Molecular and structural characterization of the L1 virus-like particles that are used as vaccine antigens in Cervarix™, the AS04-adjuvanted HPV-16 and -18 cervical cancer vaccine.

Cervarix™ is a prophylactic human papillomavirus (HPV)-16/18 vaccine developed for the prevention of cervical cancer. The vaccine antigens are HPV-16 and HPV-18 L1 virus-like particles (VLPs) made from baculovirus expression vector system (BEVS)-produced HPV-16 and HPV-18 L1 proteins, respectively. In this study, we demonstrate that truncation of the nuclear targeting and DNA binding signals at the C-terminus of the HPV-16 and HPV-18 L1 proteins prevented intranuclear formation of the VLPs in the host cells and led to cytoplasmic localization of the L1 proteins as shown by in situ immunogold detection and electron microscopy. Following purification, these L1 proteins were able to form VLPs. The characteristics of these HPV-16 and HPV-18 L1 VLPs were studied using various physicochemical and immunological techniques. Amino acid analysis, SDS-PAGE and western blotting demonstrated the high purity of the L1 proteins and batch-to-batch consistency. The structure of the VLPs was shown to be similar to that reported for the native virions, as evaluated by microscopic observations, protein tomography and disc centrifugation experiments. The presence of important conformation-dependent neutralizing epitopes, such as U4, V5 and J4, was confirmed by ELISA and surface plasmon resonance. Structural robustness and consistency among batches was also observed by differential scanning calorimetry and electron microscopy. Moreover, adsorption to aluminum was shown not to impair VLP structure. In conclusion, the BEVS-produced HPV-16 and HPV-18 L1 VLPs display key structural and immunological features, which contribute to the efficacy of Cervarix™ vaccination.

Cervarix, the GSK HPV-16/HPV-18 AS04-adjuvanted cervical cancer vaccine, demonstrates stability upon long-term storage and under simulated cold chain break conditions.

Cervarix is a recombinant human papillomavirus (HPV)-16 and -18 L1 virus-like-particle (VLP) AS04-adjuvanted vaccine designed to protect against cervical intraepithelial neoplasia and cervical cancer caused by the HPV types 16 and 18. Assessment of the stability of the vaccine during long-term storage and after transient exposure to temperatures out of normal storage range is an integrated part of vaccine quality evaluation. This assessment was done with vaccine samples stored at 2-8 degrees C for up to 36 months, with or without simulated cold chain break (either one week at 37 degrees C, or two or four weeks at 25 degrees C). Among the stability-indicating parameters, antigenicity and immunogenicity were evaluated along with L1 antigen integrity and adsorption to aluminum. Differential scanning calorimetry (DSC) was used to investigate the structural stability of the VLPs before and after vaccine formulation and over time. Cervarix was stable at 2-8 degrees C for at least three years, and the occurrence of cold chain break had no impact, as shown by unchanged product characteristics during the full storage period. DSC analysis demonstrated that the structure of the HPV-16 and -18 L1 proteins and their corresponding VLPs was not affected throughout the manufacturing process. Moreover, the structure of aluminum-adsorbed HPV-16 and -18 L1 VLPs was robust over a 14-month test period. In conclusion, Cervarix was very stable upon long-term storage at 2-8 degrees C with or without transient exposure to higher temperatures (up to 37 degrees C). The observed robust structure of the L1 VLPs contributes to the excellent stability of Cervarix.

Cationic lipid/DNA complexes induce TNF-alpha secretion in splenic macrophages.

Cationic lipids are widely used as vectors to deliver DNA into mammalian cells in vitro and in vivo. However, cationic lipid/DNA lipoplexes induce an inflammatory response, characterized by pro-inflammatory cytokine secretion, which severely limits their use. The main goal of this work is to identify the organs and the cell type involved in TNF-alpha secretion after lipoplex injection. We determined the kinetics of distribution of the cationic lipid/DNA complex in blood, lung, liver and spleen and quantified the TNF-alpha amount in organ homogenates and in the serum at different points of times. Increase in TNF-alpha production was only observed in the spleen and no significant increase of TNF-alpha production could be observed in the other organs. Fractionation of spleen cells revealed that macrophages were mainly responsible for TNF-alpha secretion. This observation was verified in vivo by using macrophage-removing agents. In conclusion, we show here that the TNF-alpha secreted in the serum after intravenous injection of lipoplexes comes mainly from the splenic macrophages.

