Aerosol Delivery of Synthetic mRNA to Vaginal Mucosa Leads to Durable Expression of Broadly Neutralizing Antibodies against HIV.

There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections.

Antibiotic Minocycline Prevents Respiratory Syncytial Virus Infection.

Treatment drugs, besides their specific activity, often have multiple effects on the body. The undesired effect of the drug may be repurposed as therapeutics, saving significant investigative time and effort. Minocycline has anti-cancer, anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Presently, minocycline is also known to show anti-viral activity against Influenza virus, Japanese encephalitis virus, Simian immunodeficiency virus, Human immunodeficiency virus and West Nile virus. Here, we investigate the effect of minocycline on Respiratory syncytial virus (RSV), a common respiratory virus that causes severe mortality and morbidity in infants, children, and older adult populations. Currently, there is no effective vaccine or treatment for RSV infection; hence, there is a critical need for alternative and effective drug choices. Our study shows that minocycline reduces the RSV-mediated cytopathic effect and prevents RSV infection. This is the first study demonstrating the anti-viral activity of minocycline against RSV.

Exploitation of Synthetic mRNA To Drive Immune Effector Cell Recruitment and Functional Reprogramming In Vivo.

Therapeutic strategies based on in vitro-transcribed mRNA (IVT) are attractive because they avoid the permanent signature of genomic integration that is associated with DNA-based therapy and result in the transient production of proteins of interest. To date, IVT has mainly been used in vaccination protocols to generate immune responses to foreign Ags. In this "proof-of-principle" study, we explore a strategy of combinatorial IVT to recruit and reprogram immune effector cells to acquire divergent biological functions in mice in vivo. First, we demonstrate that synthetic mRNA encoding CCL3 is able to recruit murine monocytes in a nonprogrammed state, exhibiting neither bactericidal nor tissue-repairing properties. However, upon addition of either Ifn-γ mRNA or Il-4 mRNA, we successfully polarized these cells to adopt either M1 or M2 macrophage activation phenotypes. This cellular reprogramming was demonstrated through increased expression of known surface markers and through the differential modulation of NADPH oxidase activity, or the superoxide burst. Our study demonstrates how IVT strategies can be combined to recruit and reprogram immune effector cells that have the capacity to fulfill complex biological tasks in vivo.

Immunogenicity of RSV F DNA Vaccine in BALB/c Mice.

Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease leading to numerous hospitalizations and deaths among the infant and elderly populations worldwide. There is no vaccine or a less effective drug available against RSV infections. Natural RSV infection stimulates the Th1 immune response and activates the production of neutralizing antibodies, while earlier vaccine trials that used UV-inactivated RSV exacerbated the disease due to the activation of the allergic Th2 response. With a focus on Th1 immunity, we developed a DNA vaccine containing the native RSV fusion (RSV F) protein and studied its immune response in BALB/c mice. High levels of RSV specific antibodies were induced during subsequent immunizations. The serum antibodies were able to neutralize RSV in vitro. The RSV inhibition by sera was also shown by immunofluorescence analyses. Antibody response of the RSV F DNA vaccine showed a strong Th1 response. Also, sera from RSV F immunized and RSV infected mice reduced the RSV infection by 50% and 80%, respectively. Our data evidently showed that the RSV F DNA vaccine activated the Th1 biased immune response and led to the production of neutralizing antibodies, which is the desired immune response required for protection from RSV infections.

Gold nanorods inhibit respiratory syncytial virus by stimulating the innate immune response.

Respiratory syncytial virus (RSV) causes severe pneumonia and bronchiolitis in infants, children and older adults. The use of metallic nanoparticles as potential therapeutics is being explored against respiratory viruses like Influenza, Parainfluenza and Adenovirus. In this study, we showed that gold nanorods (GNRs) inhibit RSV in HEp-2 cells and BALB/c mice by 82% and 56%, respectively. The RSV inhibition correlated with marked upregulated antiviral genes due to GNR mediated TLR, NOD-like receptor and RIG-I-like receptor signaling pathways. Transmission electron microscopy of lungs showed GNRs in the endocytotic vesicles and histological analyses indicated infiltration by neutrophils, eosinophils and monocytes correlating with clearance of RSV. In addition, production of cytokines and chemokines in the lungs indicates recruitment of immune cells to counter RSV replication. To our knowledge, this is the first in vitro and in vivo report that provides possible antiviral mechanisms of GNRs against RSV.

A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene delivery system.

The transport of DNA into eukaryotic cells is minimal because of the cell membrane barrier, and this limits the application of DNA vaccines, gene silencing, and gene therapy. Several available transfection reagents and techniques have been used to circumvent this problem. Alternatively, nonviral nanoscale vectors have been shown to bypass the eukaryotic cell membrane. In the present work, we developed a unique nanomaterial, pHEMA+chitosan nanospheres (PCNSs), which consisted of poly(2-hydroxyethyl methacrylate) nanospheres surrounded by a chitosan cationic shell, and we used this for encapsulation of a respiratory syncytial virus (RSV)-F gene construct (a model for a DNA vaccine). The new nanomaterial was capable of transfecting various eukaryotic cell lines without the use of a commercial transfection reagent. Using transmission electron microscopy, (TEM), fluorescence activated cell sorting (FACS), and immunofluorescence, we clearly demonstrated that the positively charged PCNSs were able to bind to the negatively charged cell membrane and were taken up by endocytosis, in Cos-7 cells. Using quantitative polymerase chain reaction (qPCR), we also evaluated the efficiency of transfection achieved with PCNSs and without the use of a liposomal-based transfection mediator, in Cos-7, HEp-2, and Vero cells. To assess the transfection efficiency of the PCNSs in vivo, these novel nanomaterials containing RSV-F gene were injected intramuscularly into BALB/c mice, resulting in high copy number of the transgene. In this study, we report, for the first time, the application of the PCNSs as a nanovehicle for gene delivery in vitro and in vivo.

Functionalized carbon nanotubes: biomedical applications.

Carbon nanotubes (CNTs) are emerging as novel nanomaterials for various biomedical applications. CNTs can be used to deliver a variety of therapeutic agents, including biomolecules, to the target disease sites. In addition, their unparalleled optical and electrical properties make them excellent candidates for bioimaging and other biomedical applications. However, the high cytotoxicity of CNTs limits their use in humans and many biological systems. The biocompatibility and low cytotoxicity of CNTs are attributed to size, dose, duration, testing systems, and surface functionalization. The functionalization of CNTs improves their solubility and biocompatibility and alters their cellular interaction pathways, resulting in much-reduced cytotoxic effects. Functionalized CNTs are promising novel materials for a variety of biomedical applications. These potential applications are particularly enhanced by their ability to penetrate biological membranes with relatively low cytotoxicity. This review is directed towards the overview of CNTs and their functionalization for biomedical applications with minimal cytotoxicity.

Functionalized Gold Nanoparticles and Their Biomedical Applications.

Metal nanoparticles are being extensively used in various biomedical applications due to their small size to volume ratio and extensive thermal stability. Gold nanoparticles (GNPs) are an obvious choice due to their amenability of synthesis and functionalization, less toxicity and ease of detection. The present review focuses on various methods of functionalization of GNPs and their applications in biomedical research. Functionalization facilitates targeted delivery of these nanoparticles to various cell types, bioimaging, gene delivery, drug delivery and other therapeutic and diagnostic applications. This review is an amalgamation of recent advances in the field of functionalization of gold nanoparticles and their potential applications in the field of medicine and biology.