Laura Bungener

Virosome-mediated delivery of protein antigens to dendritic cells.

Virosomes are reconstituted viral membranes in which protein can be encapsulated. Fusion-active virosomes, fusion-inactive virosomes and liposomes were used to study the conditions needed for delivery of encapsulated protein antigen ovalbumin (OVA) to dendritic cells (DCs) for MHC class I and II presentation. Fusion-active virosomes, but not fusion-inactive virosomes, were able to deliver OVA to DCs for MHC class I presentation at picomolar OVA concentrations. Fusion activity of virosomes was not required for MHC class II presentation of antigen. Therefore, virosomes are an efficient system for delivery of protein antigens for stimulation of both helper and CTL responses.

Alum boosts TH2-type antibody responses to whole-inactivated virus influenza vaccine in mice but does not confer superior protection

Clinical trials with pandemic influenza vaccine candidates have focused on aluminium hydroxide as an adjuvant to boost humoral immune responses. In this study we investigated the effect of aluminium hydroxide on the magnitude and type of immune response induced by whole-inactivated virus (WIV) vaccine. Balb/c mice were immunized once with a range of antigen doses (0.04-5 microg) of WIV produced from A/PR/8 virus, either alone or in combination with aluminium hydroxide. The hemagglutination inhibition (HI) titers of mice receiving WIV+aluminium hydroxide were 4-16-fold higher than HI titers in mice receiving the same dose of WIV alone, indicating the boosting effect of aluminium hydroxide. WIV induced a TH1 skewed humoral and cellular immune response, characterized by strong influenza-specific IgG2a responses and a high number of IFNgamma-secreting T cells. In contrast, immunization with WIV adsorbed to aluminium hydroxide resulted in skewing of this response to a TH2 phenotype (high IgG1 levels and a low number of IFNgamma-producing T cells). To assess the effect of the observed immune response skewing on viral clearance from the lungs mice immunized once with 1 microg WIV without or with aluminium hydroxide were challenged with A/PR/8 virus 4 weeks later. The immunized mice showed a significant decrease in viral lung titers compared to control mice receiving buffer. However, despite higher antibody titers, mice immunized with WIV adsorbed to aluminium hydroxide suffered from more severe weight loss and had significantly higher virus loads in their lung tissue than mice receiving WIV alone. Major difference between these groups of mice was the type of immune response induced, TH2 instead of TH1, indicating that a TH1 response plays a major role in viral clearance.

Delivery of protein antigens to the immune system by fusion-active virosomes : A comparison with liposomes and ISCOMs

The induction of effective cellular and humoral immune responses against protein antigens is of major importance in vaccination strategies against infectious diseases and cancer. Immunization with protein alone in general does not result in efficient induction of cytotoxic T lymphocyte (CTL) and antibody responses. Numerous other immunization strategies have been explored. In this review we will discuss a number of lipid-based antigen delivery systems suitable for the induction of CTL responses. These systems comprise reconstituted virus envelopes (virosomes), liposomes, and immune-stimulating complexes (ISCOMs). We will concentrate on delivery of the protein antigen ovalbumin (OVA) since extensive studies with this antigen have been performed for all of the systems discussed, allowing direct comparison of antigen delivery efficiency. Stimulation of CTL activity requires processing of the antigen in the cytosol of antigen-presenting cells (APCs) and presentation of antigenic peptides on surface major histocompatibility class I complexes (MHC class I). In vitro, the ability of antigen delivery systems to induce MHC class I presentation indeed correlates with their capacity to deliver antigen to the cytosol of cells. This capacity appears to be less important for the induction of cytotoxic T lymphocytes in vivo. Instead, other properties of the antigen delivery system like activation of APCs and induction of T helper cells play a more prominent role. Fusion-active virosomes appear to be a very potent system for induction of CTL activity, most likely since virosomes combine efficient delivery of antigen with general stimulation of the immune system.

Eradication of established HPV16-transformed tumours after immunisation with recombinant Semliki Forest virus expressing a fusion protein of E6 and E7

Previously, we described the efficacy of immunisation with recombinant Semliki Forest virus (SFV), expressing the human papillomavirus 16 (HPV) oncoproteins E6 and E7, in inducing HPV-specific CTLs and anti-tumour responses. Recently, we developed a novel recombinant SFV construct encoding a relatively stable fusion protein of HPV16 E6 and E7 under control of a translational enhancer derived from the SFV capsid protein. In the present study we demonstrate that immunisation of tumour-bearing mice with this improved vector results in the regression and complete elimination of established tumours. We furthermore demonstrate that a long-term high level of CTL activity, up to 340 days, accompanies the anti-tumour response. Thus, immunisation with recombinant SFV particles encoding increased levels of a fusion protein of HPV16 E6 and E7 efficiently induces CTL activity and CTL memory resulting in a potent therapeutic anti-tumour effect.

