Viral safety of APOSECTM: a novel peripheral blood mononuclear cell derived-biological for regenerative medicine.

BACKGROUND: Viral reduction and inactivation of cell-derived biologicals is paramount for patients' safety and so viral reduction needs to be demonstrated to regulatory bodies in order to obtain marketing authorisation. Allogeneic human blood-derived biological medicinal products require special attention. APOSECTM, the secretome harvested from selected human blood cells, is a new biological with promising regenerative capabilities. We evaluated the effectiveness of inactivation of model viruses by methylene blue/light treatment, lyophilisation, and gamma irradiation during the manufacturing process of APOSECTM. MATERIALS AND METHODS: Samples of intermediates of APOSECTM were acquired during the manufacturing process and spiked with bovine viral diarrhoea virus (BVDV), human immunodeficiency virus type 1 (HIV-1), pseudorabies virus (PRV), hepatitis A virus (HAV), and porcine parvovirus (PPV). Viral titres were assessed with suitable cell lines. RESULTS: Methylene blue-assisted viral reduction is mainly effective against enveloped viruses: the minimum log10 reduction factors for BVDV, HIV-1, and PRV were ≥6.42, ≥6.88, and ≥6.18, respectively, with no observed residual infectivity. Viral titres of both HAV and PPV were not significantly reduced, indicating minor inactivation of non-enveloped viruses. Lyophilisation had minor effects on the viability of several enveloped model viruses. Gamma irradiation contributes to the viral safety by reduction of enveloped viruses (BVDV: ≥2.42; HIV-1: 4.53; PRV: ≥4.61) and to some degree of non-enveloped viruses as seen for HAV with a minimum log10 reduction factor of 2.92. No significant reduction could be measured for the non-enveloped virus PPV (2.60). DISCUSSION: Three manufacturing steps of APOSECTM were evaluated under Good Laboratory Practice conditions for their efficacy at reducing and inactivating potentially present viruses. It could be demonstrated that all three steps contribute to the viral safety of APOSECTM.

Reproducibility of GMP-compliant production of therapeutic stressed peripheral blood mononuclear cell-derived secretomes, a novel class of biological medicinal products.

BACKGROUND: The recent concept of secretome-based tissue regeneration has profoundly altered the field of regenerative medicine and offers promising novel therapeutic options. In contrast to medicinal products with a single active substance, cell-derived secretomes comprise pleiotropic bioactive ingredients, representing a major obstacle for reproducible drug product efficacy and warranting patient safety. Good manufacturing practice (GMP)-compliant production guarantees high batch-to-batch consistency and reproducible efficacy of biological medicinal products, but different batches of cellular secretomes produced under GMP have not been compared yet, and suitable quality control parameters have not been established. To this end, we analyzed diverse biological and functional parameters of different batches produced under GMP of the secretome obtained from γ-irradiated peripheral blood mononuclear cells with proven tissue regenerative properties in infarcted myocardium, stroke, spinal cord injury, and skin wounds. METHODS: We quantified key secretome ingredients, including cytokines, lipids, and extracellular vesicles, and functionally assessed potency in tube formation assay, ex vivo aortic ring sprouting assay, and cell-based protein and reporter gene assays. Furthermore, we determined secretome stability in different batches after 6 months of storage at various ambient temperatures. RESULTS: We observed that inter-batch differences in the bioactive components and secretome properties were small despite considerable differences in protein concentrations and potencies between individual donor secretomes. Stability tests showed that the analytical and functional properties of the secretomes remained stable when lyophilisates were stored at temperatures up to + 5 °C for 6 months. CONCLUSIONS: We are the first to demonstrate the consistent production of cell-derived, yet cell-free secretome as a biological medicinal product. The results from this study provide the basis for selecting appropriate quality control parameters for GMP-compliant production of therapeutic cell secretomes and pave the way for future clinical trials employing secretomes in tissue regenerative medicine.

Toxicological testing of allogeneic secretome derived from peripheral mononuclear cells (APOSEC): a novel cell-free therapeutic agent in skin disease.

A cell-free approach using secretomes derived from stem cells or peripheral blood mononuclear cells is an active area of regenerative medicine that holds promise for therapies. Regulatory authorities classify these secretomes as biological medicinal products, and non- clinical safety assessment thus falls under the scope of ICH S6. A secretome of stressed peripheral blood mononuclear cells (APOSEC) was successfully tested in a toxicology program, supporting clinical use of the new drug candidate. Here, to allow for topical, dermal treatment of patients with diabetic foot ulcer, several non-clinical safety studies were performed. Acute toxicity (single dose) and neuropharmacological screening were tested intravenously in a rat model. Risk for skin sensitisation was tested in mice. A 4-week intravenous toxicity study in mice and a 4-week subcutaneous toxicity study in minipigs were conducted to cover the clinical setting and application in a rodent and a non-rodent model. Acute and repeated-dose toxicity studies show that APOSEC administered intravenously and subcutaneously does not involve major toxicities or signs of local intolerance at levels above the intended total human maximal dose of 3.3 U/kg/treatment, 200 U/wound/treatment, and 100 U/cm2/treatment. The non-clinical data support the safe topical use of APOSEC in skin diseases related to deficient wound healing.

A cautionary note on experimental artefacts induced by fetal calf serum in a viral model of pulmonary eosinophilia.

