Person:

Gelfand, Jeffrey

Background: Development of a potent vaccine adjuvant without introduction of any side effects remains an unmet challenge in the field of the vaccine research. Methodology/Principal Findings: We found that laser at a specific setting increased the motility of antigen presenting cells (APCs) and immune responses, with few local or systemic side effects. This laser vaccine adjuvant (LVA) effect was induced by brief illumination of a small area of the skin or muscle with a nondestructive, 532 nm green laser prior to intradermal (i.d.) or intramuscular (i.m.) administration of vaccines at the site of laser illumination. The pre-illumination accelerated the motility of APCs as shown by intravital confocal microscopy, leading to sufficient antigen (Ag)-uptake at the site of vaccine injection and transportation of the Ag-captured APCs to the draining lymph nodes. As a result, the number of Ag+ dendritic cells (DCs) in draining lymph nodes was significantly higher in both the 1° and 2° draining lymph nodes in the presence than in the absence of LVA. Laser-mediated increases in the motility and lymphatic transportation of APCs augmented significantly humoral immune responses directed against a model vaccine ovalbumin (OVA) or influenza vaccine i.d. injected in both primary and booster vaccinations as compared to the vaccine itself. Strikingly, when the laser was delivered by a hair-like diffusing optical fiber into muscle, laser illumination greatly boosted not only humoral but also cell-mediated immune responses provoked by i.m. immunization with OVA relative to OVA alone. Conclusion/Significance: The results demonstrate the ability of this safe LVA to augment both humoral and cell-mediated immune responses. In comparison with all current vaccine adjuvants that are either chemical compounds or biological agents, LVA is novel in both its form and mechanism; it is risk-free and has distinct advantages over traditional vaccine adjuvants.

Safe and effective immunologic adjuvants are often essential for vaccines. However, the choice of adjuvant for licensed vaccines is limited, especially for those that are administered intradermally. We show that non-tissue damaging, near-infrared (NIR) laser light given in short exposures to small areas of skin, without the use of additional chemical or biological agents, significantly increases immune responses to intradermal influenza vaccination without augmenting IgE. The NIR laser-adjuvanted vaccine confers increased protection in a murine influenza lethal challenge model as compared to unadjuvanted vaccine. We show that NIR laser treatment induces the expression of specific chemokines in the skin resulting in recruitment and activation of dendritic cells and is safe to use in both mice and humans. The NIR laser adjuvant technology provides a novel, safe, low-cost, simple-to-use, potentially broadly applicable and clinically feasible approach to enhancing vaccine efficacy as an alternative to chemical and biological adjuvants.

Background: Although dendritic cell (DC) vaccines are considered to be promising treatments for advanced cancer, their production and administration is costly and labor-intensive. We developed a novel immunotherapeutic agent that links a single-chain antibody variable fragment (scFv) targeting mesothelin (MSLN), which is overexpressed on ovarian cancer and mesothelioma cells, to Mycobacterium tuberculosis (MTB) heat shock protein 70 (Hsp70), which is a potent immune activator that stimulates monocytes and DCs, enhances DC aggregation and maturation and improves cross-priming of T cells mediated by DCs. Methods: Binding of this fusion protein with MSLN on the surface of tumor cells was measured by flow cytometry and fluorescence microscopy. The therapeutic efficacy of this fusion protein was evaluated in syngeneic and orthotopic mouse models of papillary ovarian cancer and malignant mesothelioma. Mice received 4 intraperitoneal (i.p.) treatments with experimental or control proteins post i.p. injection of tumor cells. Ascites-free and overall survival time was measured. For the investigation of anti-tumor T-cell responses, a time-matched study was performed. Splenocytes were stimulated with peptides, and IFNγ- or Granzyme B- generating CD3+CD8+ T cells were detected by flow cytometry. To examine the role of CD8+ T cells in the antitumor effect, we performed in vivo CD8+ cell depletion. We further determined if the fusion protein increases DC maturation and improves antigen presentation as well as cross-presentation by DCs. Results: We demonstrated in vitro that the scFvMTBHsp70 fusion protein bound to the tumor cells used in this study through the interaction of scFv with MSLN on the surface of these cells, and induced maturation of bone marrow-derived DCs. Use of this bifunctional fusion protein in both mouse models significantly enhanced survival and slowed tumor growth while augmenting tumor-specific CD8+ T-cell dependent immune responses. We also demonstrated in vitro and in vivo that the fusion protein enhanced antigen presentation and cross-presentation by targeting tumor antigens towards DCs. Conclusions: This new cancer immunotherapy has the potential to be cost-effective and broadly applicable to tumors that overexpress mesothelin.

