Binding to cells of virosomes containing herpes simplex virus type 1 glycoproteins and evidence for fusion

Envelope proteins and lipids were extracted from purified herpes simplex virus type 1 virions with octyl glucoside and mixed with phosphatidylcholine for preparation of virosomes by removal of the detergent. Greater than 85% of the extracted envelope proteins, including all the glycoproteins and the nonglycosylated protein designated VP16, were associated with virosomes, which ranged in density from ca. 1.07 to 1.13 g/cm3. All the glycoproteins except gC were as susceptible to degradation by added protease in virosomes as in virions, indicating similar orientations in both. Approximately 30 to 40% of radiolabel incorporated into virosomes bound to HEp-2 cells within 1.5 h at either 4 or 37 degrees C. The cell-bound virosomes were enriched for gB and deficient in other glycoproteins, in comparison with unbound or total virosomes. Binding of virosomes to HEp-2 cells could be inhibited by purified virus, heparin, and monospecific antiviral antibodies. Polyclonal and monoclonal anti-gB antibodies were more effective at inhibiting virosome binding than were anti-gD or anti-gC antibodies. Virosomes depleted of gB or gD did not bind to cells as efficiently as did virosomes containing all the extracted enveloped components; this loss of binding activity was especially pronounced on depletion of gB. The binding of herpes simplex virus type 1 virosomes to cells is discussed in relation to possible heterogeneity of the virosomes and comparisons with binding of virions to cells. We also present electron microscopic evidence that bound virosomes can fuse with the cell surface.     

Monophosphoryl Lipid A‐Adjuvanted Virosomes with Ni‐Chelating Lipids for Attachment of Conserved Viral Proteins as Cross‐Protective Influenza Vaccine

Induction of CD8⁺ cytotoxic T cells (CTLs) to conserved internal influenza antigens, such as nucleoprotein (NP), is a promising strategy for the development of cross‐protective influenza vaccines. However, influenza NP protein alone cannot induce CTL immunity due to its low capacity to activate antigen‐presenting cells (APCs) and get access to the MHC class I antigen processing pathway. To facilitate the generation of NP‐specific CTL immunity the authors develop a novel influenza vaccine consisting of virosomes with the Toll‐like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) and the metal‐ion‐chelating lipid DOGS‐NTA‐Ni incorporated in the membrane. In vitro, virosomes with incorporated MPLA induce stronger activation of APCs than unadjuvanted virosomes. Virosomes modified with DOGS‐NTA‐Ni show high conjugation efficacy for his‐tagged proteins and facilitate efficient uptake of conjugated proteins by APCs. Immunization of mice with MPLA‐adjuvanted virosomes with attached NP results in priming of NP‐specific CTLs while MPLA‐adjuvanted virosomes with admixed NP are inefficient in priming CTLs. Both vaccines induce equally high titers of NP‐specific antibodies. When challenged with heterosubtypic influenza virus, mice immunized with virosomes with attached or admixed NP are protected from severe weight loss. Yet, unexpectedly, they show more weight loss and more severe disease symptoms than mice immunized with MPLA‐virosomes without NP. Taken together, these results indicate that virosomes with conjugated antigen and adjuvant incorporated in the membrane are effective in priming of CTLs and eliciting antigen‐specific antibody responses in vivo. However, for protection from influenza infection NP‐specific immunity appears not to be advantageous.     

Preservation of influenza virosome structure and function during freeze-drying and storage

