Propionibacterium acnes susceptibility to low-level 449 nm blue light photobiomodulation.

BACKGROUND AND OBJECTIVE: Recent advances in low-level light devices have opened new treatment options for mild to moderate acne patients. Light therapies have been used to treat a variety of skin conditions over the years but were typically only available as treatments provided by professional clinicians. Clinical application of blue light has proven to be effective for a broader spectral range and at lower fluences than previously utilized. Herein, we tested the hypothesis that sub-milliwatt/cm2 levels of long-wave blue light (449 nm) effectively kills Propionibacterium acnes, a causative agent of acne vulgaris, in vitro. MATERIALS AND METHODS: Two types of LED light boards were designed to facilitate in vitro blue light irradiation to either six-well plates containing fluid culture or a petri plate containing solid medium. P. acnes. Survival was determined by counting colony forming units (CFU) following irradiation. P. acnes was exposed in the presence and absence of oxygen. Coproporphyrin III (CPIII) photoexcitation was spectrophotometrically evaluated at 415 and 440 nm to compare the relative photochemical activities of these wavelengths. RESULTS: 422 and 449 nm blue light killed P. acnes in planktonic culture. Irradiation with 449 nm light also effectively killed P. acnes on a solid agar surface. Variation of time or intensity of light exposure resulted in a fluence-dependent improvement of antimicrobial activity. The presence of oxygen was necessary for killing of P. acnes with 449 nm light. CPIII displayed clear photoexcitation at both 415 and 440 nm, indicating that both wavelengths are capable of initiating CPIII photoexcitation at low incident light intensities (50 uW/cm2 ). CONCLUSION: Herein we demonstrate that sub-milliwatt/cm2 levels of long-wave blue light (449 nm) effectively kill P. acnes. The methods and results presented allow for deeper exploration and design of light therapy treatments. Results from these studies are expanding our understanding of the mode of action and functionality of blue light, allowing for improved options for acne patients. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Glycovaccinology: The design and engineering of carbohydrate-based vaccine components.

Vaccines remain one of the most important pillars in preventative medicine, providing protection against a wide array of diseases by inducing humoral and/or cellular immunity. Of the many possible candidate antigens for subunit vaccine development, carbohydrates are particularly appealing because of their ubiquitous presence on the surface of all living cells, viruses, and parasites as well as their known interactions with both innate and adaptive immune cells. Indeed, several licensed vaccines leverage bacterial cell-surface carbohydrates as antigens for inducing antigen-specific plasma cells secreting protective antibodies and the development of memory T and B cells. Carbohydrates have also garnered attention in other aspects of vaccine development, for example, as adjuvants that enhance the immune response by either activating innate immune responses or targeting specific immune cells. Additionally, carbohydrates can function as immunomodulators that dampen undesired humoral immune responses to entire protein antigens or specific, conserved regions on antigenic proteins. In this review, we highlight how the interplay between carbohydrates and the adaptive and innate arms of the immune response is guiding the development of glycans as vaccine components that act as antigens, adjuvants, and immunomodulators. We also discuss how advances in the field of synthetic glycobiology are enabling the design, engineering, and production of this new generation of carbohydrate-containing vaccine formulations with the potential to prevent infectious diseases, malignancies, and complex immune disorders.

Profiling Germinal Center-like B Cell Responses to Conjugate Vaccines Using Synthetic Immune Organoids.

Glycoengineered bacteria have emerged as a cost-effective platform for rapid and controllable biosynthesis of designer conjugate vaccines. However, little is known about the engagement of such conjugates with naïve B cells to induce the formation of germinal centers (GC), a subanatomical microenvironment that converts naïve B cells into antibody-secreting plasma cells. Using a three-dimensional biomaterials-based B-cell follicular organoid system, we demonstrate that conjugates triggered robust expression of hallmark GC markers, B cell receptor clustering, intracellular signaling, and somatic hypermutation. These responses depended on the relative immunogenicity of the conjugate and correlated with the humoral response in vivo. The occurrence of these mechanisms was exploited for the discovery of high-affinity antibodies against components of the conjugate on a time scale that was significantly shorter than for typical animal immunization-based workflows. Collectively, these findings highlight the potential of synthetic organoids for rapidly predicting conjugate vaccine efficacy as well as expediting antigen-specific antibody discovery.

A Low-Cost, Thermostable, Cell-Free Protein Synthesis Platform for On-Demand Production of Conjugate Vaccines.

