Efficient stabilization of therapeutic hepatitis B vaccine components by amino-acid formulation maintains its potential to break immune tolerance.

Background & Aims: Induction of potent, HBV-specific immune responses is crucial to control and finally cure HBV. The therapeutic hepatitis B vaccine TherVacB combines protein priming with a Modified Vaccinia virus Ankara (MVA)-vector boost to break immune tolerance in chronic HBV infection. Particulate protein and vector vaccine components, however, require a constant cooling chain for storage and transport, posing logistic and financial challenges to vaccine applications. We aimed to identify an optimal formulation to maintain stability and immunogenicity of the protein and vector components of the vaccine using a systematic approach. Methods: We used stabilizing amino acid (SAA)-based formulations to stabilize HBsAg and HBV core particles (HBcAg), and the MVA-vector. We then investigated the effect of lyophilization and short- and long-term high-temperature storage on their integrity. Immunogenicity and safety of the formulated vaccine was validated in HBV-naïve and adeno-associated virus (AAV)-HBV-infected mice. Results: In vitro analysis proved the vaccine's stability against thermal stress during lyophilization and the long-term stability of SAA-formulated HBsAg, HBcAg and MVA during thermal stress at 40 °C for 3 months and at 25 °C for 12 months. Vaccination of HBV-naïve and AAV-HBV-infected mice demonstrated that the stabilized vaccine was well tolerated and able to brake immune tolerance established in AAV-HBV mice as efficiently as vaccine components constantly stored at 4 °C/-80 °C. Even after long-term exposure to elevated temperatures, stabilized TherVacB induced high titre HBV-specific antibodies and strong CD8+ T-cell responses, resulting in anti-HBs seroconversion and strong suppression of the virus in HBV-replicating mice. Conclusion: SAA-formulation resulted in highly functional and thermostable HBsAg, HBcAg and MVA vaccine components. This will facilitate global vaccine application without the need for cooling chains and is important for the development of prophylactic as well as therapeutic vaccines supporting vaccination campaigns worldwide. Impact and implications: Therapeutic vaccination is a promising therapeutic option for chronic hepatitis B that may enable its cure. However, its application requires functional cooling chains during transport and storage that can hardly be guaranteed in many countries with high demand. In this study, the authors developed thermostable vaccine components that are well tolerated and that induce immune responses and control the virus in preclinical mouse models, even after long-term exposure to high surrounding temperatures. This will lower costs and ease application of a therapeutic vaccine and thus be beneficial for the many people affected by hepatitis B around the world.

Algorithm-Based Liquid Formulation Development Including a DoE Concept Predicts Long-Term Viral Vector Stability.

Specifically tailored amino acid-based formulations were previously shown to have a high potential to avoid stress-mediated degradation of complex molecules such as monoclonal antibodies and viral vectors. By using adenovirus 5 (Ad5) as a model, we studied whether such formulations may also efficiently protect viral vectors in thermal stress experiments and during long-term liquid storage. Algorithm-based amino acid preselection using an excipient database and subsequent application of design of experiments (DoE) in combination with a 37°C challenging model enabled the prediction of long-term storage stability of Ad5. By statistical analysis of the Ad5 infectivity, amino acids with significant influence on Ad5 stability were detected after 2 and 3 weeks of liquid storage at 37°C. Ad5 formulations comprising positively selected amino acids did not reveal any loss of infectivity after 24 months in liquid storage at 5°C. By contrast, a 2 log reduction after 3 months and complete loss of infectivity after 18 months was observed with a standard viral vector formulation. By an optimization round, we designed a simple and well-balanced formulation avoiding MgCl2, previously considered essential in Ad5 formulations. This work demonstrates the efficacy of an algorithm-based development approach in the formulation development for viral vectors.

Bioartificial Therapy of Sepsis: Changes of Norepinephrine-Dosage in Patients and Influence on Dynamic and Cell Based Liver Tests during Extracorporeal Treatments.

Purpose. Granulocyte transfusions have been used to treat immune cell dysfunction in sepsis. A granulocyte bioreactor for the extracorporeal treatment of sepsis was tested in a prospective clinical study focusing on the dosage of norepinephrine in patients and influence on dynamic and cell based liver tests during extracorporeal therapies. Methods and Patients. Ten patients with severe sepsis were treated twice within 72 h with the system containing granulocytes from healthy donors. Survival, physiologic parameters, extended hemodynamic measurement, and the indocyanine green plasma disappearance rate (PDR) were monitored. Plasma of patients before and after extracorporeal treatments were tested with a cell based biosensor for analysis of hepatotoxicity. Results. The observed mortality rate was 50% during stay in hospital. During the treatments, the norepinephrine-dosage could be significantly reduced while mean arterial pressure was stable. In the cell based analysis of hepatotoxicity, the viability and function of sensor-cells increased significantly during extracorporeal treatment in all patients and the PDR-values increased significantly between day 1 and day 7 only in survivors. Conclusion. The extracorporeal treatment with donor granulocytes showed promising effects on dosage of norepinephrine in patients, liver cell function, and viability in a cell based biosensor. Further studies with this approach are encouraged.

