A virosomal formulated Her-2/neu multi-peptide vaccine induces Her-2/neu-specific immune responses in patients with metastatic breast cancer: a phase I study.

We have previously shown in mice that vaccination with three Her-2-peptides representing B-cell epitopes of the extracellular domain of Her-2/neu induces Her-2/neu-specific IgG antibodies with strong anti-tumor activity in vitro and in vivo. We have now finalized a phase I clinical trial with an anti-Her-2/neu vaccine-construct of immunopotentiating reconstituted influenza virosomes with the three peptides in patients with metastatic breast cancer (MBC). Ten MBC patients with low protein overexpression of Her-2/neu of MBC (+ or ++ upon immunohistochemistry, FISH negative) and positive hormone receptor status were enrolled in a single center phase I study. The virosomal formulated vaccine, consisting of 10 microg/peptide, was intramuscularly applied three times on days 1, 28, and 56. The primary endpoint of the study, which lasted 12 weeks, was safety, the secondary endpoint immunogenicity. Local erythema at the injection site was the only vaccine-related side effect occurring in four patients. In 8 of 10 patients an increase in peptide-specific antibody titer measured by ELISA was found. Importantly, the induced antibodies were also directed against the native Her-2/neu protein. Cellular immune responses, as measured by in vitro production of IL-2, IFN-c, and TNF-a of PBMCs showed a marked increase after vaccination in the majority of vaccinees. Notably, the number of CD4+CD25+Foxp3+T regulatory cells, which were significantly increased compared to healthy controls prior to vaccination, was markedly reduced following vaccination. In all, the immunological responses after vaccination indicated that the patients in stage IV of disease were immunocompetent and susceptible to vaccination. The Her-2/neu multipeptide vaccine was safe, well tolerated and effective in overcoming immunological tolerance to Her-2/neu. The induction of anti-Her-2-specific antibodies could result in clinical benefit comparable to passive anti-Her-2 antibody therapy.

Quantitative microstructure analysis of polymer-modified mortars.

Digital light, fluorescence and electron microscopy in combination with wavelength-dispersive spectroscopy were used to visualize individual polymers, air voids, cement phases and filler minerals in a polymer-modified cementitious tile adhesive. In order to investigate the evolution and processes involved in formation of the mortar microstructure, quantifications of the phase distribution in the mortar were performed including phase-specific imaging and digital image analysis. The required sample preparation techniques and imaging related topics are discussed. As a form of case study, the different techniques were applied to obtain a quantitative characterization of a specific mortar mixture. The results indicate that the mortar fractionates during different stages ranging from the early fresh mortar until the final hardened mortar stage. This induces process-dependent enrichments of the phases at specific locations in the mortar. The approach presented provides important information for a comprehensive understanding of the functionality of polymer-modified mortars.

Mucosal antibody response induced with a nasal virosome-based influenza vaccine.

A vaccination against influenza that elicits both a systemic antibody and a mucosal IgA response would improve on the protective efficacy of currently available vaccines. Previous studies have shown the safety and efficacy of virosomes as delivery systems in vaccination. This study was a controlled, randomised, double-blind, single centre, phase II trial assessing an intranasal virosome vaccine, adjuvanted with heat-labile toxin (HLT) from enterotoxigenic Escherichia coli, versus an intranasal without HLT and comparing it open to an intramuscular vaccine in a total of 88 healthy adults. The development of a new technique enabled for the first time the detection of neutralising IgA antibodies in very dilute nasal wash samples. It was demonstrated that intranasally administered inactivated influenza vaccine, adjuvanted with HLT, not only elicits a spectrum of humoral and cell-mediated responses in healthy adults, critical for the protection and recovery from influenza virus infection, but is also highly effective in eliciting IgA neutralising antibodies at the mucosa. Intranasal virosome-formulated, HLT-adjuvanted, influenza vaccine was effective and well tolerated in this study. Its potential to offer a high level of mucosal protection, not provided by conventional parenteral vaccination, could play a significant role in preventing morbidity and mortality associated with influenza.

In vivo study of the GC90/IRIV vaccine for immune response and autoimmunity into a novel humanised transgenic mouse.

