Simona Bot

Plasmid Vaccination with Insulin B Chain Prevents Autoimmune Diabetes in Nonobese Diabetic Mice

The insulin B (InsB) chain bears major type 1 diabetes-associated epitopes of significance for disease in humans and nonobese diabetic (NOD) mice. Somatic expression of InsB chain initiated early in life by plasmid inoculation resulted in substantial protection of female NOD mice against disease. This was associated with a T2 shift in spleen, expansion of IL-4-producing and, to a lesser extent, of IFN-γ-secreting T cells in pancreatic lymph nodes, as well as intermolecular Th2 epitope spreading to glutamic acid decarboxylase determinants. A critical role of IL-4 for the Ag-specific protective effect triggered by plasmid administration was revealed in female IL-4−/− NOD mice that developed diabetes and higher Th1 responses. Coadministration of IL-4-expressing plasmid or extension of the vaccination schedule corrected the unfavorable response of male NOD mice to DNA vaccination with InsB chain. Thus, plasmid-mediated expression of the InsB chain early in diabetes-prone mice has the potential to prevent transition to full-blown disease depending on the presence of IL-4.

Novel lipid-based hollow-porous microparticles as a platform for immunoglobulin delivery to the respiratory tract.

AbstractPurpose. Delivery of specific antibodies or immunoglobulin constructsto the respiratory tract may be useful for prophylaxis or active treatmentof local or systemic disorders. Therefore, we evaluated the utilityof lipid-based hollow-porous microparticles (PulmoSpheres™) as apotential delivery vehicle for immunoglobulins. Methods. Lipid-based microparticles loaded with humanimmunoglobulin (hIgG) or control peptide were synthesized by spray drying and testedfor: i) the kinetics of peptide/protein release, using ELISA and bioassays;ii) bioavailability subsequent to nonaqueous liquid instillation into therespiratory tract of BALB/c mice, using ELISA and Western blotting;iii) bioactivity in terms of murine immune response to xenotypic epitopeson human IgG, using ELISA and T cell assays; and iv) mechanismsresponsible for the observed enhancement of immune responses, usingmeasurement of antibodies as well as tagged probes. Results. Human IgG and the control peptide were both readily releasedfrom the hollow-porous microspheres once added to an aqueousenvironment, although the kinetics depended on the compound. Nonaqueousliquid instillation of hIgG formulated in PulmoSpheres into the upperand lower respiratory tract of BALB/c mice resulted in systemicbiodistribution. The formulated human IgG triggered enhanced local andsystemic immune responses against xenotypic epitopes and wasassociated with receptor-mediated loading of alveolar macrophages. Conclusions. Formulation of immunoglobulins in hollow-porousmicroparticles is compatible with local and systemic delivery via therespiratory mucosa and may be used as means to trigger or modulateimmune responses.

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.

Enhanced protection against influenza virus of mice immunized as newborns with a mixture of plasmids expressing hemagglutinin and nucleoprotein

Effective immunization of neonates is an important goal for vaccinology. We show that inoculation of newborn mice with a mixture of plasmids expressing influenza hemagglutinin (HA) and nucleoprotein (NP) genes leads to an enhanced protection subsequent to the lethal challenge with two distinct strains. In sharp contrast, neonatal injection with UV-inactivated influenza virus strain WSN, failed to induce protection against the homologous challenge. Our results show that while plasmid immunization of neonates elicits a protective immunity, the immunization with inactivated virus does not.

Receptor-mediated targeting of spray-dried lipid particles coformulated with immunoglobulin and loaded with a prototype vaccine.

