MANα1-2MAN decorated liposomes enhance the immunogenicity induced by a DNA vaccine against BoHV-1.

New technologies in the field of vaccinology arise as a necessity for the treatment and control of many diseases. Whole virus inactivated vaccines and modified live virus ones used against Bovine Herpesvirus-1 (BoHV-1) infection have several disadvantages. Previous works on DNA vaccines against BoHV-1 have demonstrated the capability to induce humoral and cellular immune responses. Nevertheless, 'naked' DNA induces low immunogenic response. Thus, loading of antigen encoding DNA sequences in liposomal formulations targeting dendritic cell receptors could be a promising strategy to better activate these antigen-presenting cells (APC). In this work, a DNA-based vaccine encoding the truncated version of BoHV-1 glycoprotein D (pCIgD) was evaluated alone and encapsulated in a liposomal formulation containing LPS and decorated with MANα1-2MAN-PEG-DOPE (pCIgD-Man-L). The vaccinations were performed in mice and bovines. The results showed that the use of pCIgD-Man-L enhanced the immune response in both animal models. For humoral immunity, significant differences were achieved when total antibody titres and isotypes were assayed in sera. Regarding cellular immunity, a significant increase in the proliferative response against BoHV-1 was detected in animals vaccinated with pCIgD-Man-L when compared to the response induced in animals vaccinated with pCIgD. In addition, upregulation of CD40 molecules on the surface of bovine dendritic cells (DCs) was observed when cells were stimulated and activated with the vaccine formulations. When viral challenge was performed, bovines vaccinated with MANα1-2MAN-PEG-DOPE elicited better protection which was evidenced by a lower viral excretion. These results demonstrate that the dendritic cell targeting using MANα1-2MAN decorated liposomes can boost the immunogenicity resulting in a long-lasting immunity. Liposomes decorated with MANα1-2MAN-PEG-DOPE were tested for the first time as a DNA vaccine nanovehicle in cattle as a preventive treatment against BoHV-1. These results open new perspectives for the design of vaccines for the control of bovine rhinotracheitis.

Targeting glioma cells in vitro with ascorbate-conjugated pharmaceutical nanocarriers.

6-Ascorbate-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (6-ascorbate-PEG-PE) was synthesized according to a two-step procedure: (1) activation of ascorbic acid with bromine, and (2) synthesis of 6-ascorbate-PEG-PE by reacting 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (poly(ethylene glycol))-2000] with an excess of 6-Br-ascorbic acid. The 6-ascorbate-PEG-PE was recovered by precipitation in diethyl ether and purified by gel permeation chromatography. The analysis of the product by 1H NMR and UV-vis spectroscopy confirmed the identity of the conjugate. Liposomes and PEG-PE-based lipid-core micelles were prepared by thin film hydration technique incorporating 6-ascorbate-PEG-PE as targeting moiety. The targeting properties of the ascorbate-decorated nanosystems were tested by fluorescence-activated cell sorting (FACS) analysis and fluorescent microscopy on a panel of tumor cell lines preliminary selected for their ability to express the SVCT2 ascorbate transporter. Cell lines had been selected on the basis of the immunological properties assessed by FACS, which showed that two glioma cell lines, C6 and F98, and fibroblasts NIH/3T3 express plasma membrane-associated SVCT2 transporter for reduced ascorbic acid. Ascorbate-decorated pharmaceutical nanocarriers were endowed with selective targeting properties toward the SVCT2 transporter expressed in glioma cell models. This study shows that SVCT2 transporter for ascorbic acid expressed both in peculiar epithelial cells of the choroid plexus responsible for the filtering of vitamin C into the central nervous system (CNS) and, in some brain tumor cell lines, can be conceivably exploited as a potential target for delivery of drug-loaded pharmaceutical nanocarriers to the brain.