Parathyroid hormone-related protein (PTH-rP), a secreted protein produced by prostate carcinoma and other epithelial cancers, is considered a key agent for the development of bone metastases. We investigated the construct GC90/IRIV, composed of immunopotentiating reconstituted influenza virosomes (IRIV) containing PTH-rP gene plasmids (GC90), as a potential tool for human anticancer immunotherapy into humanised mice transgenic for HLA-A(*)02.01, the human-beta2 microglobulin, and the human CD8alpha molecule. Intranasal administration of GC90/IRIV resulted in the induction of a PTH-rP-specific multiepitope cytotoxic T-cell (CTL) response. Cytotoxic T cells derived from vaccinated mice were capable of lysing in vitro syngenic murine PTH-rP transfectants and human HLA-A((*))02.01(+)/PTH-rP(+) prostate carcinoma LNCaP cells as well. The immune response capacity and the absence of any sign of toxicity and/or autoimmunity in vivo suggest the GC90/IRIV vaccine as a valid tool for active specific immunotherapy of human cancers and metastases overexpressing PTH-rP.

Influenza virosomes are an efficient delivery system for respiratory syncytial virus-F antigen inducing humoral and cell-mediated immunity.

In the present study we investigated the efficacy of a new potential vaccine constituted of the respiratory syncytial virus (RSV)-F protein associated with influenza virosomes (RSV-F/IRIV) in combination with the mucosal adjuvant Escheriagen (Escherichia coli heat-labile toxin), administered intranasally (i.n.) to BALB/c mice. After an intramuscular "priming" with influenza virus vaccine, group A of mice was i.n. immunized with of RSV-F/IRIV+heat-labile toxin (HLT), groups B and C were inoculated i.n. with F-RSV+HLT and IRIV+HLT, respectively. The results showed that the virosomal delivery system greatly potentiate immune responses in animals. All mice immunized with the RSV-F/IRIV+HLT developed a mucosal IgA response and a high level of serum IgG. A balanced Th1/Th2 cytokine profile was observed in mice immunized with RSV-F/IRIV+HLT, while a Th2 response was observed in mice immunized with RSV-F+HLT. Histological analysis of lung tissue of RSV challenged mice did not reveal a vaccine-enhanced pulmonary eosinophilia. These results show that i.n. immunization of BALB/c mice with RSV-F/IRIV in combination with HLT can be considered a promising approach for the development of an efficacious human vaccine.

Perspectives: towards a peptide-based vaccine against hepatitis C virus.

Hepatitis C virus (HCV) is a widespread infectious disease in humans with the negative implication of becoming chronic in most persons. Patients infected with HCV are at risk of liver cirrhosis or hepatocellular carcinoma at later stages. In contrast to hepatitis A and hepatitis B, there is no immunization yet available, neither prophylactic nor therapeutic. Thus, there is an urgent need to develop a safe, protective vaccine against this fatal disease. Developing countries are even more at risk for HCV. There are currently a number of scientific approaches aimed towards solving this problem. Taking both risks and costs of immunization into consideration, a peptide-based vaccine may be a reasonable prophylactic protection. Also, it might be of therapeutic use in already infected patients by increasing a specific CTL response against HCV. In our lab, we are focusing on immunopotentiating reconstituted influenza virosomes (IRIVs) as carriers for immunogenic HLA-A2-restricted core epitopes to induce peptide-specific cytotoxic T lymphocytes (CTLs). The IRIVs are similar to liposomes, but in addition contain influenza-derived hemagglutinin and neuraminidase on their outer surface which makes them fusogenic, thus, permitting antigen delivery to host cells. So far, virosomes have been successfully used for vaccine development and as a result a virosomal vaccine against both influenza virus (Inflexal) BERNA) and hepatitis A virus (HAV) (Epaxal) BERNA) already exist on the market. This paper focuses on the importance of development of a successful vaccine against HCV and, more specifically, we discuss the use, advantages and disadvantages of a peptide-based vaccine. A brief report of our latest findings will be included.

