Scalable Process for High-Yield Production of PfCyRPA Using Insect Cells for Inclusion in a Malaria Virosome-Based Vaccine Candidate.

Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort has been committed to the development of scalable cost-effective, robust, and high-yield PfCyRPA production processes. Despite insect cells being a suitable expression system due to their track record for protein production (including vaccine antigens), these are yet to be explored to produce this antigen. In this study, different insect cell lines, culture conditions (baculovirus infection strategy, supplementation schemes, culture temperature modulation), and purification strategies (affinity tags) were explored aiming to develop a scalable, high-yield, and high-quality PfCyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved PfCyRPA volumetric titers by 50% when added at the time of infection. In addition, from three different affinity tags (6x-His, 4x-His, and C-tag) evaluated, the 4x-His affinity tag was the one leading to the highest PfCyRPA purification recovery yields (61%) and production yield (26 mg/L vs. 21 mg/L and 13 mg/L for 6x-His and C-tag, respectively). Noteworthy, PfCyRPA expressed using High Five cells did not show differences in protein quality or stability when compared to its human HEK293 cell counterpart. When formulated in a lipid-based virosome nanoparticle, immunized rabbits developed functional anti-PfCyRPA antibodies that impeded the multiplication of P. falciparum in vitro. This work demonstrates the potential of using IC-BEVS as a qualified platform to produce functional recombinant PfCyRPA protein with the added benefit of being a non-human expression system with short bioprocessing times and high expression levels.

Bet v 1 contiguous overlapping peptides anchored to virosomes with TLR4 agonist enhance immunotherapy efficacy in mice.

BACKGROUND: Whereas sublingual allergen immunotherapy (AIT) is routinely performed without any adjuvant or delivery system, there is a strong scientific rationale to better target the allergen(s) to oral dendritic cells known to support regulatory immune responses by using appropriate presentation platforms. OBJECTIVE: To identify a safe presentation platform able to enhance allergen-specific tolerance induction. METHODS: Virosomes with membrane-integrated contiguous overlapping peptides (COPs) of Bet v 1 and TLR4 or TLR2/TLR7 agonists were assessed for induction of Bet v 1-specific IgG1, IgG2a and IgE antibodies, hypersensitivity reactions and body temperature drop following subcutaneous injection in naive CD-1 mice. The most promising candidate, Bet v 1 COPs anchored to virosomes with membrane-incorporated TLR4 agonist (Vir.A-Bet v 1 COPs), was further evaluated by the sublingual route in a therapeutic setting in BALB/c mice with birch pollen-induced allergic asthma. Airway hyperresponsiveness, pro-inflammatory cells in bronchoalveolar lavages and polarization of Th cells in the lungs and spleen were then assessed. RESULTS: Both types of adjuvanted virosomes coupled to Bet v 1 COPs triggered a boosted Th1 immunity. Given a more favourable safety profile, Vir.A-Bet v 1 COPs were further evaluated and shown to able to fully reverse asthma symptoms and lung inflammation in a sublingual therapeutic model of birch pollen allergy. CONCLUSIONS AND CLINICAL RELEVANCE: We report herein for the first time on the capacity of a novel and safe presentation platform, that is virosomes with membrane-integrated TLR4 agonist, to improve dramatically sublingual AIT efficacy in a murine model due to its intrinsic dual properties of targeting and stimulating to further promote anti-allergic immune responses. As such, our study paves the ground for further clinical development of this allergen presentation platform for patients suffering from respiratory allergies.

Vaccination with virosomally formulated recombinant CyRPA elicits protective antibodies against Plasmodium falciparum parasites in preclinical in vitro and in vivo models.