Lysosome-Dependent Activation of Human Dendritic Cells by the Vaccine Adjuvant QS-21.

The adjuvant properties of the saponin QS-21 have been known for decades. It is a component of the Adjuvant System AS01 that is used in several vaccine candidates. QS-21 strongly potentiates both cellular and humoral immune responses to purified antigens, yet how it activates immune cells is largely unknown. Here, we report that QS-21 directly activated human monocyte-derived dendritic cells (moDCs) and promoted a pro-inflammatory transcriptional program. Cholesterol-dependent QS-21 endocytosis followed by lysosomal destabilization and Syk kinase activation were prerequisites for this response. Cathepsin B, a lysosomal cysteine protease, was essential for moDC activation in vitro and contributed to the adjuvant effects of QS-21 in vivo. Collectively, these findings provide new insights into the pathways involved in the direct activation of antigen-presenting cells by a clinically relevant QS-21 formulation.

Central Role of CD169+ Lymph Node Resident Macrophages in the Adjuvanticity of the QS-21 Component of AS01.

Saponins represent a promising class of vaccine adjuvant. Together with the TLR4-ligand MPL, QS-21 is part of the Adjuvant System AS01, a key component of the malaria and zoster candidate vaccines that display demonstrated clinical efficacy. However, the mechanism of action of QS-21 in this liposomal formulation is poorly understood. Upon intra-muscular immunisation, we observed that QS-21 rapidly accumulated in CD169+ resident macrophages of the draining lymph node where it elicited a local innate immune response. Depletion of these cells abrogated QS-21-mediated innate cell recruitment to the lymph node, dendritic cell (DC) phenotypic maturation as well as the adjuvant effect on T-cell and antibody responses to co-administered antigens. DCs rather than lymph node-resident macrophages were directly involved in T-cell priming by QS-21, as revealed by the decrease in antigen-specific T-cell response in Batf3-/- mice. Further analysis showed that the adjuvant effect of QS-21 depended on the integration of Caspase-1 and MyD88 pathways, at least in part through the local release of HMGB1. Taken together, this work unravels the key role of lymph node sentinel macrophage in controlling the adjuvant effect of a molecule proven to improve vaccine response in humans.

Role of intracellular cationic liposome-DNA complex dissociation in transfection mediated by cationic lipids.

The cationic lipid-mediated gene transfer process involves sequential steps: internalization of the cationic lipid-DNA complexes inside the cells via an endocytosis-like mechanism, escape from endosomes, dissociation of the complex, and finally entry of free DNA into the nucleus. However, cationic lipid-DNA complex dissociation in the cytoplasm and the ability of the subsequently released DNA to enter the nucleus have not yet been demonstrated. In this report we showed, using confocal laser scanning analysis, that microinjection of a double fluorescent-labeled cationic lipid-pCMV-LacZ plasmid complex into the cytoplasm of HeLa cells results in efficient complex dissociation. However, the released DNA did not enter the nucleus, and no significant transfection could be detected. In contrast, nuclear microinjection of the cationic lipid-pCMV-LacZ plasmid complex resulted in efficient complex dissociation and transfection of all the cells. Taken together, the data suggest that intracellular dissociation of the cationic lipid-DNA complex is not a limiting step for transfection as previously thought.

Adjuvant System AS03 containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity.

AS03 is an Adjuvant System (AS) containing α-tocopherol and squalene in an oil-in-water (o/w) emulsion. AS03 has been considered for the development of pandemic and seasonal influenza vaccines. Key features of AS03's mode of action were investigated in vivo in mice and ex vivo in human cells. AS03's adjuvant activity was superior to that of aluminium hydroxide and required the spatio-temporal co-localisation of AS03 with the antigen. This requirement coincided with AS03 triggering a transient production of cytokines at the injection site and in the draining lymph nodes (dLNs). The nature of the cytokines produced was consistent with the enhanced recruitment of granulocytes and of antigen-loaded monocytes in the dLNs. The presence of α-tocopherol in AS03 was required for AS03 to achieve the highest antibody response. The presence of α-tocopherol also modulated the expression of some cytokines, including CCL2, CCL3, IL-6, CSF3 and CXCL1; increased the antigen loading in monocytes; and increased the recruitment of granulocytes in the dLNs. Hence, AS03's promotion of monocytes as the principal antigen-presenting cells, and its effects on granulocytes and cytokines, may all contribute to enhancing the antigen-specific adaptive immune response.

