Investigation into powder tribo-charging of pharmaceuticals. Part II: Sensitivity to relative humidity.

Environmental conditions can have a profound impact on the bulk behaviour of pharmaceutical powders, including their tribo-charging tendency. Typically, high relative humidity (RH) has been associated to a reduction in the electrostatic charge of the material. However, the occurrence of charge mitigation seems to be related to the quantity of water molecules at the powder surface, which depends on intrinsic material attributes (i.e., water sorption propensity), and external factors (i.e., RH level). In the present study, pharmaceutical powders (i.e., microcrystalline cellulose, D-mannitol, paracetamol and magnesium stearate) were conditioned at three levels of RH, relevant for pharmaceutical operations, and their bulk behaviour, including charging propensity, was analyzed. Depending on the material type, powders sorbed water from the humid atmosphere to different extents, resulting in different charging behaviours. Overall, the charge density of the materials was found to decrease after a certain RH or monotonically decrease with an increase of RH, except for D-mannitol. For this material, a contrasting trend of increase in charging was observed with an increase in RH. Moreover, the powders showed a distinct tribo-charging sensitivity to RH, with paracetamol being the most affected. These findings suggest that a careful consideration on solid material-moisture interactions is needed when using RH as strategy to minimize electrostatic effects in powder processing.

Investigation into powder tribo-charging of pharmaceuticals. Part I: Process-induced charge via twin-screw feeding.

Powder feeding is a crucial unit operation in continuous manufacturing (CM) of pharmaceutical products. Twin-screw feeders are typically employed to ensure the accurate mass flow of pharmaceutical materials throughout the production process. Here, contact and separation of particles can give rise to electrostatic charges, affecting feeder performance and final product quality. The knowledge of the material charging tendency would therefore be beneficial for both formulation and process design. At the early stage of product development, only a limited amount of material is available and the propensity of the powders to charge needs to be assessed on lab test equipment, which not necessarily represent the material state during processing. In this study, the tribo-charging behaviour of a set of common pharmaceutical materials (i.e., microcrystalline cellulose, D-mannitol, paracetamol and magnesium stearate) was experimentally evaluated. To this end, powder materials were let to flow over the stainless-steel pipes of the GranuCharge™ instrument. The resulting charge was compared to the one acquired during twin-screw feeding. In both cases, paracetamol exhibited the highest charging tendency followed by D-mannitol and microcrystalline cellulose and last by magnesium stearate. A good correlation was found for charge values obtained for both methods, despite the different tribo-charging mechanisms involved in the two set-ups. However, these differences in experimental set-ups led to diverse magnitudes and, in one case, polarity of charge. Additionally, an extensive material characterization was performed on the selected powders and results were statistically analyzed to identify critical material attributes (CMAs) affecting powder tribo-charging. A strong correlation was obtained between the measured charge and inter-particle friction. This indicated the latter as one of the most influencing material characteristic impacting the powder tribo-charging phenomenon of the selected materials.

Reconstitution of cytomegalovirus-specific T-cell response in allogeneic hematopoietic stem cell recipients: the contribution of six frequently recognized, virus-encoded ORFs.

BACKGROUND: The reactivation of human cytomegalovirus (HCMV) in immunosuppressed patients is associated with significant morbidity. Testing HCMV-specific T-cell responses can help determine which patients are at high risk of HCMV disease. We optimized selection of HCMV antigens for detection of T-cell response of patients after allogeneic hematopoietic stem cell transplantation (HSCT) with the aim of identifying patients with insufficient control of HCMV reactivation. METHODS: T-cell immune response to HCMV was monitored in 30 patients during the first year after HSCT. The HSCT recipients were classified according to their anti-HCMV T-cell response and the presence of HCMV DNA in the blood. RESULTS: We observed an inverse relationship between the magnitude of HCMV-specific T-cell responses against CMV lysate, phosphoprotein (pp) 65, immediate early-1 (IE-1), UL36, and UL55, but not to US3 and US29 detected by interferon-gamma (IFNγ)- ELISPOT and the level of HCMV DNA in the blood of patients during the 30 days following sampling. The study has revealed that patients who received a graft from a seronegative donor have a lower T-cell response against HCMV and increased probability of HCMV reactivation in comparison to the patients who had received their graft from a seropositive donor. CONCLUSION: The individual peptide pools and native HCMV antigens were useful for monitoring the time course of the anti-HCMV response by IFNγ-ELISPOT, which proved to have a prognostic value. Besides widely employed peptide pools of pp65 and IE-1, the use of antigens UL36 and UL55, but not US3 or US29, increased sensitivity of the test.

Antitumor activity and immunogenicity of recombinant vaccinia virus expressing HPV 16 E7 protein SigE7LAMP is enhanced by high-level coexpression of IGFBP-3.