Impaired cell functions of hepatocytes incubated with plasma of septic patients.

OBJECTIVE AND DESIGN: The development of liver failure is a major problem in septic patients. In this prospective clinical experimental study the hepatotoxicity of plasma from septic and non-septic patients was tested. METHODS AND SUBJECTS: The basic test components consist of human liver cells (HepG2/C3A) used in a standardized microtiter plate assay. After incubation with patient's plasma viability of cells (XTT-test), the cytochrome 1A2 activity and synthesis of micro albumin were measured. Subjects (28) enrolled comprise the septic shock group (SSG, n=10), the non-septic group (NSG, n=5) and the healthy volunteers group (HVG, n=13). RESULTS: The 28-day mortality was 30% in the SSG. The APACHE II-, SOFA-, and SAPS-scores and the values of bilirubin and prothrombin time as INR were significantly higher in the SSG than in the NSG. The cytochrome 1A2 activity and the release of albumin were significantly reduced in HepG2/C3A cells incubated with plasma of the SSG (p<0.05). The cytochrome 1A2 activities were higher in survivors compared to non-survivors at the time point 0 and were increasing in survivors and decreasing in non-survivors within 54 h in the SSG. In the SSG there was a significant decrease in IL-10 and IL-8 between inclusion and 54 h. Values of IL-6, TNF alpha and IL-10 were significantly lower in the NSG compared with the values of the SSG at inclusion and after 54 h. CONCLUSION: The plasma of patients with septic shock impaired cellular functions of HepG2/C3A cells.

Selection of proangiogenic ascorbate derivatives and their exploitation in a novel drug-releasing system for wound healing.

The pathophysiology leading to delayed wound healing is complex and efficient therapeutic approaches for accelerated wound healing currently do not exist. We developed a novel drug-eluting platform for the potential use in wound dressings. Here, we report on the potential of eluting ascorbic acid-2-phosphate (ASC-2P), a highly stable variant of ascorbic acid, to induce angiogenesis and to promote collagen synthesis by fibroblasts. The drug-eluting platform device (DEPD) consists of biocompatible polymeric layers comprising polyethylene terephtalate, polyvinyl alcohol (PVA), and polyurethane with PVA as the solvent for ASC-2P. The angiogenic potential of ASC-2P was evaluated in the endothelial cell tube formation assay (TFA) and in the chorion allantoic membrane (CAM) model. Collagen synthesis by ASC-2P-stimulated fibroblasts was determined by Sirius Red staining. ASC-2P significantly induced angiogenesis in five independent TFA and CAM assays and induced collagen synthesis in two different fibroblast cell lines. The eluting kinetics of ASC-2P was determined by the ultraviolet NanoDrop method and the functional 2,2'-Azinobis-(3-ethylbenzthiazolin-6-sulfonic acid) method. Eluting profiles showed a continuous release in the range of biologically effective concentrations >10 days. This is the first report showing the proangiogenic- and collagen-promoting features of ASC-2P. DEPD loaded with ASC-2P ought to be further evaluated as wound dressings or as supplementary pads for topical treatment of delayed wound healing in preclinical studies.

Stimulation of Fas signaling down-regulates activity of neutrophils from major trauma patients with SIRS.

Posttrauma apoptosis resistance of neutrophils (PMN) is related to overshooting immune responses, systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF). Recently, we have shown that the apoptosis resistance in circulating PMN from severely injured patients which is known to be mediated by high serum levels of pro-inflammatory cytokines can be overcome by the activation of Fas death receptor. Here, we aimed to study whether stimulation of surface Fas leads to the inactivation of hyperactivated PMN from critically ill patients with SIRS. PMN from 23 multiple trauma patients (mean injury severity score (ISS) 34±1.9) were isolated at day 1 after admission to the trauma center. PMN from 17 volunteer blood donors served as controls. Neutrophil activity has been determined after ex vivo short (1 h) and long-term (4 h) stimulation of freshly isolated PMN with immobilized agonistic anti-Fas antibodies. We found neutrophil chemotactic migration in response to IL-8, phagocytosis and oxidative burst to be significantly inhibited in control cells already after short-term (1 h) Fas stimulation. In contrast, inactivation of trauma PMN by agonistic anti-Fas antibodies was found to be efficient only after long-term (4 h) incubation of cells with agonistic antibodies. Thus, in trauma PMN down-regulation of neutrophil activity seems to be delayed when compared to cells isolated from healthy controls, suggesting impaired susceptibility for Fas stimulation in these cells. Interestingly, whereas Fas-mediated inhibition of phagocytosis and oxidative burst could be prevented by the broad range caspase inhibitor t-butoxycarbonyl-aspartyl(O-methyl)-fluoromethyl ketone (BocD-fmk), the chemotactic activity in response to IL-8 was unaffected. In conclusion, we demonstrate that stimulation of neutrophil Fas does not only initiate apoptosis but also induces inhibition of neutrophil functions, partially by non-apoptotic signaling.

