Mucosal Application of gp140 Encoding DNA Polyplexes to Different Tissues Results in Altered Immunological Outcomes in Mice

Increasing evidence suggests that mucosally targeted vaccines will enhance local humoral and cellular responses whilst still eliciting systemic immunity. We therefore investigated the capacity of nasal, sublingual or vaginal delivery of DNA-PEI polyplexes to prime immune responses prior to mucosal protein boost vaccination. Using a plasmid expressing the model antigen HIV CN54gp140 we show that each of these mucosal surfaces were permissive for DNA priming and production of antigen-specific antibody responses. The elicitation of systemic immune responses using nasally delivered polyplexed DNA followed by recombinant protein boost vaccination was equivalent to a systemic prime-boost regimen, but the mucosally applied modality had the advantage in that significant levels of antigen-specific IgA were detected in vaginal mucosal secretions. Moreover, mucosal vaccination elicited both local and systemic antigen-specific IgG(+) and IgA(+) antibody secreting cells. Finally, using an Influenza challenge model we found that a nasal or sublingual, but not vaginal, DNA prime/protein boost regimen protected against infectious challenge. These data demonstrate that mucosally applied plasmid DNA complexed to PEI followed by a mucosal protein boost generates sufficient antigen-specific humoral antibody production to protect from mucosal viral challenge

Evaluation of Mucociliary Clearance by Three Dimension Micro-CT-SPECT in Guinea Pig: Role of Bitter Taste Agonists

Different image techniques have been used to analyze mucociliary clearance (MCC) in humans, but current small animal MCC analysis using in vivo imaging has not been well defined. Bitter taste receptor (T2R) agonists increase ciliary beat frequency (CBF) and cause bronchodilation but their effects in vivo are not well understood. This work analyzes in vivo nasal and bronchial MCC in guinea pig animals using three dimension (3D) micro-CT-SPECT images and evaluates the effect of T2R agonists. Intranasal macroaggreggates of albumin-Technetium 99 metastable (MAA-Tc99m) and lung nebulized Tc99m albumin nanocolloids were used to analyze the effect of T2R agonists on nasal and bronchial MCC respectively, using 3D micro-CT-SPECT in guinea pig. MAA-Tc99m showed a nasal mucociliary transport rate of 0.36 mm/min that was increased in presence of T2R agonist to 0.66 mm/min. Tc99m albumin nanocolloids were homogeneously distributed in the lung of guinea pig and cleared with time-dependence through the bronchi and trachea of guinea pig. T2R agonist increased bronchial MCC of Tc99m albumin nanocolloids. T2R agonists increased CBF in human nasal ciliated cells in vitro and induced bronchodilation in human bronchi ex vivo. In summary, T2R agonists increase MCC in vivo as assessed by 3D micro-CT-SPECT analysis

Thy1+ Nk Cells from Vaccinia Virus-Primed Mice Confer Protection against Vaccinia Virus Challenge in the Absence of Adaptive Lymphocytes

While immunological memory has long been considered the province of T- and B- lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1+ subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1+ NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance

Microneedle arrays for the transcutaneous immunization of diphtheria and influenza in BALB/c mice

Transcutaneous immunization (TCI) is limited by poor permeation of macromolecules across the skin. Microneedle arrays form transient conduits and enhance the transport of vaccine molecules across the skin barrier without pain sensation. Here we investigated in mouse the immune responses after TO using two model antigens, diphtheria toxoid (DT) and influenza subunit vaccine. The electric applicator enabled shorter microneedle (300 mu m) to pierce mouse skin effectively, as shown by Trypan blue staining and trans-epidermal water loss measurement. The vaccines were topically applied with and without cholera toxin (CT) on microneedle-treated skin. In DT TCI, microneedle array pretreatment of the skin was essential to achieve substantial IgG and toxin-neutralizing antibody titers. Addition of CT further boosted the immune response to similar levels as observed after subcutaneous injection of AlPO(4)-adsorbed DT (DT-alum). In contrast, microneedle array pretreatment showed no effect on the immune response to plain influenza vaccine. This response was strongly improved by inclusion of CT, independent of microneedle treatment. These results indicate that TO of DT and CT with microneedle treatment results in comparable protection as injection of DT-alum, and TO of influenza vaccine adjuvanted with CT is superior to the injection of plain vaccine. (C) 2009 Elsevier B.V. All rights reserved

Pulmonary delivery of DNA encoding Mycobacterium tuberculosis latency antigen Rv1733c associated to PLGA–PEI nanoparticles enhances T cell responses in a DNA prime/protein boost vaccination regimen in mice

During persistent infection and hypoxic-stress, Mycobacterium tuberculosis (Mtb) expresses a series of Mtb latency antigens. The aim of this study was to evaluate the immunogenicity of a DNA vaccine encoding the Mtb latency antigen Rv1733c and to explore the effect of pulmonary delivery and co-formulation with poly (d,l-lactide-co-glycolide) (PLGA)-polyethyleneimine (PEI) nanoparticles (np) on host immunity. Characterization studies indicated that PLGA-PEI np kept their nanometer size after concentration and were positively charged. The np were able to mature human dendritic cells and stimulated them to secrete IL-12 and TNF-alpha comparable to levels observed after lipopolysaccharide (LPS) stimulation. Mtb latency antigen Rv1733c DNA prime combined with Rv1733c protein boost enhanced T cell proliferation and IFN-gamma secretion in mice in response to Rv1733c and Mtb hypoxic lysate. Rv1733c DNA adsorbed to PLGA-PEI np and applied to the lungs increased T cell proliferation and IFN-gamma production more potently compared to the same vaccinations given intramuscularly. The strongest immunogenicity was obtained by pulmonary priming with np-adsorbed Rv1733c DNA followed by boosting with Rv1733c protein. These results confirm that PLGA-PEI np are an efficient DNA vaccine delivery system to enhance T cell responses through pulmonary delivery in a DNA prime/protein boost vaccine regimen