Tumour-associated antigen (TAA)-specific cytotoxic T cell (CTL) response in vitro and in a mouse model, induced by TAA-plasmids delivered by influenza virosomes.

We investigated influenza virosomes as a TAA-gene delivery system for use in TAA-directed anti-cancer vaccine therapy. An engineered plasmid (GC90) expressing the parathyroid hormone-related peptide (PTH-rP), a protein secreted by prostate and lung carcinoma cells, was included in influenza virosomes (GC90V). The ability of GC90V to elicit a PTH-rP-specific cytotoxic T cell (CTL) response was demonstrated in BALB/c mice immunised with intranasal (i.n.) GC90V+/-adjuvant subcutaneous (s.c.) interleukin-2 (IL-2). A PTH-rP-specific CTL response with antitumour activity was also demonstrated in human peripheral blood mononuclear cells (PBMC) stimulated in vitro with GC90V infected autologous dendritic cells (DC). These results provide a rationale for investigating GC90V in clinical trials of anticancer vaccine therapy.

The humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus.

This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups.

Exploiting conformationally constrained peptidomimetics and an efficient human-compatible delivery system in synthetic vaccine design.

Peptide and protein mimetics are potentially of great value in synthetic vaccine design. The mimetics should function by stimulating the immune system to produce antibodies that recognize the intact parasite. Also the mimetics should be presented to the immune system in a way that leads to efficient antibody production. Here we investigate the application of cyclic peptidomimetics presented on immunopotentiating reconstituted influenza virosomes (IRIVs), a form of antigen delivery that is licensed already for human clinical use, in synthetic vaccine design. We focus on the central (NPNA)(n) repeat region of the circumsporozoite (CS) protein of the malaria parasite Plasmodium falciparum as a model system. Cyclic peptidomimetics of the NPNA repeats were incorporated into both an IRIV and (for comparison) a multiple-antigen peptide (MAP). Both IRIV and MAP delivery forms induced mimetic-specific humoral immune responses in mice, but only with the mimetic-IRIV preparations did a significant fraction of the elicited antibodies cross-react with sporozoites. The results demonstrate that IRIVs are a delivery system suitable for the efficient induction of antibody responses against conformational epitopes by use of cyclic template-bound peptidomimetics. Combined with combinatorial chemistry, this approach may have great potential for the rapid optimization of molecularly defined synthetic vaccine candidates against a wide variety of infectious agents.

Intranasal immunization with mumps virus DNA vaccine delivered by influenza virosomes elicits mucosal and systemic immunity.

To improve the efficiency of liposome-mediated DNA transfer as a tool for gene therapy or vaccinology, we have further developed a new delivery system based on the modified immunopotentiating reconstituted influenza virus (IRIV). In this study, we engineered a plasmid DNA vector expressing the mumps virus hemagglutinin or the fusion protein. The administration of this DNA vaccine delivered by influenza virosomes, in combination with the mucosal adjuvant Escheriagen via the intranasal route, was efficient for inducing an immune response, both mucosally and systemically, in mice. The production of IgG2a mumps virus-specific antibodies and the secretion of interleukin 10 (IL-10) by antigen-specific T cells indicated that not only Th1 but also Th2 responses were induced by this DNA vaccine formulation. These results suggest that cationic virosomes in combination with Escheriagen may have great potential as an efficient delivery system for intranasal DNA immunization and provide an immune barrier at the mucosal sites.

DNA vaccine encoding nucleocapsid and surface proteins of wild type canine distemper virus protects its natural host against distemper.

Canine distemper virus (CDV), a member of the genus Morbillivirus induces a highly infectious, frequently lethal disease in dogs and other carnivores. Current vaccines against canine distemper consisting of attenuated viruses have been in use for many years and have greatly reduced the incidence of distemper in the dog population. However, certain strains may not guarantee adequate protection and others can induce post vaccinal encephalitis. We tested a DNA vaccine for its ability to protect dogs, the natural host of CDV, against distemper. We constructed plasmids containing the nucleocapsid, the fusion, and the attachment protein genes of a virulent canine distemper virus strain. Mice inoculated with these plasmids developed humoral and cellular immune responses against CDV antigens. Dogs immunized with the expression plasmids developed virus-neutralizing antibodies. Significantly, vaccinated dogs were protected against challenge with virulent CDV, whereas unvaccinated animals succumbed to distemper.