The Plasmodium falciparum (Pf) cysteine-rich protective antigen (PfCyRPA) has emerged as a promising blood-stage candidate antigen for inclusion into a broadly cross-reactive malaria vaccine. This highly conserved protein among various geographical strains plays a key role in the red blood cell invasion process by P. falciparum merozoites, and antibodies against PfCyRPA can efficiently prevent the entry of the malaria parasites into red blood cells. The aim of the present study was to develop a human-compatible formulation of the PfCyRPA vaccine candidate and confirming its activity in preclinical studies. Recombinant PfCyRPA expressed in HEK 293 cells was chemically coupled to phosphoethanolamine and then incorporated into the membrane of unadjuvanted influenza virosomes approved as antigen delivery system for humans. Laboratory animals were immunised with the virosome-based PfCyRPA vaccine to determine its immunogenic properties and in particular, its capacity to elicit parasite binding and growth-inhibitory antibodies. The vaccine elicited in mice and rabbits high titers of PfCyRPA-specific antibodies that bound to the blood-stage parasites. At a concentration of 10 mg/mL, purified total serum IgG from immunised rabbits inhibited parasite growth in vitro by about 80%. Furthermore, in a P. falciparum infection mouse model, passive transfer of 10 mg of purified total IgG from PfCyRPA vaccinated rabbits reduced the in vivo parasite load by 77%. Influenza virosomes thus represent a suitable antigen delivery system for the induction of protective antibodies against the recombinant PfCyRPA, designating it as a highly suitable component for inclusion into a multivalent and multi-stage virosomal malaria vaccine.

CD90+CD146+ identifies a pulmonary mesenchymal cell subtype with both immune modulatory and perivascular-like function in postnatal human lung.

Our understanding of mesenchymal cell subsets and their function in human lung affected by aging and in certain disease settings remains poorly described. We use a combination of flow cytometry, prospective cell-sorting strategies, confocal imaging, and modeling of microvessel formation using advanced microfluidic chip technology to characterize mesenchymal cell subtypes in human postnatal and adult lung. Tissue was obtained from patients undergoing elective surgery for congenital pulmonary airway malformations (CPAM) and other airway abnormalities including chronic obstructive pulmonary disease (COPD). In microscopically normal postnatal human lung, there was a fivefold higher mesenchymal compared with epithelial (EpCAM+) fraction, which diminished with age. The mesenchymal fraction composed of CD90+ and CD90+CD73+ cells was enriched in CXCL12 and platelet-derived growth factor receptor-α (PDGFRα) and located in close proximity to EpCAM+ cells in the alveolar region. Surprisingly, alveolar organoids generated from EpCAM+ cells supported by CD90+ subset were immature and displayed dysplastic features. In congenital lung lesions, cystic air spaces and dysplastic alveolar regions were marked with an underlying thick interstitium composed of CD90+ and CD90+PDGFRα+ cells. In postnatal lung, a subset of CD90+ cells coexpresses the pericyte marker CD146 and supports self-assembly of perfusable microvessels. CD90+CD146+ cells from COPD patients fail to support microvessel formation due to fibrinolysis. Targeting the plasmin-plasminogen system during microvessel self-assembly prevented fibrin gel degradation, but microvessels were narrower and excessive contraction blocked perfusion. These data provide important new information regarding the immunophenotypic identity of key mesenchymal lineages and their change in a diverse setting of congenital lung lesions and COPD.

Secretory Aspartyl Proteinases Cause Vaginitis and Can Mediate Vaginitis Caused by Candida albicans in Mice.

