We developed a new class of vaccines, based on killed but metabolically active (KBMA) bacteria, that simultaneously takes advantage of the potency of live vaccines and the safety of killed vaccines. We removed genes required for nucleotide excision repair (uvrAB), rendering microbial-based vaccines exquisitely sensitive to photochemical inactivation with psoralen and long-wavelength ultraviolet light. Colony formation of the nucleotide excision repair mutants was blocked by infrequent, randomly distributed psoralen crosslinks, but the bacterial population was able to express its genes, synthesize and secrete proteins. Using the intracellular pathogen Listeria monocytogenes as a model platform, recombinant psoralen-inactivated Lm DeltauvrAB vaccines induced potent CD4(+) and CD8(+) T-cell responses and protected mice against virus challenge in an infectious disease model and provided therapeutic benefit in a mouse cancer model. Microbial KBMA vaccines used either as a recombinant vaccine platform or as a modified form of the pathogen itself may have broad use for the treatment of infectious disease and cancer.

Amotosalen/UVA treatment more effectively inactivates T cells than the current standard of gamma irradiation (2500 cGy) for the prevention of TA-GVHD.

BACKGROUND Pathogen inactivation methods are increasingly used to reduce the risk of infections after transfusion of blood products. Photochemical treatment (PCT) of platelets (PLTs) and plasma with amotosalen and ultraviolet A (UVA) light inactivates pathogens and white blood cells through formation of adducts between amotosalen and nucleic acid that block replication, transcription, and translation. The same adducts block the amplification of nucleic acids using polymerase chain reaction (PCR) in a manner that correlates with the number of adducts formed, providing a direct quality control (QC). Current QC measures for PCT rely on indirect methods that measure the delivered UVA dose or percent residual amotosalen after illumination, rather than directly measuring nucleic acid modification. STUDY DESIGN AND METHODS Endogenous mitochondrial DNA (mtDNA), which is detectable in PLT and plasma units, was chosen as a target for the quantification of photochemically induced modifications. DNA was extracted from untreated or amotosalen and UVA–treated PLTs or plasma, and mtDNA fragments of variable lengths were quantified using a real‐time PCR inhibition assay. RESULTS PCT induced increasing real‐time PCR inhibition of mtDNA amplification for larger amplicon sizes. Amplification was unaffected by treatment with amotosalen or UVA alone, whereas up to 3 log inhibition was observed after PCT. Blinded PCR testing of a panel of 110 samples each, from PLT or plasma components prepared for routine use within a blood center, allowed 100% discrimination between untreated and treated units. CONCLUSION Our initial findings indicate that an adequately sensitive, quantitative real‐time PCR inhibition assay targeting mtDNA could provide a valuable tool to confirm and monitor PCT.

Under defined conditions, PCT with amotosalen combined with UVA light can be used to inactivate B19, a clinically significant virus that can be transmitted through blood transfusion, and heretofore has been demonstrated to be refractory to inactivation.

Background S-303 was developed to inactivate viruses, bacteria, protozoa, and leukocytes in red blood cell concentrates (RBC). S-303 is a modular FRALE compound (FRangible Anchor Linker Effector) designed to bind to nucleic acids with its Anchor, to react through its Effector, and to form cross-links. S-303 spontaneously decomposes to the non-reactive compound S-300 by hydrolysis of the Linker, to minimize protein adducts. Pathogen inactivation (PI) treatment utilized the co-addition of S-303 and unbuffered GSH to quench non-specific S-303 reactions. The treatment process was optimized to maintain RBC function and maximize PI. Pre-clinical dog and rabbit chronic transfusion studies with allogeneic S-303 RBC showed no detectable antibodies to S-303 RBC. In Phase 1 studies, transfusion of healthy subjects with autologous human S-303 RBC demonstrated acceptable post-transfusion recovery and life span. Repeated transfusion (n=5) of 28 healthy subjects with autologous S-303 RBC showed no detectable antibodies to S-303 RBC. In a Phase 3 trial evaluating chronic transfusion of allogeneic S-303 RBC to patients with thalassemia or sickle cell anemia, 2 of 26 patients developed low titer positive Indirect Antiglobulin Tests (IAT) to S-303 RBC (one of the 2 patients also had a direct reacting IgM agglutinin). For both patients Direct Antiglobulin Tests (DAT) were negative. However, pretreatment RBC from the same unit remained compatible. S-303-related Anchor derivatives inhibited the positive IAT. Following this observation, clinical trials of S-303 RBC were stopped, studies were initiated to define the immunologic response to S-303 RBC, and an improved S-303 treatment process was developed. Methods The original PI process utilized 200 μM S-303 and 2 mM unbuffered GSH. The process was modified to use 10-fold more neutralized GSH (20 mM) added to RBC 10 minutes prior to addition of S-303 (200μM). High titer anti-Anchor sera (RaS) were elicited by immunizing rabbits with a stable Anchor-KLH construct. A FACS assay to detect decoration of RBC with S-303 was developed using RaS and FITC goat anti-rabbit (GAR) IgG. IAT assays were performed with two methods: High titer RaS were tested with buffer gel cards (MTS), S-303 RBC suspended in low ionic strength solution (LISS) and GAR IgG. Reactive patient sera were tested with S-303 RBC and anti-IgG gel cards (MTS) Results S-303 RBC prepared with the original clinical process were positive for IAT by gel card for both the RaS (1:100) and for the patient sera (1:3). FACS analysis using RaS (1:100) with FITC GAR IgG (1:64) demonstrated a high level of labeling. Under the modified conditions (S-303m), S-303m RBC exhibited minimal labeling above background by FACS with RaS. Sera from the 2 patients with positive IAT against S-303 RBC were negative against S-303m RBC. In addition, high titer RaS were negative against S-303m RBC in IAT by gel card. Potent inactivation of bacteria in S303m RBC (S. epidermis, S. marcescens) and viruses (Vesicular stomatitis virus) was retained. Storage of S-303m RBC for 42 days exhibited hemolysis and K+ levels comparable to S303 RBC and higher ATP levels than S-303 RBC. Conclusions An improved PI process has been developed that significantly reduces RBC decoration by S-303 while maintaining PI and RBC in vitro function. The new process eliminated the positive IAT reactivity with sera from patients previously alloimmunized to S-303 RBC.