Immunoinformatic screening of Marburgvirus epitopes and computational investigations of epitope-allele complexes.

Marburgvirus (MARV), a member of the Filovirus family, causes severe hemorrhagic fever in humans. Currently, there are no approved vaccines or post exposure treatment methods available against MARV. With the aim of identifying vaccine candidates against MARV, we employ different sequence-based computational methods to predict the MHC-I and MHC-II T-cell epitopes as well as B-cell epitopes for the complete MARV genome. We analyzed the variations in the predicted epitopes among four MARV variants, the Lake Victoria, Angola, Musoke, and Ravn. We used a consensus approach to identify several epitopes, including novel epitopes, and narrowed down the selection based on different parameters such as antigenicity and IC50 values. The selected epitopes can be used in various vaccine constructs that give effective antibody responses. The MHC-I epitope-allele complexes for GP and NP with favorably low IC50 values were investigated using molecular dynamics computations to determine the molecular details of the epitope-allele complexes. This study provides information for further experimental validation of the potential epitopes and the design and development of MARV vaccines.

Mucosal Delivery of HIV-1 Glycoprotein Vaccine Candidate Enabled by Short Carbon Nanotubes.

The HIV-1 envelope glycoprotein spike is the target of antibodies, and therefore represents the main viral antigen for antibody-based vaccine design. One of the challenges in HIV-1 vaccine development is finding efficient ways for the immune system to recognize and respond to HIV-1 without establishing an infection. Since HIV-1 enters the body at mucosal surfaces, induction of immune response at these sites is a preferred preventive approach. Nasal administration is a very effective route for mucosal immunization since it can stimulate mucosal immune responses both locally and distantly. In this paper, Luna develops a safe, short carbon nanotube (CNT)-based, needle-free delivery platform known as "CNTVac". The size of short CNT was controlled to possess HIV-1 particle-like morphology (100-200 nm) capable of efficiently delivering a broad range of antigens intranasally. PEG-Lipid served as the antigen conformation protector and mucosal barrier penetration enhancer (Schematic Figure) was localized between V1V2 antigens, which caused highly enhanced local IgA and systemic antibody IgG responses in mice and rabbits. The short CNT incorporated with PEG-Lipid could not only serve as efficient delivery system but also reduce the amount of lipid usage in order to balance the vaccine dosage in order to eliminate the potential adverse effect. These data suggest a promising platform technology for vaccine delivery.

Layer-by-Layer Delivery of Multiple Antigens Using Trimethyl Chitosan Nanoparticles as a Malaria Vaccine Candidate.

Developing a safe and effective malaria vaccine is critical to reducing the spread and resurgence of this deadly disease, especially in children. In recent years, vaccine technology has seen expanded development of subunit protein, peptide, and nucleic acid vaccines. This is due to their inherent safety, the ability to tailor their immune response, simple storage requirements, easier production, and lower expense compared to using attenuated and inactivated organism-based approaches. However, these new vaccine technologies generally have low efficacy. Subunit vaccines, due to their weak immunogenicity, often necessitate advanced delivery vectors and/or the use of adjuvants. A new area of vaccine development involves design of synthetic micro- and nano-particles and adjuvants that can stimulate immune cells directly through their physical and chemical properties. Further, the unique and complex life cycle of the Plasmodium organism, with multiple stages and varying epitopes/antigens presented by the parasite, is another challenge for malaria vaccine development. Targeting multistage antigens simultaneously is therefore critical for an effective malaria vaccine. Here, we rationally design a layer-by-layer (LbL) antigen delivery platform (we called LbL NP) specifically engineered for malaria vaccines. A biocompatible modified chitosan nanoparticle (trimethyl chitosan, TMC) was synthesized and utilized for LbL loading and release of multiple malaria antigens from pre-erythrocytic and erythrocytic stages. LbL NP served as antigen/protein delivery vehicles and were demonstrated to induce the highest Plasmodium falciparum Circumsporozoite Protein (PfCSP) specific T-cell responses in mice studies as compared to multiple controls. From immunogenicity studies, it was concluded that two doses of intramuscular injection with a longer interval (4 weeks) than traditional malaria vaccine candidate dosing would be the vaccination potential for LbL NP vaccine candidates. Furthermore, in PfCSP/Py parasite challenge studies we demonstrated protective efficacy using LbL NP. These LbL NP provided a significant adjuvant effect since they may induce innate immune response that led to a potent adaptive immunity to mediate non-specific anti-malarial effect. Most importantly, the delivery of CSP full-length protein stimulated long-lasting protective immune responses even after the booster immunization 4 weeks later in mice.

Manipulating DNA probe presentation via enzymatic cleavage of diluent strands.

We previously reported a system for the controlled redispersion of DNA-linked aggregates using secondary, competitive hybridization events and found that complete redispersion is contingent upon dilution of the active 20 base-long probe strands with 20 base-long nonhybridizing strands. Here, to reduce the steric interference of nonhybridizing or diluent strands on probe activity, we investigate the effect of shorter diluent strands on the hybridization activity of immobilized probes using the following two approaches: (1) simultaneously coupling shorter diluent strands and longer probe strands to microspheres and (2) simultaneously coupling diluent and probe strands of the same base length to microspheres and then clipping diluent strands with the restriction endonuclease AluI. Results indicate that one can reduce the duplex density down by 50-70% of its initial value, depending on the location of the recognition motif along the hybridization segment. In addition, tighter control over the number of probe-target duplexes is achieved with the enzyme-based approach.