Virosomes derived from influenza virus are reconstituted viral envelopes, which retain the receptor-binding and cell entry properties of the native virus, but lack the viral genetic material. These virosomes are of interest because of their potential use as vaccines or cellular delivery systems. However, in aqueous dispersion influenza virosomes have a relatively poor stability. Although freeze-drying of the virosomes could improve their stability, a lyoprotectant is required to preserve the structure and function of the virosomes during the lyophilization process as well as during subsequent storage of the dry powder formulation. In this study, inulin, a medium-chain oligosaccharide, was identified as an effective stabilizer of influenza virosomes. When inulin was added to an aqueous virosomal dispersion, the vesicular structure of the virosomes, with spike proteins protruding from the virosomal surface, as well as their membrane fusion activity were completely preserved during freeze-drying. When the freeze-drying process was performed from dispersions lacking a lyoprotectant, both structure and fusogenic properties of the virosomes were lost. Moreover, it was shown that the immunogenicity of inulin-stabilized virosomes was preserved. For example, dry powder formulations of virosomes retained HA potency for at least 12 weeks at 20 degrees C. Virosomes with encapsulated pDNA encoding for the eGFP reporter gene were also found to be stabilized by inulin. The fusion capacity and the transfection efficacy (determined in BHK-21 cells) could be preserved for 12 weeks during storage at 4 degrees C. It is concluded that freeze-drying in the presence of inulin as a lyoprotectant completely preserves the structure and function of influenza virosomes.     

Preservation of Influenza Virosome Structure and Function During Freeze-Drying and Storage

Virosomes derived from influenza virus are reconstituted viral envelopes, which retain the receptor-binding and cell entry properties of the native virus, but lack the viral genetic material. These virosomes are of interest because of their potential use as vaccines or cellular delivery systems. However, in aqueous dispersion influenza virosomes have a relatively poor stability. Although freeze-drying of the virosomes could improve their stability, a lyoprotectant is required to preserve the structure and function of the virosomes during the lyophilization process as well as during subsequent storage of the dry powder formulation. In this study, inulin, a medium-chain oligosaccharide, was identified as an effective stabilizer of influenza virosomes. When inulin was added to an aqueous virosomal dispersion, the vesicular structure of the virosomes, with spike proteins protruding from the virosomal surface, as well as their membrane fusion activity were completely preserved during freeze-drying. When the freeze-drying process was performed from dispersions lacking a lyoprotectant, both structure and fusogenic properties of the virosomes were lost. Moreover, it was shown that the immunogenicity of inulin-stabilized virosomes was preserved. For example, dry powder formulations of virosomes retained HA potency for at least 12 weeks at 20°C. Virosomes with encapsulated pDNA encoding for the eGFP reporter gene were also found to be stabilized by inulin. The fusion capacity and the transfection efficacy (determined in BHK-21 cells) could be preserved for 12 weeks during storage at 4°C. It is concluded that freeze-drying in the presence of inulin as a lyoprotectant completely preserves the structure and function of influenza virosomes.     

Virosomes in vaccine development: induction of cytotoxic T lymphocyte activity with virosome-encapsulated protein antigens

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.     

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.     

Role of hemagglutinin surface density in the initial stages of influenza virus fusion: lack of evidence for cooperativity

Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pH 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Shangdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost-binding activities, and the low-pH-induced conformational change of A/Shangdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per virosome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.     

Interactions of Semliki Forest virus spike glycoprotein rosettes and vesicles with cultured cells

Semliki Forest virus (SFV)-derived spike glycoprotein rosettes (soluble octameric complexes), virosomes (lipid vesicles with viral spike glycoproteins), and liposomes (protein-free lipid vesicles) have been used to investigate the interaction of subviral particles with BHK-21 cells. Cell surface binding, internalization, degradation, and low pH- dependent membrane fusion were quantitatively determined. Electron microscopy was used to visualize the interactions. Virosomes and rosettes, but not liposomes, bound to cells. Binding occurred preferentially to microvilli and was inhibited by added SFV; it increased with decreasing pH but was, in all cases, less efficient than intact virus. At 37 degrees C the cell surface-bound rosettes and virosomes were internalized via coated pits and coated vesicles. After a lag period of 45 min the protein components of the internalized ligands were degraded and appeared, as acid-soluble activity, in the medium. The uptake of rosettes and virosomes was found to be similar to the adsorptive endocytosis of SFV except that their average residence times on the cell surface were longer. The rosettes and the liposomes did not show low pH-induced membrane fusion activity. The virosomes, however, irrespective of the lipid compositions used, displayed hemolytic activity at mildly acidic pH and were able to fuse with the plasma membrane of cells with an efficiency of 0.25 that observed with intact viruses. Cell-cell fusion activity was not observed with any of the subviral components. The results indicated that subviral components possess some of the entry properties of the intact virus.     

Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of various macromolecules. To further advance the use of virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of virosomes. Analysis of the virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for virosomes produced by either method. Moreover, both virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of virosomes is straightforward and robust and allows further exploitation of virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings.     

Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of various macromolecules. To further advance the use of virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of virosomes. Analysis of the virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for virosomes produced by either method. Moreover, both virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of virosomes is straightforward and robust and allows further exploitation of virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings.     

Depletion of glycoprotein gp85 from virosomes made with Epstein-Barr virus proteins abolishes their ability to fuse with virus receptor-bearing cells.

Entry of an enveloped virus such as Epstein-Barr virus (EBV) into host cells involves fusion of the virion envelope with host cell membranes either at the surface of the cell or within endocytic vesicles. Previous work has indirectly implicated the EBV glycoprotein gp85 in this fusion process. A neutralizing monoclonal antibody to gp85, F-2-1, failed to inhibit binding of EBV to its receptor but interfered with virus fusion as measured with the self-quenching fluorophore octadecyl rhodamine B chloride (R18) (N. Miller and L. M. Hutt-Fletcher, J. Virol. 62:2366-2372, 1988). To test further the hypothesis that gp85 functions as a fusion protein, EBV virion proteins including or depleted of gp85 were incorporated into lipid vesicles to form virosomes. Virosomes were labeled with R18, and those that were made with undepleted protein were shown to behave in a manner similar to that of R18-labeled virus. They bound to receptor-positive but not to receptor-negative cells and fused with Raji cells but not with receptor-positive, fusion-incompetent Molt 4 cells; monoclonal antibodies that inhibited binding or fusion of virus inhibited binding and fusion of virosomes, and virus competed with virosomes for attachment to cells. In contrast, virosomes made from virus proteins depleted of gp85 by immunoaffinity chromatography remained capable of binding to receptor-positive cells but failed to fuse. These results are compatible with the hypothesis that gp85 is actively involved in the fusion of EBV with lymphoblatoid cell lines and suggest that the ability of antibody F-2-1 to neutralize infectivity of EBV represents a direct effect on the function of gp85 as a fusion protein.     

Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly

Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIbeta. One can prevent the interaction by introducing a C(11)-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase IIalpha/beta in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIalpha and IIbeta antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases IIalpha/beta in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.     

Gating Movements of Colicin A and Colicin Ia Are Different

The effect of temperature on fusion of Sendai virus with target membranes and mobility of the viral glycoproteins was studied with fluorescence methods. When intact virus was used, the fusion threshold temperature (20–22°C) was not altered regardless of the different types of target membranes. Viral glycoprotein mobility in the intact virus increased with temperature, particularly sharply at the fusion threshold temperature. This effect was suppressed by the presence of erythrocyte ghosts and/or dextran sulfate in the virus suspension. In these cases also, no change in the fusion threshold temperature was observed. On the other hand, reconstituted viral envelopes (virosomes) bearing viral glycoproteins but lacking matrix proteins were capable of fusing with erythrocyte ghosts even at temperatures lower than the fusion threshold temperature and no fusion threshold temperature was observed over the range of 10–40°C. The mobility of viral glycoproteins on virosomes was much greater and virtually temperature-independent. The intact virus treated with an actin-affector, jasplakinolide, reduced the extent of fusion with erythrocyte ghosts and the mobility of viral glycoproteins, while the treatment of virosomes with the same drug did not affect the extent of fusion of virosomes with erythrocyte ghosts and the mobility of the glycoproteins. These results suggest that viral matrix proteins including actins affect viral glycoprotein mobility and may be responsible for the temperature threshold phenomenon observed in Sendai virus fusion.This revised version was published online in August 2005 with a corrected cover date.     

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.     

Are anti-HIV IgAs good guys or bad guys?