Cell-free protein synthesis systems that can be lyophilized for long-term, non-refrigerated storage and transportation have the potential to enable decentralized biomanufacturing. However, increased thermostability and decreased reaction cost are necessary for further technology adoption. Here, we identify maltodextrin as an additive to cell-free reactions that can act as both a lyoprotectant to increase thermostability and a low-cost energy substrate. As a model, we apply optimized formulations to produce conjugate vaccines for ∼$0.50 per dose after storage at room temperature (∼22 °C) or 37 °C for up to 4 weeks, and ∼$1.00 per dose after storage at 50 °C for up to 4 weeks, with costs based on raw materials purchased at the laboratory scale. We show that these conjugate vaccines generate bactericidal antibodies against enterotoxigenic Escherichia coli (ETEC) O78 O-polysaccharide, a pathogen responsible for diarrheal disease, in immunized mice. We anticipate that our low-cost, thermostable cell-free glycoprotein synthesis system will enable new models of medicine biosynthesis and distribution that bypass cold-chain requirements.

A low-cost recombinant glycoconjugate vaccine confers immunogenicity and protection against enterotoxigenic Escherichia coli infections in mice.

Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of traveler's diarrhea and a major cause of diarrheal disease and death worldwide, especially in infants and young children. Despite significant efforts over the past several decades, an affordable vaccine that appreciably decreases mortality and morbidity associated with ETEC infection among children under the age of 5 years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic E. coli or their cell-free extracts for producing conjugate vaccine candidates against two of the most prevalent O serogroups of ETEC, O148 and O78. Specifically, we demonstrate site-specific installation of O-antigen polysaccharides (O-PS) corresponding to these serogroups onto licensed carrier proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. The resulting conjugates stimulate strong O-PS-specific humoral responses in mice and elicit IgG antibodies that possess bactericidal activity against the cognate pathogens. We also show that one of the prototype conjugates decorated with serogroup O148 O-PS reduces ETEC colonization in mice, providing evidence of vaccine-induced mucosal protection. We anticipate that our bacterial cell-based and cell-free platforms will enable creation of multivalent formulations with the potential for broad ETEC serogroup protection and increased access through low-cost biomanufacturing.

Synthesis, Characterization, and Antimicrobial Investigation of a Novel Chlorhexidine Cyclamate Complex.

The synthesis, crystal structure, and antimicrobial efficacy are reported for a novel material comprising a 1:2 ratio of chlorhexidine (CHX) to N-cyclohexylsulfamate (i.e., artificial sweetener known as cyclamate). The chemical structure is unambiguously identified by incorporating a combination of single-crystal X-ray diffraction (SC-XRD), electrospray ionization mass spectrometry (ESI-MS), 1H nuclear magnetic resonance (NMR) spectroscopy, correlation spectroscopy (COSY), and attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). The new material: 1) is amongst only several reported structures identified to date incorporating the vital chlorhexidine antimicrobial drug; 2) exhibits broad spectrum antimicrobial activity at concentrations less than 15 µg/mL; and 3) provides a unique delivery method for the essential active pharmaceutical ingredient (API). Furthermore, substitution of inactive gluconate with bioactive cyclamate counterion potentially provides the additional benefit of improving the taste profile of chlorhexidine.

Synthesis, Characterization, and Investigation of the Antimicrobial Activity of Cetylpyridinium Tetrachlorozincate.

Cetylpyridinium tetrachlorozincate (referred to herein as (CP)2ZnCl4) was synthesized and its solid-state structure was elucidated via single-crystal X-ray diffraction (SC-XRD), revealing a stoichiometry of C42H76Cl4N2Zn with two cetylpyridinium (CP) cations per [ZnCl4]2- tetrahedra. Crystal structures at 100 and 298 K exhibited a zig-zag pattern with alternating alkyl chains and zinc units. The material showed potential for application as a broad-spectrum antimicrobial agent, to reduce volatile sulfur compounds (VSCs) generated by bacteria, and in the fabrication of advanced functional materials. Minimum inhibitory concentration (MIC) of (CP)2ZnCl4 was 60, 6, and 6 µg mL-1 for Salmonella enterica, Staphylococcus aureus, and Streptococcus mutans, respectively. The MIC values of (CP)2ZnCl4 were comparable to that of pure cetylpyridinium chloride (CPC), despite the fact that approximately 16% of the bactericidal CPC is replaced with bacteriostatic ZnCl2 in the structure. A modified layer-by-layer deposition technique was implemented to synthesize mesoporous silica (i.e., SBA-15) loaded with approximately 9.0 wt % CPC and 8.9 wt % Zn.