET imaging to characterize HIV reservoirs and identify anatomical foci of persistent viral transcriptional activity using a radiolabeled HIV Env-specific broadly neutralizing antibody, 89Zr-VRC01, in HIV-infected individuals with detectable viremia and on suppressive ART compared to uninfected controls (NCT03729752). We also assess the relationship between PET tracer uptake in tissues and timing of ART initiation and direct HIV protein expression in CD4 T cells obtained from lymph node biopsies. We observe significant increases in 89Zr-VRC01 uptake in various tissues (including lymph nodes and gut) in HIV-infected individuals with detectable viremia (N = 5) and on suppressive ART (N = 5) compared to uninfected controls (N = 5). Importantly, PET tracer uptake in inguinal lymph nodes in viremic and ART-suppressed participants significantly and positively correlates with HIV protein expression measured directly in tissue. Our strategy may allow non-invasive longitudinal characterization of residual HIV infection and lays the framework for the development of immunoPET imaging in a variety of other infectious diseases.
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