An estimated 90% of all HIV transmissions occur mucosally. Immunoglobulin A (IgA) molecules are important components of mucosal fluids. In a vaccine efficacy study, in which virosomes displaying HIV gp41 antigens protected most rhesus monkeys (RMs) against simian-human immunodeficiency virus (SHIV), protection correlated with vaginal IgA capable of blocking HIV transcytosis in vitro. Furthermore, vaginal IgG exhibiting virus neutralization and/or antibody-dependent cellular cytotoxicity (ADCC) correlated with prevention of systemic infection. In contrast, plasma IgG had neither neutralizing nor ADCC activity. More recently, a passive mucosal immunization study provided the first direct proof that dimeric IgAs (dIgAs) can prevent SHIV acquisition in RMs challenged mucosally. This study compared dimeric IgA1 (dIgA1), dIgA2, or IgG1 versions of a human neutralizing monoclonal antibody (nmAb) targeting a conserved HIV Env epitope. While the nmAb neutralization profiles were identical in vitro, dIgA1 was significantly more protective in vivo than dIgA2. Protection was linked to a new mechanism: virion capture. Protection also correlated with inhibition of transcytosis of cell-free virus in vitro. While both of these primate model studies demonstrated protective effects of mucosal IgAs, the RV144 clinical trial identified plasma IgA responses to HIV Env as risk factors for increased HIV acquisition. In a secondary analysis of RV144, plasma IgA decreased the in vitro ADCC activity of vaccine-induced, Env-specific IgG with the same epitope specificity. Here we review the current literature regarding the potential of IgA – systemic as well as mucosal – in modulating virus acquisition and address the question whether anti-HIV IgA responses could help or harm the host.     

Independence of H-2 and viral antigens on the cell surface and absence of H-2 antigens on murine leukemia virus and mouse mammary tumor virus particles

By indirect immunoelectron microscopy we tested for the presence of H-2 antigens on murine mammary tumor virus (MMTV) and murine leukemia virus (MuLV) particles. The association of H-2 antigens and viral antigens on the virus-infected cell surface was investigated with antibody-induced redistribution. Mammary tumor cells and leukemia cell lines with different H-2 genotypes and carrying different MuMTV or MuLV were used. No H-2 antigens could be demonstrated on the envelope of MMTV and MuLV particles, even after the permeabilization of their envelopes with saponin. On the surface of virus-infected cells antibody-induced patching or capping of the viral antigens did not result in copatching or cocapping of the H-2 antigens. In the reciprocal tests no co-redistribution of viral antigens with H-2 antigens was seen. Our experiments failed to show any physical association between H-2 antigens and MMTV or MuLV antigens on the cell surface.Abbreviations used in this paper MMTV mammary tumor virus - MuLV murine leukemia virus - MHC major histocompatibility complex - IEM immunelectron microscopy     

保护性病毒抗原的筛选策略及其在新型疫苗研发中的应用

随着疫苗研发技术的发展,新型疫苗在传染病的预防中得到了广泛应用。由于新型疫苗安全性良好,因此其在烈性病疫苗的应用中有着得天独厚的优势,然而研制新型疫苗的前提是筛选出保护性抗原。随着各种组学研究的发展,针对真核生物的多种生物信息学方法代表着最前沿的技术手段。相对于真核细胞,病毒具有更为简单的结构,对应着相对简单的研究方法,未来的保护性抗原筛选策略,需要结合生物信息学和传统分子生物学方法的优势。本文分别从宿主和病毒入手,论述了病毒保护性抗原的筛选策略,列举了一系列基于真核细胞开发的可能用于保护性抗原筛选的生物信息学方法,并总结了应用保护性抗原进行新型疫苗设计的案例,以便加深对病毒保护性抗原筛选策略的认知,为新型疫苗的研发提供借鉴。     

Functional reconstitution of influenza virus envelopes.