UNLABELLED: Vaginal inflammation (vaginitis) is the most common disease caused by the human-pathogenic fungus Candida albicans. Secretory aspartyl proteinases (Sap) are major virulence traits of C. albicans that have been suggested to play a role in vaginitis. To dissect the mechanisms by which Sap play this role, Sap2, a dominantly expressed member of the Sap family and a putative constituent of an anti-Candida vaccine, was used. Injection of full-length Sap2 into the mouse vagina caused local neutrophil influx and accumulation of the inflammasome-dependent interleukin-1ß (IL-1ß) but not of inflammasome-independent tumor necrosis factor alpha. Sap2 could be replaced by other Sap, while no inflammation was induced by the vaccine antigen, the N-terminal-truncated, enzymatically inactive tSap2. Anti-Sap2 antibodies, in particular Fab from a human combinatorial antibody library, inhibited or abolished the inflammatory response, provided the antibodies were able, like the Sap inhibitor Pepstatin A, to inhibit Sap enzyme activity. The same antibodies and Pepstatin A also inhibited neutrophil influx and cytokine production stimulated by C. albicans intravaginal injection, and a mutant strain lacking SAP1, SAP2, and SAP3 was unable to cause vaginal inflammation. Sap2 induced expression of activated caspase-1 in murine and human vaginal epithelial cells. Caspase-1 inhibition downregulated IL-1ß and IL-18 production by vaginal epithelial cells, and blockade of the IL-1ß receptor strongly reduced neutrophil influx. Overall, the data suggest that some Sap, particularly Sap2, are proinflammatory proteins in vivo and can mediate the inflammasome-dependent, acute inflammatory response of vaginal epithelial cells to C. albicans. These findings support the notion that vaccine-induced or passively administered anti-Sap antibodies could contribute to control vaginitis. IMPORTANCE: Candidal vaginitis is an acute inflammatory disease that affects many women of fertile age, with no definitive cure and, in its recurrent forms, causing true devastation of quality of life. Unraveling the fungal factors causing inflammation is important to be able to devise novel tools to fight the disease. In an experimental murine model, we have discovered that aspartyl proteinases, particularly Sap2, may cause the same inflammatory signs of vaginitis caused by the fungus and that anti-Sap antibodies and the protease inhibitor Pepstatin A almost equally inhibit Sap- and C. albicans-induced inflammation. Sap-induced vaginitis is an early event during vaginal infection, is uncoupled from fungal growth, and requires Sap and caspase-1 enzymatic activities to occur, suggesting that Sap or products of Sap activity activate an inflammasome sensor of epithelial cells. Our data support the notion that anti-Sap antibodies could help control the essence of candidal vaginitis, i.e., the inflammatory response.

Virosomes can enter cells by non-phagocytic mechanisms.

Phagocytosis of fine particles (1 microm) by macrophages is a ligand-receptor-mediated, actin-based process, whereas the entering of smaller particles (< or = 0.2 microm) in macrophages occurs also by other mechanisms. Virosomes with a diameter of 0.12-0.18 microm are widely used as carrier systems for drugs, vectors, and plasmids in cancer therapy or for vaccines. We investigated their interactions with airway cells, in particular penetration into monocyte-derived macrophages. The microscopic analysis of phagocytic cells incubated with virosomes and polystyrene particles showed that virosomes and particles penetrated cells even in the presence of cytochalasin D, a drug inhibiting actin-based phagocytosis. The charge of the virosomes and particles did not influence their penetration. Also, different inhibitors of endocytotic pathways did not prevent the particles and virosomes from penetrating into the cells. Additionally, to study the ability of virosomes to overcome the epithelial airway barrier, a triple cell co-culture model composed of epithelial cells, monocyte-derived macrophages and dendritic cells of the respiratory tract was used. We found virosomes and polystyrene particles in both populations of antigen-presenting cells, monocyte-derived macrophages, and dendritic cells, in the latter even if they were not directly exposed. In conclusion, virosomes are readily taken up by monocyte-derived macrophages, both by conventional phagocytosis and by actin-independent mechanisms. Further, they can penetrate the airway barrier and reach resident dendritic cells. Therefore, virosomes are promising vaccine candidates.

Identification of a synthetic peptide inducing cross-reactive antibodies binding to Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Hyalomma anatolicum anatolicum and Rhipicephalus appendiculatus BM86 homologues.

The BM86 antigen, originally identified in Rhipicephalus (Boophilus) microplus, is the basis of the only commercialized anti-tick vaccine. The long-term goal of our study is to improve BM86 based vaccines by induction of high levels of tick gut binding antibodies that are also cross-reactive with a range of BM86 homologues expressed in other important tick species. Here we have used a BD86 derived synthetic peptide, BD86-3, to raise a series of mouse monoclonal antibodies. One of these mAbs, named 12.1, recognized BM86 homologues in immuno-histochemical analyses in four out of five tick species including R. (B.) microplus, Rhipicephalus (Boophilus) decoloratus, Hyalomma anatolicum anatolicum and Rhipicephalus appendiculatus. Our results indicate that broadly cross-reactive tick gut binding antibodies can be induced after immunization with a synthetic peptide derived from the protein BD86.

