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.

Effect of additional warning sounds on pedestrians' detection of electric vehicles: An ecological approach.

Virtually silent electric vehicles (EVs) may pose a risk for pedestrians. This paper describes two studies that were conducted to assess the influence of different types of external sounds on EV detectability. In the first study, blindfolded participants had to detect an approaching EV with either no warning sounds at all or one of three types of sound we tested. In the second study, designed to replicate the results of the first one in an ecological setting, the EV was driven along a road and the experimenters counted the number of people who turned their heads in its direction. Results of the first study showed that adding external sounds improve EV detection, and modulating the frequency and increasing the pitch of these sounds makes them more effective. This improvement was confirmed in the ecological context. Consequently, pitch variation and frequency modulation should both be taken into account in future AVAS design.

Gene transfer of interleukin-18-binding protein attenuates cardiac allograft rejection.

Interleukin (IL) 18 is a potent pro-inflammatory Th1 cytokine that exerts pleiotropic effector functions in both innate and acquired immune responses. Increased IL-18 production during acute rejection has been reported in experimental heart transplantation models and in kidney transplant recipients. IL-18-binding protein (IL-18BP) binds IL-18 with high affinity and neutralizes its biologic activity. We have analyzed the efficacy of an adenoviral vector expressing an IL-18BP-Ig fusion protein in a rat model of heart transplantation. IL-18BP-Ig gene transfer into Fisher (F344) rat donor hearts resulted in prolonged graft survival in Lewis recipients (15.8 +/- 1.4 days vs. 10.3 +/- 2.5 and 10.1 +/- 2.1 days with control virus and buffer solution alone, respectively; P < 0.001). Immunohistochemical analysis revealed decreased intra-graft infiltrates of monocytes/macrophages, CD4(+), CD8alpha(+) and T-cell receptor alphabeta(+) cells after IL-18BP-Ig versus mock gene transfer (P < 0.05). Real-time reverse transcriptase polymerase chain reaction analysis showed decreased cytokine transcripts for the RANTES chemokine and transforming growth factor-beta after IL-18BP-Ig gene transfer (P < 0.05). IL-18BP-Ig gene transfer attenuates inflammatory cell infiltrates and prolongs cardiac allograft survival in rats. These results suggest a contributory role for IL-18 in acute rejection. Further studies aiming at defining the therapeutic potential of IL-18BP are warranted.

Gene transfer of a soluble IL-1 type 2 receptor-Ig fusion protein improves cardiac allograft survival in rats.

OBJECTIVE: Interleukin-1 (IL-1) mediates ischemia-reperfusion injury and graft inflammation after heart transplantation. IL-1 affects target cells through two distinct types of transmembrane receptors, type-1 receptor (IL-1R1), which transduces the signal, and the non-signaling type-2 receptor (IL-1R2), which acts as a ligand sink that subtracts IL-1beta from IL-1R1. We analyzed the efficacy of adenovirus (Ad)-mediated gene transfer of a soluble IL-1R2-Ig fusion protein in delaying cardiac allograft rejection and the mechanisms underlying the protective effect. METHODS: IL-1 inhibition by IL-1R2-Ig was tested using an in vitro functional assay whereby endothelial cells preincubated with AdIL-1R2-Ig or control virus were stimulated with recombinant IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and urokinase-type plasminogen activator (u-PA) induction was measured by zymography. AdIL-1R2-Ig was delivered to F344 rat donor hearts ex vivo, which were placed in the abdominal position in LEW hosts. Intragraft inflammatory cell infiltrates and proinflammatory cytokine expression were analyzed by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: IL-1R2-Ig specifically inhibited IL-1beta-induced u-PA responses in vitro. IL-1R2-Ig gene transfer reduced intragraft monocytes/macrophages and CD4(+) cell infiltrates (p<0.05), TNF-alpha and transforming growth factor-beta (TGF-beta) expression (p<0.05), and prolonged graft survival (15.6+/-5.7 vs 10.3+/-2.5 days with control vector and 10.1+/-2.1 days with buffer alone; p<0.01). AdIL-1R2-Ig combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone (19.4+/-3.0 vs 15.9+/-1.8 days; p<0.05). CONCLUSIONS: Soluble IL-1 type-2 receptor gene transfer attenuates cardiac allograft rejection in a rat model. IL-1 inhibition may be useful as an adjuvant therapy in heart transplantation.