Lentiviral vectors (LVVs) are powerful genetic tools that are being used with greater frequency in biomedical laboratories and clinical trials. Adverse events reported from initial clinical studies provide a basis for risk assessment of occupational exposures, yet many questions remain about the potential harm that LVVs may cause. We review those risks and provide a framework for principal investigators, Institutional Biosafety Committees, and occupational health professionals to assess and communicate the risks of exposure to staff. We also provide recommendations to federal research and regulatory agencies for tracking LVV exposures to evaluate long-term outcomes. U.S. Food and Drug Administration approved antiviral drugs for HIV have theoretical benefits in LVV exposures, although evidence to support their use is currently limited. If treatment is appropriate, we recommend a 7-day treatment with an integrase inhibitor with or without a reverse transcriptase inhibitor within 72 hours of exposure.

Antimicrobial photodynamic inactivation (aPDI) employs photosensitizing dyes activated by visible light to produce reactive oxygen species. aPDI is independent of the antibiotic resistance status of the target cells, and is thought unlikely to produce resistance itself. Among many PS that have been investigated, tetracyclines occupy a unique niche. They are potentially dual-action compounds that can both kill bacteria under illumination, and prevent bacterial regrowth by inhibiting ribosomes. Tetracycline antibiotics are regarded as bacteriostatic rather than bactericidal. Doxycycline (DOTC) is excited best by UVA light (365 nm) while demeclocycline (DMCT) can be efficiently activated by blue light (415 nm) as well as UVA. Both compounds were able to eradicate Gram-positive (methicillin-resistant Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria (>6 log(10) steps of killing) at concentrations (10–50μM) and fluences (10-20J/cm2). In contrast to methylene blue, MB plus red light, tetracyclines photoinactivated bacteria in rich growth medium. When ~3 logs of bacteria were killed with DMCT/DOTC+light and the surviving cells were added to growth medium, further bacterial killing was observed, while the same experiment with MB allowed complete regrowth. MIC studies were carried out either in the dark or exposed to 0.5mW/cm2 blue light. Up to three extra steps (8-fold) increased antibiotic activity was found with light compared to dark, with MRSA and tetracycline-resistant strains of E. coli. Tetracyclines can accumulate in bacterial ribosomes, where they could be photoactivated with blue/UVA light producing microbial killing via ROS generation.

Drug-resistant urinary tract infections (UTIs) are difficult and sometimes impossible to treat. Many UTIs are caused by uropathogenic Escherichia coli (UPEC). We developed an intact rat model of UTI, by catheterizing female rats and introducing a bioluminescent UPEC strain into the female rat bladder which lasted for up to six days. We recently showed that antimicrobial photodynamic inactivation (aPDI) of a bacterial infection mediated by the well-known phenothiazinium salt, methylene blue (MB) could be strongly potentiated by addition of the non-toxic salt potassium iodide (KI). In the intact rat model we introduced MB into the bladder by catheter, followed by KI solution and delivered intravesicular illumination with a diffusing fiber connected to a 1 W 660 nm laser. Bioluminescent imaging of the bacterial burden was carried out during the procedure and for 6 days afterwards. Light-dose dependent loss of bioluminescence was observed with the combination of MB followed by KI, but recurrence of infection was seen the next day in some cases. aPDT with MB + KI gave a significantly shorter duration of infection compared to untreated controls. aPDT with MB alone was the least effective. No signs of aPDT damage to the bladder lining were detected. This procedure to treat urinary tract infections without antibiotics by using already approved pharmaceutical substances (MB and KI) may have clinical applicability, either initially as a stand-alone therapy, or as an adjunct to antibiotic therapy by a rapid and substantial reduction of the bacterial burden.