We have examined several procedures for the reconstitution of influenza virus envelopes, based on detergent removal from solubilized viral membranes. With octylglucoside, no functionally active virosomes are formed, irrespective of the rate of detergent removal: in the final preparation the viral spike proteins appear predominantly as rosettes. Protein incorporation in reconstituted vesicles is improved when a method based on reverse-phase evaporation of octylglucoside-solubilized viral membranes in an ether/water system is employed. However, the resulting vesicles do not fuse with biological membranes, but exhibit only a non-physiological fusion reaction with negatively charged liposomes. Functional reconstitution of viral envelopes is achieved after solubilization with octaethyleneglycol mono(n-dodecyl)ether (C12E8), and subsequent detergent removal with Bio-Beads SM-2. The spike protein molecules are quantitatively incorporated in a single population of virosomes of uniform buoyant density and appear on both sides of the membrane. The virosomes display hemagglutination activity and a strictly pH-dependent hemolytic activity. The virosomes fuse with erythrocyte ghosts, as revealed by a fluorescence resonance energy transfer assay. The rate and the pH dependence of fusion are essentially the same as those of the intact virus. The virosomes also fuse with cultured cells, either at the level of the endosomal membrane or directly with the cellular plasma membrane upon a brief exposure to low pH.     

Immunogenicity and protective capacity of a virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid A in mice

Respiratory Syncytial Virus (RSV) is a major cause of viral brochiolitis in infants and young children and is also a significant problem in elderly and immuno-compromised adults. To date there is no efficacious and safe RSV vaccine, partially because of the outcome of a clinical trial in the 1960s with a formalin-inactivated RSV vaccine (FI-RSV). This vaccine caused enhanced respiratory disease upon exposure to the live virus, leading to increased morbidity and the death of two children. Subsequent analyses of this incident showed that FI-RSV induces a Th2-skewed immune response together with poorly neutralizing antibodies. As a new approach, we used reconstituted RSV viral envelopes, i.e. virosomes, with incorporated monophosphoryl lipid A (MPLA) adjuvant to enhance immunogenicity and to skew the immune response towards a Th1 phenotype. Incorporation of MPLA stimulated the overall immunogenicity of the virosomes compared to non-adjuvanted virosomes in mice. Intramuscular administration of the vaccine led to the induction of RSV-specific IgG2a levels similar to those induced by inoculation of the animals with live RSV. These antibodies were able to neutralize RSV in vitro. Furthermore, MPLA-adjuvanted RSV virosomes induced high amounts of IFNγ and low amounts of IL5 in both spleens and lungs of immunized and subsequently challenged animals, compared to levels of these cytokines in animals vaccinated with FI-RSV, indicating a Th1-skewed response. Mice vaccinated with RSV-MPLA virosomes were protected from live RSV challenge, clearing the inoculated virus without showing signs of lung pathology. Taken together, these data demonstrate that RSV-MPLA virosomes represent a safe and efficacious vaccine candidate which warrants further evaluation.     

Delivery of Foreign Substances to Cells Mediated by Fusion-Active Reconstituted Influenza Virus Envelopes (Virosomes)

Abstract

This paper presents a survey of the properties and applications of reconstituted influenza virus envelopes (virosomes). Influenza virosomes can be reconstituted from the original viral membrane lipids and spike glycoproteins, after solubilization of intact virus with octaethyleneglycol monododecyl ether (C₁₂E₈) and removal of this detergent with a hydrophobic resin (BioBeads SM-2). These virosomes are functionally active, i.e their membrane fusion activity closely mimics the well-defined low-pH-dependent membrane fusion activity of the intact virus, which is solely mediated by the viral hemagglutinin (HA). By virtue of their fusion activity, virosomes represent a powerful carrier system for cellular delivery of foreign substances, encapsulated in their aqueous interior or co-reconstituted in their membranes. Delivery of an encapsulated, water-soluble, compound is illustrated with data on the toxin gelonin. Protein synthesis in BHK-21 cells in culture is efficiently inhibited when gelonin-containing virosomes fuse from within endosomes, after internalization via receptor-mediated endocytosis, or are induced to fuse with the plasma membrane by a transient lowering of the pH in the medium. The results indicate that delivery is quite efficient; as much as 6 × 10³ molecules of gelonin can readily be delivered to the cytoplasm of a single cell by fusion with gelonin-containing virosomes.