Gene transfer of soluble interleukin-17 receptor prolongs cardiac allograft survival in a rat model.

OBJECTIVE: Interleukin-17 (IL-17), a potent proinflammatory cytokine, has been implicated in allograft rejection. We analyzed the efficacy of an adenoviral vector expressing an IL-17 inhibitor in delaying acute allograft rejection in a rat model of heart transplantation, and the biological mechanisms underlying the protective effect. METHODS: We constructed an adenoviral vector expressing a soluble IL-17 receptor-immunoglobulin (IL-17R-Ig) fusion protein. IL-17R-Ig activity was assessed by inhibition of IL-17-induced IL-6 release in HeLa cells preincubated with the vector. Intracoronary vector administration was performed in F344 donor hearts that were placed as vascularized grafts into Lewis hosts. Inflammatory cells infiltrating the graft were analyzed by immunohistology. Cytokine transcripts in the graft were determined by real-time RT-PCR. RESULTS: IL-17R-Ig gene transfer resulted in prolonged allograft survival (16.1+/-3.1 days vs 10.3+/-2.5 days with control virus and 10.1+/-2.1 days with virus dilution buffer alone; p<0.001). IL-17R-Ig gene transfer reduced inflammatory cell infiltrates, especially monocytes/macrophages and CD4+ T cells (p<0.05). It also reduced intragraft cytokine transcripts for interferon-gamma and transforming growth factor-beta (p<0.05) and, to a lesser extent, IL-1beta and tumor necrosis factor-alpha (p=0.083). CONCLUSIONS: Local expression of soluble IL-17 receptor-immunoglobulin attenuates T helper type 1 (Th1) cytokine responses and leukocyte infiltration in rat cardiac allografts, thereby mediating prolonged graft survival. Intragraft IL-17 inhibition may be useful as an adjuvant therapy to systemic immunosuppression in heart transplantation.

Helper-dependent adenovirus vectors devoid of all viral genes cause less myocardial inflammation compared with first-generation adenovirus vectors.

BACKGROUND: First-generation, E1-deleted (deltaE1) adenovirus vectors currently used in cardiovascular gene therapy trials are limited by tissue inflammation, mainly due to immune responses to viral gene products. Recently, helper-dependent (HD; also referred to as "gutless") adenovirus vectors devoid of all viral coding sequences have been shown to cause low inflammation when injected intravenously or into skeletal muscles. However, HD vectors have not been evaluated in cardiovascular tissues. METHODS AND RESULTS: HD and deltaE1 vectors containing a cytomegalovirus-driven expression cassette for the green fluorescent protein (GFP) gene were administered intramyocardially to adult rats (n = 54). GFP expression was measured by ELISA at varying time intervals after gene transfer. HD and deltaE1 vectors were equally efficient at transducing the myocardium. Tissue inflammation was assessed by immunostaining for leukocytes and quantitative real-time RT-PCR for cytokine mRNA expression. Monocyte/macrophages, CD4(+) and CD8(+) lymphocytes infiltrating the myocardium were less abundant with HD than deltaE1 vectors. Transcripts levels for pro-inflammatory cytokines such as IL-1beta, tumor necrosis factor-alpha, and RANTES were decreased with HD vectors. However, both vectors were associated with a decline in GFP expression over time, although low-level expression was occasionally detectable 10 weeks after HD vector administration. The two vectors transduced endothelial cells in rat arteries (n = 11) with comparable efficiencies. Vascular GFP expression was not detectable at 10 weeks. CONCLUSIONS: HD vectors are as efficient as deltaE1 vectors at transducing the myocardium and vascular endothelium, while causing less myocardial inflammation. Thus, HD vectors may be superior to earlier-generation adenovirus vectors for cardiovascular gene therapy applications.