Sequence-optimised E2 constructs from BVDV-1b and BVDV-2 for DNA immunisation in cattle.

We report DNA immunisation experiments in cattle using plasmid constructs that encoded glycoprotein E2 from bovine viral diarrhoea virus (BVDV)-1 (E2.1) and BVDV-2 (E2.2). The coding sequences were optimised for efficient expression in mammalian cells. A modified leader peptide sequence from protein gD of BoHV1 was inserted upstream of the E2 coding sequences for efficient membrane export of the proteins. Recombinant E2 were efficiently expressed in COS7 cells and they presented the native viral epitopes as judged by differential recognition by antisera from cattle infected with BVDV-1 or BVDV-2. Inoculation of pooled plasmid DNA in young cattle elicited antibodies capable of neutralising viral strains representing the major circulating BVDV genotypes.

Free diC14-amidine liposomes inhibit the TNF-alpha secretion induced by CpG sequences and lipopolysaccharides: role of lipoproteins.

It has been shown that a preinjection of diC14-amidine cationic liposomes decreased TNF-alpha secretion induced by lipoplexes intravenous injection. We showed here that free cationic liposomes inhibit CpG sequences- or lipopolysaccharides-induced TNF-alpha secretion by macrophages. Surprisingly, this effect was strictly dependent on serum. Free cationic liposomes alone did not reveal any anti-inflammatory activity. Low-density lipoproteins and triglyceride-rich lipoproteins were identified as the serum components that confer to the liposomes an anti-inflammatory activity. Lipid fractions of these lipoproteins were able to reproduce the effect of the total lipoproteins and could inhibit, in association with diC14-amidine liposomes, the CpG-induced TNF-alpha secretion. Serum components confer to cationic liposomes new properties that can be used to modulate the inflammatory response directed against CpG sequences and lipopolysaccharides.

Formation and intracellular trafficking of lipoplexes and polyplexes.

Cationic lipid/DNA lipoplexes and cationic polymer/DNA polyplexes represent an attractive alternative to viral vectors for cell transfection in vitro and in vivo but still suffer from a relatively low efficiency. Optimization of their transfection efficiency may be attempted by using a trial and error approach consisting of synthesizing and testing a large number of derivatives. On the other hand, rational design of highly efficient cationic lipids and polymers requires a deeper understanding of the interactions between the vector and the DNA as well as the cellular pathways and mechanisms involved in DNA entry into the cell and ultimately the nucleus. In the present review, the pathways and mechanisms involved in lipoplex- and polyplex-mediated transfection are comparatively addressed and unresolved questions are highlighted.

Lipid mixing between lipoplexes and plasma lipoproteins is a major barrier for intravenous transfection mediated by cationic lipids.

It has been previously shown that transfection activity of cationic liposome/DNA lipoplexes delivered systemically is drastically inhibited by lipoproteins (Tandia, B. M., Vandenbranden, M., Wattiez, R., Lakhdar, Z., Ruysschaert, J. M., and Elouahabi, A. (2003) Mol Ther. 8, 264-273). In this work, we have compared the binding/uptake and transfection activities of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) and diC14-amidine (3-tetradecylamino-N-tert-butyl-N'-tetra-decylpropionamidine)-containing lipoplexes in the presence or absence of purified low density lipoproteins and high density lipoprotein. Binding/uptake of both lipoplexes by the mouse lung endothelial cell line was inhibited to a similar extent in the presence of lipoproteins. In contrast, transfection activity of diC14-amidine-containing lipoplexes was almost completely inhibited (approximately by 95%), whereas approximately 40% transfection activity of DOTAP-containing lipoplexes was preserved in the presence of lipoproteins. Interestingly, the ability of lipoproteins to inhibit the transfection efficiency of lipoplexes was well correlated with their ability to undergo lipid mixing with the cationic lipid bilayer as revealed by fluorescence resonance energy transfer assay. Incubation of lipoplexes with increased doses of lipoproteins resulted in enhanced lipid mixing and reduced transfection activity of the lipoplexes in mouse lung endothelial cells. The role of lipid mixing in transfection was further demonstrated using lipid-mixing inhibitor, lyso-phosphatidylcholine, or activator (dioleoylphosphatidylethanolamine). Incorporation of Lyso-PC into diC14-amidine-containing lipoplexes completely abolished their capacity to undergo lipid mixing with lipoproteins and allowed them to reach a high transfection efficiency in the presence of lipoproteins. On the other hand, the incorporation of dioleoylphosphatidylethanolamine into DOTAP/DNA lipoplex activated lipid mixing with the lipoproteins and was shown to be detrimental toward the transfection activity of these lipoplexes. Taken together, these results indicate that fusion of lipoplexes with lipoproteins is a limiting factor for in vivo transfection.

