Adrian 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.

Genetic immunization of neonates.

The vaccination of neonates is generally difficult due to immaturity of the immune system, higher susceptibility to tolerance and potential negative interference of maternal antibodies. Studies carried out in rodents and non-human primates showed that plasmid vaccines expressing microbial antigens, rather than inducing tolerance, triggered significant humoral and cellular immunity with a Th1 component. The ability of bacterial CpG motifs to activate immature antigen-presenting cells is critical for the neonatal immunogenicity of DNA vaccines. In addition, the endogenous production of antigen subsequent to transfection of antigen-presenting cells may explain the lack of inhibition by maternal antibodies of cellular responses. Together, these features make the plasmid vaccines an appealing strategy to prime immune responses against foreign pathogens, during early life. In combination with subsequent boosting using conventional vaccines, DNA vaccine-based regimens may provide a qualitatively superior immunity against microbes. Thorough understanding of immunomodulatory properties of plasmid-vectors may extend their use for early prophylaxis of inflammatory disorders.

Engineering of doubly antigenized immunoglobulins expressing T and B viral epitopes

BACKGROUND Concomitant with the advent of molecular biology techniques and the ability of immunoglobulins (Ig) to recognize proteins, carbohydrates, lipopeptides and nucleic acids, vaccinologists have taken advantage to develop a variety of prophylactic and therapeutic vaccine prototypes. Presentation of epitopes to the immune system by Ig molecules as a carrier platform offers several advantages: (i) long exposure of the antigen to antigen processing cells (APCs) by virtue of their long half life, (ii) lack of the immune response to self Ig, focusing the immune response to protective epitopes rather than irrelevant epitopes, (iii) it takes advantage of the properties of Fc fragment of various isotypes like crossing the placenta (IgG) or homing in epithelia (IgA), and (iv) targeting various antigens by virtue of their binding specificity. OBJECTIVES This study was aimed to genetically and enzymatically engineer immunoglobulins (Igs) able to express and to deliver concomitantly immunodominant T and B viral epitopes. STUDY DESIGN Using a genetic engineering approach we replaced the complementary determining region 3 (CDR3) and complementary determining region 2 (CDR2) of an anti-arsonate 91A3 mAb with the immunodominant HA110-120 T cell epitope and HA150-159 B cell epitope of hemagglutinin (HA) of influenza A/PR8 virus, respectively. The second doubly antigenized Ig (Ig-HA-Gal-B) was constructed on an Ig in which CDR3 was replaced with HA110-120 T cell epitope while the HA150-159 B cell epitope was enzymatically assembled through an imidic bond on the galactose (Gal) residues of the carbohydrate moiety. RESULTS AND CONCLUSIONS Both genetically and genetically/enzymatically doubly antigenized Ig constructs (dAIg) were properly folded and they were able to activate peptide-specific T cells and to elicit anti-viral antibody response in mice. This demonstrates that the CDR loops as well as carbohydrate moieties of immunoglobulins represent permissive sites for grafting foreign epitopes without altering the structural integrity of immunoglobulins and the immunogenicity of the viral peptides.

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.

Immune response of neonates elicited by somatic transgene vaccination with naked DNA.

Neonates display lower immune responsiveness and higher susceptibility for high-dose tolerance. Quantitative as well as functional differences between the neonatal and adult lymphocytes or antigen presenting cells (APC) respectively, explain the particular immune responsiveness during the early stages of the postnatal development. Reduced numbers of lymphocytes and APCs as well as a modified responsiveness of T cells in neonates, are the main factors that account for the low threshold of tolerance in newborns. Taking into account these particularities, the design of effective vaccines for neonates poses significant difficulties. We hypothesized that a continuous exposure to low doses of antigens may avoid high-zone tolerance and may lead instead, to effective expansion of effector and memory cells. Indeed, inoculation of newborn mice with plasmids encoding nucleoprotein (NP) or hemagglutinin (HA) of influenza virus, led to the priming of specific cytotoxic (CTL), helper (Th) and B cells, rather than induction of unresponsiveness. Mice immunized as neonates with naked DNA and challenged later with lethal doses of influenza virus, displayed significant protection. Thus, DNA immunization may be a promising strategy for vaccination against serious infectious diseases of infants and children.

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.

DNA Vaccination and the Immune Responsiveness of Neonates

Neonates often respond poorly to conventional vaccines or microbial infections. Immaturity of the immune system has been considered to play a role in this regard. However, accumulating evidence shows that in certain conditions, neonatal inoculation of antigens leads to protective immunity. In the particular case of DNA vaccines administered to neonates, the rule is immunity rather than tolerance. Exceptions to the rule give opportunities to further understand the neonatal responsiveness and the mechanism of DNA vaccination. Due to the very nature of the vaccine vector, inhibition of neonatal DNA vaccination by maternal antibodies may be limited to the humoral immunity.