Gene transfer of cytoprotective and immunomodulatory molecules for prevention of cardiac allograft rejection.

Current treatments of heart transplantation are limited by incomplete effectiveness, significant toxicity, and failure to prevent chronic rejection. Genetic manipulation of the donor heart at the time of removal offers the unique opportunity to produce a therapeutic molecule within the graft itself, while minimizing systemic effects. Cytoprotective approaches including gene transfer of heme oxygenase (HO)-1, endothelial nitric oxide synthase, and antisense oligodeoxynucleotides specific for nuclear factor (NF)-kappa B or intercellular adhesion molecule (ICAM)-1 reduced ischaemia-reperfusion injury and delayed cardiac allograft rejection in small animals. Exogenous overexpression of immunomodulatory cytokines such as interleukin (IL)-4, IL-10 and transforming growth factor-beta, as well as gene transfer of inhibitors of pro-inflammatory cytokines also delayed graft rejection. Gene transfer-based blockade of T-cell costimulatory activation with CTLA4-Ig or CD40-Ig resulted in long-lasting graft survival and donor-specific unresponsiveness, as manifested by acceptance of a second graft from the original donor strain but rejection of third-party grafts. Similar results were obtained with donor major histocompatibility complex class I gene transfer into bone marrow cells. Gene therapy approaches to chronic rejection included gene transfer of HO-1, soluble Fas, tissue plasminogen activator and antisense oligodeoxynucleotides specific for the anti-apoptotic mediator Bcl-x or the E2F transcription factor. Despite major experimental advances, however, gene therapy for heart transplantation has not entered the clinical arena yet. Fundamental questions regarding the most suitable vector, the best gene, and safety issues remain unanswered. Well-controlled studies that compare gene therapy with established treatments in non-human primates are needed before clinical trials can be started.

Multiply attenuated, self-inactivating lentiviral vectors efficiently deliver and express genes for extended periods of time in adult rat cardiomyocytes in vivo.

BACKGROUND: Among retroviral vectors, lentiviral vectors are unique in that they transduce genes into both dividing and nondividing cells. However, their ability to provide sustained myocardial transgene expression has not been evaluated. METHODS AND RESULTS: Multiply attenuated, self-inactivating lentivectors based on human immunodeficiency virus-1 contained the enhanced green fluorescent protein (EGFP) gene under the transcriptional control of either the cytomegalovirus (CMV) immediate-early enhancer/promoter, the elongation factor-1alpha (EF-1alpha) promoter, or the phosphoglycerate-kinase (PGK) promoter. Lentivectors transduced adult rat cardiomyocytes in a dose-dependent manner (transduction rates, >90%; multiplicity of infection, approximately 5). The CMV promoter achieved higher EGFP expression levels than the EF-1alpha and PGK promoters. Insertion of the central polypurine tract pol sequence improved gene transfer efficiency by approximately 2-fold. In vivo gene transfer kinetics was studied by measuring the copy number of integrated lentivirus DNA and EGFP concentrations in cardiac extracts by real-time polymerase chain reaction and ELISA, respectively. With CMV promoter-containing lentivectors, vector DNA peaked at day 3, declined by approximately 4-fold at day 14, but then remained stable up to week 10. Similarly, EGFP expression peaked at day 7, decreased by approximately 7-fold at day 14, but was essentially stable thereafter. In contrast, vector DNA and EGFP expression declined rapidly with EF-1alpha promoter-containing lentivectors. Peak EGFP expression with titer-matched adenovectors was approximately 35% higher than with CMV lentivectors but was lost rapidly over time. CONCLUSIONS: Lentivectors efficiently transduce and express genes for extended periods of time in cardiomyocytes in vivo. Lentivectors provide a useful tool for studying myocardial biology and a potential system for gene heart therapy.