Novel polysaccharide-protein conjugates provide an immunogenic 13-valent pneumococcal conjugate vaccine for S. pneumoniae.

Pneumonia remains the single leading cause of childhood death worldwide. Despite the commercial availability of multiple pneumococcal conjugate vaccines (PCVs), high dosage cost and supply shortages prevent PCV delivery to much of the developing world. The current work presents high-yield pneumococcal conjugates that are immunogenic in animals and suitable for use in human vaccine development. The 13-valent pneumococcal conjugate vaccine (PCV-13) investigated in this research incorporated serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. Pneumococcal polysaccharides (PnPSs) and CRM197 carrier protein were produced and purified in-house, and used to prepare PnPS-CRM conjugates using unique, cyanide-free, in vacuo glycation conjugation methods. In vitro characterization confirmed the generation of higher molecular weight PnPS-CRM conjugates low in free protein. In vivo animal studies were performed to compare PnuVax's PCV-13 to the commercially available PCV-13, Prevnar®13 (Pfizer, USA). A boost dose was provided to all groups post-dose 1 at t = 14 days. Post-dose 2 results at t = 28 days showed that all 13 serotypes in PnuVax's PCV-13 were boostable. Per serotype IgG GMCs demonstrated that PnuVax's PCV-13 is immunogenic for all 13 serotypes, with 10 of the 13 serotypes statistically the same or higher than Prevnar®13 post-dose 2. As a result, the novel polysaccharide-protein conjugates developed in this work are highly promising for use in human PCV development. The in vacuo conjugation technique applied in this work could also be readily adapted to develop many other conjugate vaccines.

The performance of decellularized adipose tissue microcarriers as an inductive substrate for human adipose-derived stem cells.

With the aim of developing a clinically-translatable cell expansion and delivery vehicle for adipose tissue engineering, the adipogenic differentiation of human adipose-derived stem cells (ASCs) was investigated on microcarriers fabricated from human decellularized adipose tissue (DAT). ASCs seeded on the DAT microcarriers and cultured in adipogenic differentiation medium within a low-shear spinner culture system demonstrated high levels of adipogenic differentiation, as measured by the expression of adipogenic genes, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and intracellular lipid accumulation. In contrast, gelatin microcarrier controls did not demonstrate significant adipogenesis, emphasizing the role of the native matrix in mediating ASC differentiation. Interestingly, ASCs cultured on the DAT microcarriers in proliferation medium expressed elevated levels of the adipogenic markers, suggesting that the DAT provided an adipo-inductive substrate for the human ASCs. In vivo testing of the DAT and gelatin microcarriers in a subcutaneous Wistar rat model confirmed injectability and demonstrated stable volume retention over 28 days. Under histological analysis, the DAT microcarriers demonstrated no evidence of immunogenicity or cytotoxicity, with the DAT supporting cellular infiltration and tissue remodeling. Pre-seeding the DAT microcarriers with allogenic rat ASCs enhanced cellularity and angiogenesis within the implant region.

Design and characterization of tissue-specific extracellular matrix-derived microcarriers.

The three-dimensional (3D) extracellular matrix (ECM) environment plays a critical role in mediating normal cellular behavior and tissue organization. While commercially available microcarriers have shown promise, limited research has been conducted on the design of tissue-specific, custom-fabricated microcarriers, engineered to mimic the composition of the native ECM of cells or tissues of interest. Moving toward this goal, methods were developed to fabricate microcarriers from decellularized adipose tissue (DAT) via minimally-cytotoxic protocols. Characterization by microscopy confirmed the production of stable spherical microcarriers, with a microporous surface topography and porous interior. The mean diameter of the DAT microcarriers was 934±51 µm, while the porosity was estimated as 29%±4% using liquid displacement. Stability and swelling behavior over 4 weeks indicated that the DAT microcarriers were effectively stabilized with the photochemical crosslinking agent rose bengal, with total protein release in a simulated physiological environment remaining below 10 µg/mL at all time points. Preliminary cell culture studies with human adipose-derived stem cells (ASCs) in a spinner flask system indicated enhanced cell attachment and proliferation of ASCs on DAT microcarriers over 14 days, as compared with gelatin control microcarriers fabricated using similar methods. Testing confirmed injectability of the DAT microcarriers, further supporting the clinical potential of the approach for localized cell delivery and small volume augmentation in plastic and reconstructive surgery. Overall, tissue-specific microcarriers prepared from solubilized DAT were found to be highly supportive of human ASCs cultured in a 3D dynamic environment.