Efficient vaccination by intradermal or intramuscular inoculation of plasmid DNA expressing hepatitis B surface antigen under desmin promoter/enhancer control.

The small surface antigen of the hepatitis B virus (HB5Ag) was cloned into expression plasmid pCI under either a viral (CMV) promoter;enhancer sequence control (plasmid pCI/S), or a human desmin promoter/enhancer sequence control (plasmid pDes/S). Cells of different species and tissue origin transiently transfected in vitro with pCI/S or pDes/S plasmid DNA expressed readily detectable amounts of HBsAg, either intracellularly (precipitated from cell lysates), or as secreted products (detectable in ELISA). When these plasmids were used in DNA vaccination, both efficiently primed humoral and/or cellular immune responses to HBsAg after a single injection in Balb/c mice. Intramuscular injection of a high dose of DNA (100 rig/mouse) of both plasmids primed MHC-I-restricted cytotoxic T lymphocyte (CTL) responses and Thi serum antibody responses (IgGlIgG2a ratio O.4C0.7) of comparable magnitude in all vaccinated mice. Intradermal injection of low doses of (particle-coated) DNA (1 microgm/mouse) of both plasmids with the gene gun primed Th2 serum antibody responses (IgGl/IgG2a ratio > 100) but no CTL responses. The data indicate that antigens can be efficiently expressed under viral or eukaryotic promoter/enhancer control for immunogenic in vivo presentation, but that the technique, dose and/or route of DNA injection have a decisive role in determining the type of immune response elicited.

IRIV-adjuvanted hepatitis A vaccine: in vivo absorption and biophysical characterization.

Immunopotentiating reconstituted influenza virosomes (IRIV) are 150-nm proteoliposomes composed of influenza surface glycoproteins and a mixture of natural and synthetic phospholipids. Due to size, structure and composition of the IRIVs, they serve as an antigen carrier system for efficacious vaccination, as was demonstrated for hepatitis A and influenza. This paper reviews the unique properties of IRIVs and describes the in vivo biodistribution of model antigens using 14C-labeled IRIVs and 125I-labeled streptavidin. IRIV formulated streptavidin induced a strong depot effect after intra muscular (i.m.) vaccination of mice, whereas soluble streptavidin was soon eliminated via the kidney of the animals. A mixture of antigen and IRIVs yielded higher antibody titers after i.m. inoculation than streptavidin alone. The highest immunostimulation was achieved by the binding of the antigen to the investigated adjuvant. The potential penetration of inactivated hepatitis A virions into lipid membranes was assessed by measuring the area increase of a lipid monolayer kept at a constant surface pressure corresponding to that of lipid bilayer vesicles. The monolayers were composed of phosphatidylcholine (POPC) and phosphatidylethanolamine (POPE) (75/25 mol/mol), thus resembling the lipid composition of the IRIV. The results suggested that the hepatitis A antigen may spontaneously bind to the reconstituted IRIV membranes.

Towards clinical testing of a single-administration tetanus vaccine based on PLA/PLGA microspheres.

The availability of single-administration vaccines would assist in the control of global mortality caused by infectious diseases where protection can be achieved only upon repeated immunisations with appropriate vaccines. Biodegradable microspheres of poly(lactide-co-glycolide) have been studied pre-clinically for this purpose and shown to be promising for several protein and sub-unit antigens. In view of preparing a microsphere-based tetanus vaccine for clinical trials, final candidate vaccine-formulations were pre-clinically optimised here. Specifically, the importance of particular materials and processing for the induction of neutralising antibodies in guinea pigs were examined. The most efficacious vaccines were small-sized (<5 microm), co-adjuvanted with admixed alum and fabricated from fast-degrading polymers. Interestingly, the immunogenicity was less influenced by the type of antigen-stabilising excipient, the number of microsphere populations mixed together, or the microencapsulation technology, i.e. spray-drying versus coacervation, used. On the basis of these, we plan to prepare clinical samples for safety and immunogenicity testing in man.