Measuring stem cell dimensionality in tissue scaffolds.

Many scaffold systems have evolved for tissue engineering and in vitro tissue models to provide a 3D (three-dimensional) microenvironment that enables cells to behave more physiologically. We hypothesized that cells would adopt morphologies with more 3D character during culture in scaffolds as compared to planar substrates. Cell shape and function are tightly linked and effects of scaffold niche properties on cell shape and dimensionality are important for directing cell function. Herein, primary human bone marrow stromal cells (hBMSCs) were cultured in 6 different scaffolds and on a planar control substrate. hBMSCs were imaged using 3D confocal microscopy, and 3D image analyses were used to assess hBMSC shape and dimensionality. A characteristic gyration tensor ellipsoid was calculated for hBMSCs in the different scaffolds which enabled hBMSC dimensionality to be classified based on shape. A "Dimensionality Matrix" was developed that showed that hBMSC shape and dimensionality were influenced by scaffold properties, and that scaffolds could drive hBMSCs into 1D, 2D or 3D shapes. In addition, the hBMSC Z-Depth was measured to determine if hBMSCs became less flat during culture in scaffolds. Z-Depth results showed that all 6 scaffolds caused an increase in cell Z-Depth compared to the 2D planar substrate. These results demonstrate that hBMSCs take on morphologies with greater 3D character in scaffolds than on a planar substrate and that scaffold properties can be adjusted to modify cell dimensionality. In addition, biomaterialists can use this measurement approach to assess and compare scaffold design modifications as they strive to create optimal cell niches that provide a 3D microenvironment.

Nanofiber scaffold gradients for interfacial tissue engineering.

We have designed a 2-spinnerette device that can directly electrospin nanofiber scaffolds containing a gradient in composition that can be used to engineer interfacial tissues such as ligament and tendon. Two types of nanofibers are simultaneously electrospun in an overlapping pattern to create a nonwoven mat of nanofibers containing a composition gradient. The approach is an advance over previous methods due to its versatility - gradients can be formed from any materials that can be electrospun. A dye was used to characterize the 2-spinnerette approach and applicability to tissue engineering was demonstrated by fabricating nanofibers with gradients in amorphous calcium phosphate nanoparticles (nACP). Adhesion and proliferation of osteogenic cells (MC3T3-E1 murine pre-osteoblasts) on gradients was enhanced on the regions of the gradients that contained higher nACP content yielding a graded osteoblast response. Since increases in soluble calcium and phosphate ions stimulate osteoblast function, we measured their release and observed significant release from nanofibers containing nACP. The nanofiber-nACP gradients fabricated herein can be applied to generate tissues with osteoblast gradients such as ligaments or tendons. In conclusion, these results introduce a versatile approach for fabricating nanofiber gradients that can have application for engineering graded tissues.

The determination of stem cell fate by 3D scaffold structures through the control of cell shape.

Stem cell response to a library of scaffolds with varied 3D structures was investigated. Microarray screening revealed that each type of scaffold structure induced a unique gene expression signature in primary human bone marrow stromal cells (hBMSCs). Hierarchical cluster analysis showed that treatments sorted by scaffold structure and not by polymer chemistry suggesting that scaffold structure was more influential than scaffold composition. Further, the effects of scaffold structure on hBMSC function were mediated by cell shape. Of all the scaffolds tested, only scaffolds with a nanofibrous morphology were able to drive the hBMSCs down an osteogenic lineage in the absence of osteogenic supplements. Nanofiber scaffolds forced the hBMSCs to assume an elongated, highly branched morphology. This same morphology was seen in osteogenic controls where hBMSCs were cultured on flat polymer films in the presence of osteogenic supplements (OS). In contrast, hBMSCs cultured on flat polymer films in the absence of OS assumed a more rounded and less-branched morphology. These results indicate that cells are more sensitive to scaffold structure than previously appreciated and suggest that scaffold efficacy can be optimized by tailoring the scaffold structure to force cells into morphologies that direct them to differentiate down the desired lineage.

Reversing DNA-mediated adhesion at a fixed temperature.

Recognition-based assembly of micron- to nano-sized colloidal particles functionalized with DNA has generated great interest in the past decade; however, reversing the assembly process is typically achieved by thermal denaturation of the oligonucleotide duplexes. Here, we report an alternative disassembly approach at a fixed temperature using competitive hybridization events between immobilized and soluble oligonucleotide strands. Microspheres are first aggregated via primary hybridization events between immobilized DNA strands with a weak, but sufficient, affinity for partner strands to link complementary surfaces together. To reverse the aggregation process, soluble oligonucleotides are then added to competitively displace the original hybridization partners through secondary hybridization events. Using flow cytometry to quantify hybridization events and microscopy to examine DNA-mediated aggregation and redispersion, we found that the efficiency of competitive displacement is based upon (1) the difference in base pair matches between the primary and secondary target for the same probe sequence and (2) the concentration of hybridizing oligonucleotides participating in microsphere aggregation. To the best of our knowledge, this study is the first to employ DNA hybridization events to mediate reversible adhesion between colloidal particles at a fixed temperature.