AbstractPurpose. Spray-dried lipid-based microparticles (SDLM) serve as a platform for delivery of a wide variety of compounds including peptides, proteins, and vaccines to the respiratory mucosa. In the present study, we assessed the impact of IgG-mediated targeting to phagocytic cells of inactivated influenza virus formulated in SDLM, on subsequent immune responses. Methods. SDLM were produced containing inactivated influenza virus strain A/WSN/32/H1N1 (WSN), with or without IgG. Using phagocytic antigen presenting cells (APC) and a T cell hybridoma (TcH) line specific for a dominant influenza virus epitope, we compared the in vitro responses elicited by ligand-formulated (SDLM-IgG-WSN) and non-ligand particles (SDLM-WSN). The effect of including the IgG ligand in the formulation was further characterized by measuring the immune responses of rodents vaccinated with SDLM. Results. SDLM-IgG-WSN were internalized in an Fc receptor (FcR)-dependent manner by phagocytic APC that were then able to effectively present a dominant, class II-restricted epitope to specific T cells. While SDLM-WSN elicited a lower response than administration of plain inactivated virus in saline, the level of the T cell response was restored both in vitro and in vivo by incorporating the APC FcR ligand, IgG, in the SDLM. Conclusions. Incorporation of FcR ligand (IgG) in SDLM restored the limited ability of formulated virus to elicit T-cell immunity, by receptor-mediated targeting to phagocytes.

Protective cellular immunity against influenza virus induced by plasmid inoculation of newborn mice.

Neonate organisms display an intrinsic disability to mount effective immune responses to infectious agents or conventional vaccines. Whereas low. doses of antigens trigger a suboptimal response, higher doses are frequently associated with tolerance induction. We investigated the ability of a plasmid-expressing nucleoprotein of influenza virus to prime a specific cellular immune response when administered to newborn mice. We found that persistent exposure to antigen following plasmid inoculation of neonates leads to a vigorous priming of specific CTLs rather than tolerance induction. The CTLs were cross-reactive against multiple strains of type A influenza viruses and produced IFNγ but no IL-4. The immunity triggered by plasmid inoculation of neonates was protective in terms of pulmonary virus clearance as well as survival rate following lethal challenge with influenza virus. Whereas the persistence of the plasmid at the site of injection was readily demonstrable in adult mice at 3 months after inoculation, mice immunized as newborns displayed no plasmid at 3 months and very little at 1 month after injection. Thus, DNA-based immunization of neonates may prove an effective and safe vaccination strategy for induction of cellular immunity against microbes that cause serious infectious diseases in the early period of life.

Infection-Triggered Regulatory Mechanisms Override the Role of STAT 4 in Control of the Immune Response to Influenza Virus Antigens

ABSTRACT Accurate control of the balance of the T1 and T2 cells during antiviral immunity is essential for optimizing immune effector functions and for avoiding potentially severe immunopathology. We examined the in vivo role of the signal transducer and activator of transcription (STAT) 4 in regulating the T1/T2 balance during the response to live influenza virus and isolated viral proteins. We found that the differentiation of gamma interferon (IFN-γ)-producing Th1 and Tc1 cells after inoculation of live virus occurred independently of STAT 4 expression. Influenza virus-specific T2 and Tc2 responses were well controlled in such STAT 4-deficient mice unless IFN-γ was eliminated as well. In contrast, the STAT 4-dependent signaling pathway played a more essential role in regulating the T1/T2 balance after immunization with viral proteins and, in particular, inactivated nonreplicating virus. Pulmonary infection was cleared even in the absence of both functional STAT 4 genes and functional IFN-γ genes because virus-neutralizing antibodies were still generated, consistent with a substantial redundancy in different antiviral effector pathways. Thus, replicating agents such as live influenza virus can elicit IFN-γ and control T2 immunity independently of STAT 4, whereas the profile of immunity to isolated proteins is more reliant on an intact STAT 4 signaling pathway.

Induction of immunological memory in baboons primed with DNA vaccine as neonates

DNA immunization is a potential vaccination strategy for neonates and infants. We tested the ability of a prototype DNA vaccine against influenza virus to prime lasting immunity when administered to newborn non-human primates. Neonatal DNA vaccination triggered virus-specific and neutralizing antibodies of titers and persistence depending on the vaccine dose. Subsequent exposure to influenza virus, revealed significantly increased recall responses in the baboons vaccinated with DNA during the neonatal stage. The humoral and cellular responses were enhanced in the baboons primed with DNA vaccine as neonates. Thus, neonatal DNA vaccination of non-human primates triggered immune memory that persisted beyond infancy.

Immunologic Control of Tumors by In Vivo Fcγ 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+) FcγR, but kept in check by ITIM motif (ITIM+) FcγRIIB-mediated mechanisms. In addition to interfering with the functionality of ITIM+ FcγR, effective anti-idiotypic and antitumoral immunity can be achieved by FcγR-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 FcγR-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+ FcγR, 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.