Use of reconstituted influenza virus virosomes as an immunopotentiating delivery system for a peptide-based vaccine.

Immunopotentiating reconstituted influenza virosomes (IRIV) were used as a delivery system for the synthetic peptide-based malaria vaccine SPf66. The reduced SPf66 peptide molecules containing terminal cysteine residues were covalently attached to phosphatidylethanolamine with the heterobifunctional crosslinker gamma-maleimidobutyric acid N-hydroxysuccinimide ester. The SPf66-phosphatidylethanolamine was incorporated into IRIV and BALB/c mice were immunized twice by intramuscular injection with peptide-loaded virosomes. Titres of elicited anti-SPf66 IgG were determined by ELISA. These titres were significantly higher and the required doses of antigen were lower, when mice had been preimmunized with a commercial whole virus influenza vaccine. After preimmunization with the influenza vaccine, SPf66-IRIV elicited far more consistently anti-SPf66 antibody responses than SPf(66)n adsorbed to alum. MoAb produced by four B cell hybridoma clones derived from a SPf66-IRIV-immunized mouse cross-reacted with Plasmodium falciparum blood stage parasites in immunofluorescence assays. All four MoAbs were specific for the merozoite surface protein-1 (MSP-1)-derived 83.1 portion of SPf66. Sequencing of their functionally rearranged kappa light chain variable region genes demonstrated that the four hybridomas were generated from clonally related splenic B cells. Biomolecular interaction analyses (BIA) together with these sequencing data provided evidence for the selection of somatically mutated affinity-matured B cells upon repeated immunization with SPf66-IRIV. The results indicate that IRIV are a suitable delivery system for synthetic peptide vaccines and thus have a great potential for the design of molecularly defined combined vaccines targeted against multiple antigens and development stages of one parasite, as well as against multiple pathogens.

Immunogenicity of IRIV- versus alum-adjuvanted diphtheria and tetanus toxoid vaccines in influenza primed mice.

The immunogenicity and protectivity of two different toxoid vaccines were compared in mice. In one formulation, toxoids (diphtheria or tetanus) were adsorbed to alumoxid, whereas in the other formulation the toxoids were crosslinked to immunopotentiating reconstituted influenza virosomes (IRIVs). A preimmunization with influenza antigens is necessary for a good anti-toxoid antibody response when the IRIV formulation was administered. After two immunizations with the IRIV- or alum-based vaccines, the IRIV-based formulation induced a higher humoral immune response than toxoids adsorbed to alum. Using an in vitro test, diphtheria toxin neutralizing antibodies were tested. Di-IRIV induced a significantly (p = 0.002) higher titer of diphtheria toxin neutralizing antibodies than Di-alum. Tetanus challenge experiments showed, that the IRIV-based tetanus vaccine induced a threefold higher titer of protective antibodies than the tetanus toxoid adsorbed to alum. Therefore, the IRIV-based formulations appeared to be superior to the alum-based vaccines in terms of immunogenicity and protectivity.

Purification of a dichlorophenol-indophenol oxidoreductase from rat and bovine synaptic membranes: tight complex association of a glyceraldehyde-3-phosphate dehydrogenase isoform, TOAD64, enolase-gamma and aldolase C.