Respiratory syncytial virus subunit vaccine based on a recombinant fusion protein expressed transiently in mammalian cells.

Although respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in infants and adults at risk, no RSV vaccine is currently available. In this report, efforts toward the generation of an RSV subunit vaccine using recombinant RSV fusion protein (rRSV-F) are described. The recombinant protein was produced by transient gene expression (TGE) in suspension-adapted human embryonic kidney cells (HEK-293E) in 4 L orbitally shaken bioreactors. It was then purified and formulated in immunostimulating reconstituted influenza virosomes (IRIVs). The candidate vaccine induced anti-RSV-F neutralizing antibodies in mice, and challenge studies in cotton rats are ongoing. If successful in preclinical and clinical trials, this will be the first recombinant subunit vaccine produced by large-scale TGE in mammalian cells.

Preclinical profiling of the immunogenicity of a two-component subunit malaria vaccine candidate based on virosome technology.

Presentation of synthetic peptides on immunopotentiating reconstituted influenza virosomes is a promising technology for subunit vaccine development. An optimized virosomally delivered peptide representing 5 NPNA repeats of P. falciparum circumsporozoite protein is highly immunogenic in mice. Antibodies against this peptide (UK-39) inhibit sporozoite invasion of human hepatocytes. A second peptide (AMA49-C1) based on domain III of apical membrane antigen 1, induces antibodies that inhibit blood-stage parasite growth in vitro. Here we show a detailed pre-clinical profiling of these virosomally formulated peptides alone and in combination in mice and rabbits. Two immunizations with virosomally formulated UK-39 or AMA49-C1 were enough to elicit high titers of parasite cross-reactive antibodies in both species. A low dose of 10 microg UK-39 was enough to induce maximal titers in rabbits. Higher doses of peptide did not increase antibody titers. In contrast, AMA49-C1 induced higher antibody titers with 25 and 50 microg peptide. Combination of UK-39 and AMA49- C1 on separate virosomes did not have any negative effect on anti-peptide antibody titers in mice or rabbits. No MHC restriction was observed in the development of humoral responses in outbred rabbits with different immunogenetic backgrounds. All vaccine formulations were safe in toxicity studies in rabbits and rats. Taken together, low amounts of synthetic peptides delivered on virosomes induced high antibody titers in mice and rabbits. Moreover, different peptides could be combined without interfering with individual anti-peptide responses, augmenting the value of this system for the development of a multivalent malaria vaccine.

Anti-angiogenetic effects of immune-reconstituted influenza virosomes assembled with parathyroid hormone-related protein derived peptide vaccine.

BACKGROUND: C-IRIV/PTR-4 is a novel anticancer vaccine construct composed of immune-reconstituted influenza virosomes (IRIV) assembled with the PTH-rP derived peptide (PTR)-4, a synthetic CTL epitope with HLA-A(*)02.01 amino acid binding motifs. This peptide is able to generate a human PTH-rP specific CTL response with anti-tumor activity in vitro and in mice. MATERIALS AND METHODS: We have investigated the immunological and preventive anti-tumor activity of C-IRIV/PTR-4 compared with the soluble PTR-4 peptide, in HHD mice inoculated with autologous PTH-rP+ tumor cells. RESULTS: Peptide vaccination with either a soluble and an IRIV formulation showed similar immunological activity and the ability to purge the tumor tissue of tumor cell clones able to produce the target antigen (PTR-rP). The most efficient protection from tumor growth was however observed in animals vaccinated with C-IRIV/PTR-4 in which an additional IRIV related anti-angiogenetic effect was detected in the tumor tissue. CONCLUSIONS: These results confirm the immunological activity of PTR-4 vaccination and suggest a more efficacious therapeutic potential of C-IRIV/PTR-4 against bone metastases and malignancies like breast, prostate and lung which very often over-express PTH-rP.

