Noncoding RNA danger motifs bridge innate and adaptive immunity and are potent adjuvants for vaccination.

The adaptive immune response is triggered by recognition of T and B cell epitopes and is influenced by "danger" motifs that act via innate immune receptors. This study shows that motifs associated with noncoding RNA are essential features in the immune response reminiscent of viral infection, mediating rapid induction of proinflammatory chemokine expression, recruitment and activation of antigen-presenting cells, modulation of regulatory cytokines, subsequent differentiation of Th1 cells, isotype switching, and stimulation of cross-priming. The heterogeneity of RNA-associated motifs results in differential binding to cellular receptors, and specifically impacts the immune profile. Naturally occurring double-stranded RNA (dsRNA) triggered activation of dendritic cells and enhancement of specific immunity, similar to selected synthetic dsRNA motifs. Based on the ability of specific RNA motifs to block tolerance induction and effectively organize the immune defense during viral infection, we conclude that such RNA species are potent danger motifs. We also demonstrate the feasibility of using selected RNA motifs as adjuvants in the context of novel aerosol carriers for optimizing the immune response to subunit vaccines. In conclusion, RNA-associated motifs produced during viral infection bridge the early response with the late adaptive phase, regulating the activation and differentiation of antigen-specific B and T cells, in addition to a short-term impact on innate immunity.

Rational design of solid aerosols for immunoglobulin delivery by modulation of aerodynamic and release characteristics.

Parenteral administration of immunoglobulins (Ig) for prevention or treatment of respiratory diseases achieves only modest concentrations of antibody in the pulmonary interstitial tissue and airways. Aerosols, including spray-dried particles, must overcome two limiting factors in order to be effective vehicles for pulmonary delivery of Ig: (i) Fc receptor (FcR)-mediated scavenging by macrophages and (ii) clearance by the mucociliary system. Ig-incorporated spray-dried lipid microparticles (SDLM), coformulated with or without a biocompatible surfactant (1% w:w) to modulate protein release, were designed and tested for their capability to deliver Ig to the respiratory tract. To determine efficacy, rodents were immunized with SDLM containing antiinfluenza antibody followed by virus challenge and clinical parameters measured. Control of the release kinetics resulted in enhanced delivery of immunoglobulins to the respiratory tract and interstitial tissue with slow translocation into the systemic circulation. As much as 60% of the IgG delivered from nonretentive SDLM could be recovered from the lung interstitial tissue within 1 h after aerosol administration at a dose of 1 mg of Ig/kg of body weight. In addition, nonretentive rather than slow-release particles loaded with antiinfluenza antibody were effective in curbing virus replication with a resulting positive clinical outcome. Thus, controlled release of Ig by manipulating aerosol characteristics and composition allows for a significant increase in the efficiency of pulmonary delivery of antibodies.

Immunologic control of tumors by in vivo Fc gamma receptor-targeted antigen loading in conjunction with double-stranded RNA-mediated immune modulation.

Despite the expression of non-self or neo-epitopes, many tumors such as lymphoid malignancies or cancers induced by oncogenic viruses are able to gradually overcome the immune defense mechanisms and spread. Using a preclinical model of hematological malignancy, we show that Ig-associated idiotypic determinants are recognized by the immune system in a fashion that results in immune deviation, allowing tumor progression and establishment of metastases. Using gene-targeted mice, we show that anti-idiotypic MHC class I-restricted immunity is promoted by ITAM motif (ITAM+) FcgammaR, but kept in check by ITIM motif (ITIM+) FcgammaRIIB-mediated mechanisms. In addition to interfering with the functionality of ITIM+ FcgammaR, effective anti-idiotypic and antitumoral immunity can be achieved by FcgammaR-targeted delivery of epitope in conjunction with administration of stimulatory motifs such as dsRNA, correcting the ineffective response to idiotypic epitopes. The immune process initiated by FcgammaR-mediated targeting of epitope together with dsRNA, resulted in control of tumor growth, establishment of immune memory and protection against tumors bearing antigenic variants. In summary, targeted delivery of MHC class I-restricted epitopes via ITAM+ FcgammaR, in conjunction with use of TLR-binding immune stimulatory motifs such as dsRNA, overcomes suboptimal responses to idiotypic determinants and may constitute a novel approach for the treatment of a broad range of malignancies. Finally, the results shed light on the mechanisms regulating the idiotypic network and managing the diversity associated with immune receptors.

Evaluation of novel aerosol formulations designed for mucosal vaccination against influenza virus.

Influenza viruses are among the most significant human pathogens, responsible for increased seasonal morbidity and mortality particularly in immunodepressed and chronically ill. Conventional vaccination with non-replicative vaccine is currently performed by injection. In the present study, we explore simple spray-dried lipid formulations containing whole inactivated virus or split-subunit vaccine that allow aerosolization and thus, mucosal vaccination of the pulmonary tract. We show that by using biocompatible excipients already approved for human use, one could engineer microparticles that induce substantial local and systemic immunity subsequent to pulmonary administration. Exposure of the bronchial-associated lymphoid tissue (BALT) to vaccine was more effective than parenteral or nasal administration in triggering specific immunity. Co-formulation of a biocompatible surfactant detergent greatly ameliorated the immune profile of microparticles containing a whole inactivated virus vaccine. In addition, mere formulation of a licensed split-subunit vaccine significantly enhanced its immunogenicity. Together, our data underline a simple strategy to convert conventional parenteral vaccination of currently available non-replicative vaccines against influenza virus, into one that is more effective and practical upon respiratory administration.