VIROSOMES IN VACCINE DEVELOPMENT: INDUCTION OF CYTOTOXIC T LYMPHOCYTE ACTIVITY WITH VIROSOME-ENCAPSULATED PROTEIN ANTIGENS

ABSTRACT Virosomes are reconstituted viral envelopes which lack the genetic material but retain the cell entry and membrane fusion characteristics of the virus they are derived from. Thus, influenza virosomes are taken up by cells via receptor-mediated endocytosis, which directs the particles to the endosomal cell compartment. Subsequently, the virosomal membrane fuses with the endosomal membrane induced by the mildly acidic pH within the endosomes. This fusion process establishes continuity between the lumen of the virosome and the cell cytosol. Upon interaction of virosomes with antigen-presenting cells (APCs), protein antigens encapsulated within virosomes will be delivered to the cell cytosol, and thus, into the MHC class I presentation pathway. Indeed, virosome-mediated delivery of antigens in vivo results in efficient priming of a class I MHC-restricted cytotoxic T lymphocyte (CTL) response.

Incorporation of LpxL1, a detoxified lipopolysaccharide adjuvant, in influenza H5N1 virosomes increases vaccine immunogenicity

The increasing number of human influenza H5N1 infections accentuates the need for the development of H5N1 vaccine candidates to prevent a potential influenza pandemic. The use of adjuvants in such vaccines can contribute significantly to antigen dose-sparing. In this study, we evaluated the capacity of the non-toxic Neisseria meningitidis lipopolysaccharide analog LpxL1 to function as an adjuvant for an influenza H5N1 virosomal vaccine. Inactivated influenza H5N1 virus (NIBRG-14) was used to construct virosomes (reconstituted virus envelopes) with LpxL1 incorporated in the virosomal membrane thus combining the influenza hemagglutinin (HA) antigen and the adjuvant in the same particle. Mice were immunized in a one- or two-dose immunization regimen with H5N1 virosomes with or without incorporated LpxL1. After a single immunization, H5N1 virosomes with incorporated LpxL1 induced significantly enhanced H5N1-specific total IgG titers as compared to non-adjuvanted virosomes but hemagglutination inhibition (HI) titers remained low. In the two-dose immunization regimen, LpxL1-modified H5N1 virosomes induced HI titers above 40 which were significantly higher than those obtained with non-adjuvanted virosomes. Incorporation of LpxL1 had little effect on virosome-induced IgG1 levels, but significantly increased IgG2a levels in both the one- and two-dose immunization regimen. Compared to non-adjuvanted virosomes, LpxL1-modified virosomes induced similar numbers of IFNgamma-producing T cells but decreased numbers of IL-4-producing T cells irrespective of the number of immunizations. We conclude that LpxL1 incorporated in H5N1 influenza virosomes has the capacity to function as a potent adjuvant particularly stimulating Th1-type immune reactions.

Preclinical evaluation of the saponin derivative GPI-0100 as an immunostimulating and dose-sparing adjuvant for pandemic influenza vaccines

With the current global influenza vaccine production capacity the large demand for vaccines in case of a pandemic can only be fulfilled when antigen dose sparing strategies are employed. Here we used a murine challenge model to evaluate the potential of GPI-0100, a semi-synthetic saponin derivative, to serve as a dose-sparing adjuvant for influenza subunit vaccine. Balb/c mice were immunized with different doses of A/PR8 (H1N1) subunit antigen alone or in combination with varying doses of GPI-0100. The addition of GPI-0100 significantly stimulated antibody and cellular immune responses, especially of the Th1 phenotype. Furthermore, virus titers detected in the lungs of mice challenged one week after the second immunization were significantly reduced among the animals that received GPI-0100-adjuvanted vaccines. Remarkably, adjuvantation of subunit vaccine with GPI-0100 allowed a 25-fold reduction in hemagglutinin dose without compromising the protective potential of the vaccine.

VIROSOMES AS AN ANTIGEN DELIVERY SYSTEM

Abstract Live, replicating, vaccines have the advantage that they closely mimick the actual infection and therefore induce a broad and physiologically relevant immune response, involving both a humoral immune response (antibody production) and cell-mediated immunity (cytotoxic T lymphocytes). However, there is an increasing concern about the adverse side effects that may occur as a result of vaccination with replicating pathogen preparations. Therefore, in general killed whole pathogens or (recombinant) subunit vaccines are used for vaccination. These preparations induce satisfying antibody responses although less efficient than live, replicating, vaccines. This is due to the way in which the antigens are processed and presented to the immune system. The development of antigen delivery systems to introduce nonreplicating antigens into presentation pathways that result in activation of the humoral arm of the immune response, but also the cytotoxic T-cell arm is therefore of major interest. Virosomes represent such a unique system for presentation of antigens to the immune system. First, virosomes closely resemble the envelope of the virus they are derived from and therefore constitute an antigen-presentation form superior to isolated surface antigens. In addition, properly assembled virosomes retain the membrane fusion activity of the native virus and, therefore, virosomes may be used to deliver encapsulated, unrelated, antigens to the cytosol of antigen-presenting cells. In this respect, virosomes differ from conventional liposomes which will target enclosed antigens primarily to the phagolysosomal system of macrophages. We have recently exploited both aspects of virosomes, derived from influenza virus, to induce CTL activity against a virosome-encapsulated antigenic peptide and whole protein. Here we will present a short overview of our own investigations on virosomes followed by a number of conclusions and perspectives on the poten tial application of virosomes in new-generation vaccines.