A new and versatile virosomal antigen delivery system to induce cellular and humoral immune responses.

The purpose of a vaccine is the induction of effective cellular and/or humoral immune responses against antigens. Because defined antigens are often poor immunogens when administered alone, an adjuvant is required to potentiate the immune response. Most of these adjuvants are designed to induce humoral immune responses, including immunopotentiating reconstituted influenza virosomes (IRIVs). IRIVs are one of the few adjuvants currently licensed for human use with the advantage of an excellent safety profile. To induce a potent cytotoxic T lymphocyte (CTL) immune response CTL epitopes have to be encapsulated into IRIVs. However, the existing encapsulation methods are inefficient or rather laborious. We have developed and characterised a new generation of influenza virosomes (TIRIVs) that induced both, strong CTL and antibody responses against specific antigens of choice. In addition, these virosomes were stabilised and offer the possibility of lyophilisation while retaining all their structural, functional and immunogenic properties after reconstitution. TIRIVs induce strong cellular and humoral immune responses and are a versatile and efficient carrier system with adjuvant properties for a variety of antigens. TIRIVs are not only stabilised but also allow easy formulation of new and/or labile T cell and B cell antigens. Considering their immunogenic properties, their flexibility and their superior storage characteristics TIRIVs provide a versatile technology platform for any vaccination strategy.

Virosome-mediated delivery of tumor antigen to plasmacytoid dendritic cells.

Cytotoxic T lymphocytes (CTL) are crucial in viral clearance and tumor growth control. Thus the induction of CTL activity is an important aim in vaccine development. We investigate an innovative delivery system for peptide transfer to the MHC class I processing pathway of APC with the aim to trigger CTL in the context of an antitumoral response. The strategy relies on a novel antigen delivery system termed "chimeric immunopotentiating reconstituted influenza virosomes" (CIRIV) targeting plasmacytoid dendritic cells (PDC). By using virosomes containing encapsulated Melan-A peptide and a PDC line developed in our laboratory, we evaluated the response of Melan-A specific T cells. Virosomes have the capacity to bind PDC and are endocyted within vesicles in the cytosol. This endocytosis is inhibited by neuraminidase, suggesting that it is mediated by sialic acid present on cell surface. Furthermore, PDC loaded with Melan-A virosomes can induce a Melan-A specific T cell activation. Interestingly, they activate T cells with a better efficiency than PDC loaded with a free peptide and when PDC where previously activated by a TLR ligand. These results indicate that virosomes could be a suitable delivery system for tumor peptide in immunotherapy of cancer.

Advanced liposomal vectors as cancer vaccines in melanoma immunotherapy.

Malignant tumors represent a major source of disability and account for more than one of five deaths in Western countries. Among the different cancers, melanoma harbors two distinctive features. First, its has long been recognized as an immunogenic tumor, and second, an unprecedented rise in incidence is currently observed, in face of few therapeutic options. Thus, melanoma represent an ideal target for a cancer immunotherapy program. To date, a number of immunodominant epitopes from tumor associated antigens (TAA) are used as cancer vaccines in clinical trials, in spite of an acknowledged rapid degradation in vivo and low immunogenicity. However, most of the immunotherapy trials reported so far do not achieve consistent clinical results. Hence, there is an urgent need for the development of a carrier system and strong adjuvants suitable for a TAA-based cancer immunotherapy. Liposomes and their further development as virosomes with added adjuvancy may address both these issues. We report here our experience in the tailoring of dedicated advanced liposomal vectors that were developed in the context of an upcoming immunotherapy clinical trial for melanoma.

Efficient induction of tumoricidal cytotoxic T lymphocytes by HLA-A0201 restricted, melanoma associated, L(27)Melan-A/MART-1(26-35) peptide encapsulated into virosomes in vitro.