In BALB/c mice, sensitization with the attachment protein (G) of respiratory syncytial virus (RSV) leads to CD4(+) T cell-mediated lung eosinophilia during subsequent challenge with RSV. In this study, we originally intended to test whether activation of RSV-specific cytotoxic T cells by peptide-pulsed dendritic cells (DC) after G protein sensitization could prevent this eosinophilic response. Peptide-pulsed dendritic cells activated CTL, which could mediate protective immunity to RSV. However, DC vaccination aggravated, rather than prevented, pulmonary eosinophilia in G-sensitized mice and also enhanced weight loss upon RSV infection. This was accompanied by preferential pulmonary recruitment of CD4(+) T cells secreting IL-5. The same enhanced Th2-mediated eosinophilic response could be observed in mice that received unloaded dendritic cells and this response occurred even in the absence of prior G sensitization. Since both dendritic cells and RSV were grown in fetal calf serum (FCS)-containing medium, we suspected that FCS had provoked this response. Indeed, neither eosinophilia nor enhanced pathology were observed in mice treated with DC raised in mouse serum. This observation calls for meticulous controls for artefacts induced by fetal calf serum particularly in mouse models of allergic responses of the respiratory tract.

Pulmonary T cells induced by respiratory syncytial virus are functional and can make an important contribution to long-lived protective immunity.

The contribution of T cell responses to immunity against respiratory syncytial virus (RSV) is not fully defined, but this is an important issue for vaccine design. Recent studies demonstrating RSV-induced pulmonary T cell suppression suggest that RSV may have evolved strategies to escape T cell immunity. Here we evaluated potential consequences of RSV-mediated immunosuppression for protective memory T cell responses in vivo. Surprisingly, we found strong ex vivo cytolytic activity and interferon production of pulmonary RSV-specific CD8(+) T cells both in the acute and the memory phase of primary murine RSV infection. More significantly, T cell memory made an important contribution to immunity against RSV independent of antibodies. Thus, RSV-primed mice were protected against challenge with RSV-recombinant vaccinia viruses, which can be controlled by RSV-specific T cells, but not by RSV-specific antibodies. In conclusion, RSV does not appear to impair acute and protective memory T cell responses induced by a primary infection. These findings further support that induction of T cell immunity should be a relevant goal for RSV vaccines.

Virus clearance and immunopathology by CD8(+) T cells during infection with respiratory syncytial virus are mediated by IFN-gamma.

CD8(+) T cells (CTL) are important effector cells for virus control and immunopathology after primary infection with respiratory syncytial virus (RSV). To investigate the effector mechanisms involved, we set up an adoptive transfer model, in which effector CTL specific for p82-90 of RSV M2 were generated in vivo, followed by short-term restimulation in vitro and transfusion into infected recipients. A total of 4 x 10(4) donor-derived p82-specific CTL homing to the lung within 4 days after transfusion were sufficient to completely eliminate a virusinoculum of 1.5 x 10(6) pfu. This was accompanied by significant lung pathology. Surprisingly, virus control and immunopathology proceeded unimpaired when donor cells lacking perforin, CD95 ligand or TNF were transfused. By contrast, treatment of recipient mice with a neutralizing antibody against IFN-gamma or transfusion of IFN-gamma-deficient effector CTL largely abolished virus control and significantly reduced CD8(+) T cell-mediated pathology. In IFN-gamma-deficient mice, high-dose primary infection experiments revealed attenuated immunopathology, but only slightly delayedvirus clearance, suggesting that other cells and molecules can partly substitute for the effects of CTL-derived IFN-gamma on virus clearance. These experiments identify IFN-gamma as a key molecule in RSV-induced immunopathology and in CD8(+) T cell-mediated control of RSV infection.

"Bystander" recruitment of systemic memory T cells delays the immune response to respiratory virus infection.

The concept of heterologous T cell immunity postulates that nonspecific memory T cells recruited and reactivated in the context of an unrelated virus infection may contribute to protective antiviral immunity. Pulmonary infection with respiratory syncytial virus (RSV) of mice immune to lymphocytic choriomeningitis virus (LCMV) leads to substantial recruitment of systemic LCMV-specific memory CD8 T cells to the lung. Using a sensitive TCR-transgenic model, we show that such "bystander" recruitment to the RSV-infected lung could induce a single round of cell division among LCMV-specific T cells. However, it did not change their activation status as assessed by expression of surface markers, the extent or kinetics of IFN-gamma production and cytolytic effector function. Moreover,recruitment of LCMV-specific bystander T cells not only failed to enhance immunity to RSV, but significantly delayed virus elimination and slightly enhanced RSV-induced weight loss in non-transgenic BALB/c mice. This correlated with a delay in the recruitment of RSV-specific T cells to the lung. These data show that bystander recruitment of heterologous T cells is not necessarily accompanied by bystander activation. More importantly, bystander recruitment of systemic memory T cells can impair antiviral immunity, presumably by interference with the recruitment of specific T cells.

The role of Toll-like receptor 4 versus interleukin-12 in immunity to respiratory syncytial virus.

Toll-like receptors (TLR) and IL-12 represent key elements of innate immunity. Using C57BL/10 ScCr mice it was shown that TLR4 is important for control of infection with respiratory syncytial virus (RSV). Since these mice have an additional defect in the IL-12R, we reinvestigated immunity to RSV in several C57BL/10 and BALB/c mouse strains lacking a functional TLR4, a functional IL-12-IL-12R interaction or both. In the absence of a functional IL-12 axis, early virus control was impaired in C57BL/10 mice, but not in BALB/c mice. By contrast, TLR4 had no impact on RSV elimination. Pulmonary NK cell recruitment was impaired in IL-12 deficient BALB/c mice and NK cytotoxicity was reduced in IL-12/IL-12R-deficient mice of both genetic backgrounds. Absence of TLR4 had no impact on NK cell recruitment or NK activity nor on recruitment of other pulmonary inflammatory cells. Activation of RSV-specific T cell immunity, including T cell mediated immunopathology, was normal in all mutant strains. These findings clearly argue against a significant role for TLR4 and define a limited role for IL-12 in primary murine RSV infection.