Short Carbon Nanotube-Based Delivery of mRNA for HIV-1 Vaccines.

Developing a safe and effective preventive for HIV-1 remains the hope for controlling the global AIDS epidemic. Recently, mRNA vaccines have emerged as a promising alternative to conventional vaccine approaches, primarily due to their rapid development and potential for low-cost manufacture. Despite the advantages of mRNA vaccines, challenges remain, especially due to the adverse effects of the delivery vehicle and low delivery efficiency. As a result, Luna Labs is developing a short carbon nanotube-based delivery platform (NanoVac) that can co-deliver mRNA and HIV-1 glycoproteins to the immune system efficiently with negligible toxicity. Surface chemistries of NanoVac were optimized to guide antigen/mRNA loading density and presentation. Multiple formulations were engineered for compatibility with both intramuscular and intranasal administration. NanoVac candidates demonstrated immunogenicity in rabbits and generated human-derived humoral and cellular responses in humanized mice (HIS). Briefly, 33% of the HIV-1-infected HIS mice vaccinated with NanoVac-mRNA was cleared of virus infection by 8-weeks post-infection. Finally, NanoVac stabilized the loaded mRNA against degradation under refrigeration for at least three months, reducing the cold chain burden for vaccine deployment.

Transposon tagging in diploid strawberry.

Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T1 progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T0 launch pads, putative transposants in the T1 generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T1 plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T1 plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T0 generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3' UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T2 transposon-tagged plants. The mutant collection has been catalogued in an on-line database.

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.