Cancer immunotherapy requires the induction of HLA class I restricted cytotoxic T lymphocytes (CTL) specific for tumor associated antigens (TAA). While a number of TAA have been identified, there is an urgent need for the development of adjuvants capable of stimulating CTL responsiveness. Previously, we reported the capacity of immunopotentiating reconstituted influenza virosomes (IRIV) to enhance CTL responses specific for synthetic peptides simultaneously added to cultures in soluble form. This effect was based on IRIV mediated activation of CD4(+) T cells. Here we investigated the "in vitro" immunogenicity of a novel virosome formulation coupling in a single reagent the adjuvant power of IRIV to the capacity of liposomes to efficiently encapsulate synthetic peptides. As a model epitope we chose L(27)Melan-A/Mart-1(26-35) HLA-A0201 restricted peptide from a melanoma-associated antigen widely used in tumor immunotherapy. The reagent thus developed induced the proliferation of CD4(+) T cells characterized by a T helper 1 cytokine profile and CXCR3 expression. Most importantly, it significantly enhanced the generation of L(27)Melan-A/Mart-1(26-35) specific CTL, as compared to soluble peptides, in particular at low nominal epitope concentrations (<1 microg/ml). These effector cells were able to efficiently kill HBL melanoma cells expressing Melan-A/MART-1 and HLA-A0201. The adjuvant effects observed were also detectable in the absence of CD4(+) T cells. Taken together our results suggest that this highly immunogenic antigenic formulation might qualify for clinical use in active, antigen-specific, melanoma immunotherapy.

Peptide-loaded chimeric influenza virosomes for efficient in vivo induction of cytotoxic T cells.

Virus-specific CD8(+) T cells are thought to play an important role in resolving acute hepatitis C virus (HCV) infection as viral clearance has been associated with a strong and sustained CD8(+) T cell response. During the chronic state of HCV infection virus-specific T cells have a low frequency and a reduced responsiveness. Based on this, a therapeutic vaccine increasing the frequency of specific T cells is a promising alternative for the treatment of chronic HCV infection. We improved an existing vaccine platform based on immunopotentiating reconstituted influenza virosomes (IRIVs) for efficient delivery of peptide epitopes to the MHC class I antigen presentation pathway. IRIVs are proteoliposomes composed of phospholipids and influenza surface glycoproteins. Due to their fusogenic activity, IRIVs are able to deliver encapsulated macromolecules, e.g. peptides to immunocompetent cells. We developed a novel method based on chimeric virosomes [chimeric immunopotentiating reconstituted influenza virosomes (CIRIVs)] combining the high peptide-encapsulation capacity of liposomes and the fusion activity of virosomes. This new approach resulted in a 30-fold increase of the amount of incorporated soluble peptide compared with current preparation methods. To study the immunogenicity of chimeric virosomes HLA-A2.1 transgenic mice were immunized with CIRIVs containing the HCV Core132 peptide. Core132-CIRIVs efficiently induced specific cytotoxic and IFNgamma-producing T cells already with low peptide doses. Vaccine formulations, which include combinations of different HCV-derived CTL epitopes could be used to induce not only a strong but also a multi-specific CTL response, making them potential candidates for therapeutic and maybe prophylactic T cell vaccines in humans.

Gene expression analysis using single molecule detection.

Recent developments of single molecule detection techniques and in particular the introduction of fluorescence correlation spectroscopy (FCS) led to a number of important applications in biological research. We present a unique approach for the gene expression analysis using dual-color cross-correlation. The expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target gene. The counting of the dual-labeled molecules within the solution allows the quantification of the expressed gene copies in absolute numbers. As detection and analysis by FCS can be performed at the level of single molecules, there is no need for any type of amplification. We describe the gene expression assay and present data demonstrating the capacity of this novel technology. In order to prove the gene specificity, we performed experiments with gene-depleted total cDNA. The biological application was demonstrated by quantifying selected high, medium and low abundant genes in cDNA prepared from HL-60 cells.

Detection of promoter activity by flow cytometric analysis of GFP reporter expression.

Low efficiency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially difficult to efficiently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quantified by flow cytometry. In this system, green fluorescent protein (GFP) reporter constructs are co-transfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection efficiency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color fluorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low efficiency.