NADH-dichlorophenol-indophenol oxidoreductases (PMOs) were purified from synaptic plasma membranes or synaptic vesicles (small recycling vesicles) from both bovine and rat brains and from a neuroblastoma cell line, NB41A3. Several isoforms could be identified in purified plasma membranes and vesicles. Purification of the enzyme activity involved protein extraction with detergents, (NH4)2SO4 precipitation, chromatography under stringent conditions and native PAGE. PMO activity could be attributed to a very tight complex of several proteins that could not be separated except by SDS/PAGE. SDS/PAGE resolved the purified complex into at least five proteins, which could be micro-sequenced and identified unambiguously as hsc70, TOAD64 and glyceraldehyde-3-phosphate dehydrogenase tightly associated with the brain-specific proteins aldolase C and enolase-gamma. Enzyme activity could be purified from both synaptic plasma membranes and recycling vesicles, yields being much greater from the latter source. Highly purified plasma membranes (prepared from a neuroblastoma cell line NB41A3 by iminobiotinylation of intact cells and affinity purification with avidin and anti-avidin antibodies under very stringent conditions) also displayed PMO activity tightly associated with TOAD64. The association of PMO in a tight complex was confirmed by its immunoprecipitation from cellular and membrane extracts of NB41A3 using antibodies directed against any component protein of the complex followed by immunodetection with antibodies directed against the other members. Antibodies also inhibited the enzyme activity synergistically. In addition, induction of the different components of the complex during dichlorophenol-indophenol stress was demonstrated by the S1 RNase-protection assay in synchronized NB41A3 cells. The role of the complex in membrane fusion and cellular response to extracellular oxidative stress during growth and development is discussed.

The plasma membrane NADH-diaphorase is active during selective phases of the cell cycle in mouse neuroblastoma cell line NB41A3. Its relation to cell growth and differentiation.

Plasma membrane oxidoreductases have been described in all cells and use extracellular impermeant electron acceptors (DCIP, Ferricyanide) that are reduced by NADH. They appear to regulate the overall cell activity in response to oxidative stress from the cellular environment. An NADH-DCIP reductase has been described at the plasma membrane of NB41A3, a neuroblastoma cell line (Zurbriggen and Dryer (1993) Biochim. Biophys. Acta 1183, 513-520) whose activation with extracellular impermeant substrates promotes cell growth. Elutriation was performed to separate cells and the various fractions were analysed for enzyme activity on intact cells combined with flow cytometry. These studies showed that the enzyme is mostly induced and activated during the G1 and during the G2/M-phases. These observations were further corroborated with specific inhibitors of the cell cycle. A three-fold increase in enzyme activity was observed in the presence of alpha-amanitin, a specific cell cycle inhibitor of the G1-phase. Taxol, a specific inhibitor of the M-phase, also induces a significant increase in enzyme activity. FACS analysis of taxol -treated and alpha-amanitin-treated cells corroborated these data. The cells have been synchronized and the enzyme activity was measured at different time intervals. An activity increase was observed after ca. 2-3 h, that corresponds to a raise in the M-phase, according to FACS data. Furthermore, NTera-2 cells - a human neuroblastoma cell line that differentiates into fully mature neurones in the presence of retinoic acid - exhibit a 50% decrease in the enzyme activity during the G0-phase upon differentiation, compared to undifferentiated cells. Together the data presented in this paper show that this plasma membrane NADH-diaphorase affects cell growth and differentiation and is strongly modulated at various phases of the cell cycle.

An NADH-diaphorase is located at the cell plasma membrane in a mouse neuroblastoma cell line NB41A3.

Plasma membranes from most mammalian cells display significant transplasma membrane oxidoreductase (PMO) activity. The enzymes use an extracellular, impermeant electron acceptor as substrate and intracellular reduced pyridine nucleotide as electron donor. The plasma membrane from a neuroblastoma cell line, NB41A3, has been biotinylated and purified by immunoprecipitation with avidin and antiavidin-antibodies. The protein recovery of an immunopurified membrane preparation was < 0.15% of the protein content in the cell extract. The preparation displays an increase in the specific activity of PMO's of 15- to 20-fold compared to the activity in whole cells. With this approach the presence of a NADH-diaphorase within the cell plasma membrane can be demonstrated. This activity accounts for about one third of the total cellular diaphorase activity. The PMO activity cannot be attributed to an increased permeabilization of the plasma membrane induced upon biotinylation nor to intracellular activity from lysed cells. Activation of basal metabolism (glycolysis) stimulates PMO activity up to approx. 54%, presumably through a raise of the intracellular NADH store. PMO also promotes cell growth at low substrate concentrations (0.1-1 microM). Native gel electrophoresis of iminobiotinylated and affinity purified plasma membrane extracts displays two diaphorase-positive bands, indicating that a homogeneous cell population may express several PMO activities at the plasma membrane.