Vaccination with the recombinant allergen ProDer p 1 complexed with the cationic lipid DiC14-amidine prevents allergic responses to house dust mite.

The present study evaluated the prophylactic potential of ProDer p 1, the recombinant precursor form of the major mite allergen Der p 1, combined with the cationic lipid diC14-amidine in a murine model of house dust mite allergy. Naive mice vaccinated with the amidine/allergen complex developed a Th1-biased immune response characterized by the absence of specific IgE, the production of specific IgG2a, and the presence of IFN-gamma in splenocyte cultures. In contrast, ProDer p 1 adjuvanted with alum induced typical strictly Th2-biased allergic responses with strong IgG1 and IgE titers and IL-5 secretion. Removal of negatively charged sialic acids in ProDer p 1 or increasing the ionic strength reduced the binding of ProDer p 1 to the cationic liposomes and resulted in a decrease of the allergen immunogenicity, suggesting that complexation is required for triggering an optimal immune response. Finally, prophylactic vaccination with ProDer p 1-diC14-amidine reduced drastically the production of specific IgE and airway eosinophilia following subsequent immunization with Der p 1-alum and challenge with aerosolized house dust mite extracts. In conclusion, recombinant ProDer p 1 complexed with diC14-amidine could represent an efficient prophylactic vaccine against house dust mite allergy.

Identification of human plasma proteins that bind to cationic lipid/DNA complex and analysis of their effects on transfection efficiency: implications for intravenous gene transfer.

Interaction of cationic lipid/DNA complex with the plasma is a limiting step for the cationic lipid-mediated intravenous gene transfer and expression process. Most of the plasma components that interact with the complex and inhibit its transfection efficiency are still unknown. In the present work, human plasma proteins and lipoproteins that bind to a cationic lipid/DNA complex were isolated on a sucrose density gradient and identified by 2-D gel electrophoresis. Protein binding did not result in complex dissociation or DNA degradation. The effects of several complex-binding plasma components on the transfection efficiency were studied using lung endothelial cells cultured in vitro. Lipoprotein particles caused a drastic loss of the transfection efficiency of the complex. Surprisingly, fibrinogen was found to activate the transfection process. The roles of these complex-binding plasma components on the complex uptake efficiency were quantitatively assessed using radiolabeled plasmid DNA and qualitatively evaluated using fluorescence microscopy. A good correlation was found between the effects of the complex-binding plasma components on the transfection and on cell uptake efficiencies. In contrast to what was generally believed, our data suggest that disruption of the complex does not occur when it is in contact with the plasma and therefore could not be responsible for the loss of transfection activity. Instead, coating of complexes with plasma components seems to be responsible for reduced uptake by cells, which in turn results in reduced transfection.

Free cationic liposomes inhibit the inflammatory response to cationic lipid-DNA complex injected intravenously and enhance its transfection efficiency.

In this report, we show that intravenous (i.v.) injection into mice of a complex made of the cationic lipid diC14-amidine and the luciferase reporter plasmid (pCMV-luc) results in efficient gene expression in several organs but elicits an inflammatory response characterized by a release of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) into the serum of treated animals. A single preinjection of free diC14-amidine liposomes improves the i.v. transfection efficiency of the diC14-amidine/protamine/pCMV-luc complex as much as 40 times. This improvement is correlated with the ability of free liposomes to inhibit TNF-alpha but not IFN-gamma production resulting from complex injection. TNF-alpha-rich serum obtained from mice injected with diC14-amidine/protamine/pCMV-luc complex inhibits luciferase expression in transfected mouse lung endothelial (MLE) cells cultured in vitro, whereas IFN-gamma has no effect. This inhibitory effect can be partly abolished by treating the mouse serum with a specific anti-TNF-alpha antibody. These data point out that cationic lipids are potent inhibitors of the inflammatory response to the CpG motifs in plasmid DNA. This property is shown to enhance the in vivo transfection efficiency.