Neutrophil-derived circulating free DNA (cf-DNA/NETs), a potential prognostic marker for mortality in patients with severe burn injury.

The predictive value of circulating free DNA/neutrophil extracellular traps (cf-DNA/NETs) has recently been shown in patients with major trauma for sepsis, multiple organ failure, and mortality. Here we report on the predictive potential of cf-DNA/NETs for mortality in patients with severe burn injury. In a prospective study 32 patients with severe burn injury were included. Blood samples were sequentially obtained on day 1, 3, 5, and 7 after admission. cf-DNA/NETs was directly quantified from plasma by means of rapid fluorescence assay. Time kinetics of cf-DNA/NETs were correlated with clinical data, C-reactive protein (CRP), procalcitonin (PCT), and interleukin (IL)-6. Furthermore sensitivity, specificity, and positive and negative predictive value, as well as receiver operation characteristic (ROC) curves were calculated. Seven patients died within the first month after burn injury. cf-DNA/NETs values from these patients were significantly increased already on day 1 and 3 after admission compared with patients who survived (p < 0.01). In contrast, PCT levels of nonsurvivors were significantly elevated on day 3 and 5 (p < 0.01), while CRP and IL-6 did not show any significant difference between survivors and nonsurvivors. At a cutoff of 255 ng/ml, cf-DNA/NETs had sensitivity of 0.8 and specificity of 0.74. ROC revealed largest areas under the curve (AUC) for cf-DNA/NETs on day 1 (0.851) and 3 (0.883) after admission. For all values between day 1 and 7, AUC was 0.815. cf-DNA/NETs seems to be a rapid, valuable marker for prediction of mortality in burn patients. A larger confirmation trial ought to be carried out.

Safety evaluation for a cell-based immune support system in an ex vivo rat model of gram-negative sepsis.

Granulocyte dysfunction is a central component of immunodeficiency in septic patients. Granulocyte transfusions appear to be pathophysiologically useful; however, they cause unwanted side-effects in the lungs and other organs. This study evaluates the safety of an extracorporeal immune support system with granulocytic cells in a rat model of Gram-negative sepsis. Three groups of male CD rats received either saline (control group, I), a dose of Escherichia coli O7:K1 lethal to 90% of the animals (LD90) (septic group, II), or an LD90 dose of E. coli that was incubated with the human promyelocytic leukemia cell line (HL-60) (differentiated into the granulocytic direction) for 20 min prior to infusion (second septic group, III). The animals were observed for seven days. Pre-treatment with HL-60 cells resulted in no adverse effects in the group III animals. Significantly lower bacterial counts and endotoxin levels in the plasma were detected after 24 h as compared to group II (P < 0.05). Group III animals had better weight gain and more stable hemodynamics than group II animals (P < 0.01). Seven day survival was 0/8 in group II, 6/8 in group III, and 8/9 in group I (log-rank test: II-III: P < 0.001). The data suggest that extracorporeal use of granulocytes allows the therapeutic use of these cells while avoiding unwanted effects resulting from direct contact to internal organs.

Mcl-1-mediated impairment of the intrinsic apoptosis pathway in circulating neutrophils from critically ill patients can be overcome by Fas stimulation.

The systemic inflammatory response syndrome and subsequent organ failure are mainly driven by activated neutrophils with prolonged life span, which is believed to be due to apoptosis resistance. However, detailed underlying mechanisms leading to neutrophil apoptosis resistance are largely unknown, and possible therapeutic options to overcome this resistance do not exist. Here we report that activated neutrophils from severely injured patients exhibit cell death resistance due to impaired activation of the intrinsic apoptosis pathway, as evidenced by limited staurosporine-induced mitochondrial membrane depolarization and decreased caspase-9 activity. Moreover, we found that these neutrophils express high levels of antiapoptotic Mcl-1 and low levels of proapoptotic Bax protein. Mcl-1 up-regulation was dependent on elevated concentrations of GM-CSF in patient serum. Accordingly, increased Mcl-1 protein stability and GM-CSF serum concentrations were shown to correlate with staurosporine-induced apoptosis resistance. However, cross-linking of neutrophil Fas by immobilized agonistic anti-Fas IgM resulted in caspase-dependent mitochondrial membrane depolarization and apoptosis induction. In conclusion, the observed impairment of the intrinsic pathway and the resulting apoptosis resistance may be overcome by immobilized agonistic anti-Fas IgM. Targeting of neutrophil Fas by immobilized agonistic effector molecules may represent a new therapeutic tool to limit neutrophil hyperactivation and its sequelae in patients with severe immune disorders.

Diagnostic accuracy of neutrophil-derived circulating free DNA (cf-DNA/NETs) for septic arthritis.

The release of "neutrophil extracellular traps" (NETs) has been identified as a novel immune response in innate immunity. NETs are composed of neutrophil-derived circulating free DNA (cf-DNA) and neutrophil cytoplasm-derived proteins such as proteases. In this study, we analyzed the putative diagnostic value of synovial cf-DNA/NETs for identification of septic arthritis. Forty-two patients with a joint effusion who had undergone arthrocentesis were included. From synovial fluid, cf-DNA/NETs (j-cf-DNA) levels were directly quantified. Diagnostic value of j-cf-DNA was compared with white blood cells (WBC), synovial white blood cells (j-WBC), C-reactive protein (CRP), j-IL-6, j-TNF alpha, j-IL-1 beta, and myeloperoxidase (j-MPO). Sensitivity, specificity, positive and negative predictive value, as well as ROC-curves for each parameter were calculated. Synovial fluid cf-DNA/NETs values from patients with septic arthritis (3,286 +/- 386 ng/ml, n = 9) were significantly increased compared to patients with noninfectious joint inflammation (1,040 +/- 208 ng/ml, n = 17) or osteoarthritis (278 +/- 34 ng/ml, n = 16, p < 0.01). In conjunction with j-cf-DNA, j-IL-6 and j-IL-1 beta were significantly elevated (p < 0.01), but WBC, CRP, and j-WBC were not. At a cut-off of 300 ng/ml, j-cf-DNA had a sensitivity of 0.89, a specificity of 1.0, a positive predictive value of 1.0, and a negative predictive value of 0.97. Receiver operation curves revealed largest areas under the curve for cf-DNA/NETs (0.933) and j-IL-6 (0.951). cf-DNA/NETs seem to be a valuable additional marker for the diagnosis of septic arthritis or periprosthetic infections. However, this result should be confirmed in a large clinical trial.

Neutrophil-derived circulating free DNA (cf-DNA/NETs): a potential prognostic marker for posttraumatic development of inflammatory second hit and sepsis.

The release of "neutrophil extracellular traps" (NETs) has been identified as a novel immune response in innate immunity. Neutrophil extracellular traps are composed of neutrophil-derived circulating free DNA (cf-DNA), histones, and neutrophil cytoplasm-derived proteins such as proteases. Here, we studied the putative predictive value of plasma cf-DNA/NETs for the development of sepsis and mortality after multiple trauma. In a prospective pilot study with 45 multiple trauma (Injury Severity Score>16) patients, cf-DNA was directly quantified in plasma. Blood samples were sequentially obtained daily from admission to our Trauma Center until day 10. Because of limited intensive care unit (ICU) stay of less than 3 days, 8 patients have been excluded, resulting in 37 patients that were evaluated. Time kinetics of cf-DNA/NETs was compared with C-reactive protein (CRP), interleukin (IL) 6, leukocyte counts, and myeloperoxidase. The severity of the injury was calculated on the basis of the Injury Severity Score, as well as Multiple Organ Dysfunction Score, Sequential Organ Failure Assessment, and Simplified Acute Physiology Score II on ICU. Initially high cf-DNA/NETs values (>800 ng/mL) with recurrent increased values between days 5 to 9 were associated with subsequent sepsis, multiple organ failure, and death. In conjunction with cf-DNA/NETs, IL-6 was significantly elevated after admission. However, the development of a second hit was not indicated by IL-6. In contrast to cf-DNA/NETs, no difference in CRP kinetics was observed between patients with and without development of sepsis. Circulating free DNA/NETs kinetics rather followed kinetics of Multiple Organ Dysfunction Score, Sepsis-related Organ Failure Assessment, leukocyte counts, and partially of myeloperoxidase. Circulating free DNA/NETs seems to be a valuable additional marker for the calculation of injury severity and/or prediction of inflammatory second hit on ICU. However, a large clinical trial with severely injured patients should confirm the prognostic value of neutrophil-derived cf-DNA/NETs.