We constructed recombinant vaccinia viruses (VACVs) coexpressing the insulin-like growth factor-binding protein-3 (IGFBP-3) gene and the fusion gene encoding the SigE7Lamp antigen. The expression of the IGFBP-3 transgene was regulated either by the early H5 promoter or by the synthetic early/late (E/L) promoter. We have shown that IGFBP-3 expression regulated by the H5 promoter yielded higher amount of IGFBP-3 protein when compared with the E/L promoter. The immunization with P13-SigE7Lamp-H5-IGFBP-3 virus was more effective in inhibiting the growth of TC-1 tumors in mice and elicited higher T-cell response against VACV-encoded antigen than the P13-SigE7Lamp-TK(-) control virus. We found that high-level production of IGFBP-3 enhanced virus replication both in vitro and in vivo, resulting in more profound antigen stimulation. Production of IGFBP-3 was associated with a higher adsorption rate of P13-SigE7Lamp-H5-IGFBP-3 to CV-1 cells when compared with P13-SigE7Lamp-TK(-). Intracellular mature virions (IMVs) of the IGFBP-3-expressing virus P13-SigE7Lamp-H5-IGFBP-3 have two structural differences: they incorporate the IGFBP-3 protein and they have elevated phosphatidylserine (PS) exposure on outer membrane that could result in increased uptake of IMVs by macropinocytosis. The IMV PS content was measured by flow cytometry using microbeads covered with immobilized purified VACV virions.

Expression of soluble TGF-ß receptor II by recombinant Vaccinia virus enhances E7 specific immunotherapy of HPV16 tumors.

Therapeutic immunization with double recombinants of vaccinia virus (VACV) co-expressing sTßRII increased rejection of established TC-1 tumors in C57BL/6 mice in comparison with single recombinant expressing SigE7LAMP. Recombinant VACV derived from vaccination strain Praha expressed either the sTßRII (ectodomain) or chimeric protein fused to immunoglobulin Fc fragment (sTßRII-Fc-Jun) under control of two different promotors together with the immunogenic tumor associated antigen HPV16 E7 oncoprotein in a form of SigE7LAMP fusion molecule. The ability of soluble receptors to bind TGF-ß in vitro was proved. Immunization of mice with double recombinant viruses and virus expressing SigE7LAMP only led to eliciting similar response of E7 specific CD8+ T cells as detected by IFN-γ ELISPOT.

The expression of the soluble isoform of hFlt3 ligand by recombinant vaccinia virus enhances immunogenicity of the vector.

Recombinant vaccinia viruses (rVACV) expressing various tumor-associated antigens have been shown to elicit anti-tumor effect in numerous experimental models and clinical trials. We tested the hypotheses that rVACV expressing biologically active fms-like tyrosine kinase 3 ligand (Flt3L) would show higher immunogenicity than control viruses expressing only model antigen and that coexpression of Flt3L would influence anti-tumor activity of rVACV in the preventive and therapeutic arrangements of the in vivo experiment. To answer these questions, we took advantage of the well-described model of transplanted tumor cells expressing HPV16 E6 and E7 oncoproteins. To determine the effects of hFlt3L on the induction of anti-tumor immunity, we generated live vaccinia viruses that express human Flt3L regulated by the early H5 or strong synthetic E/L promoter together with fusion protein SigE7LAMP, which is a highly immunogenic form of HPV E7 oncoprotein. We tested Flt3L production in vitro and in vivo. Despite higher expression of Flt3L from the synthetic E/L promoter in vitro, the P13-E/L-FL-SigE7LAMP induced lower levels of Flt3L in the serum of mice than P13-H5-FL-SigE7LAMP. The Flt3L expression under the strong early VACV H5 promoter is able to inhibit expansion of CD11b+Gr-1+ myeloid suppressor cells (MSC) and increase the amount of CD11b+ CD11c+ dendritic cells in the spleen of mice immunized with vaccinia virus. Determination of viral DNA isolated from the ovaries of infected animals did not reveal differences in replication between rVACVs in this organ. Coexpression of Flt3L by replication-competent virus P13-FL-SigE7LAMP induced enhancement of the cellular immune response against HPV16 E7 and VACV E3 proteins as well as increased anti-tumor efficacy in both the protective and therapeutic immunization schemes. On the other hand, the short-time Flt3L coexpression by MVA-H5-FL-SigE7LAMP was not sufficient to enhance anti-tumor effect of immunization.

Combination of intratumoral injections of vaccinia virus MVA expressing GM-CSF and immunization with DNA vaccine prolongs the survival of mice bearing HPV16 induced tumors with downregulated expression of MHC class I molecules.

Downregulation of MHC class I molecules is believed to be often the cause of tumor immune escape and at the same time it is the major obstacle to T-cell based immunotherapy of tumors. In our experimental model, the C57BL/6 mice bearing tumors induced by TC-1/A9 cells characterized by expression of HPV16 oncogenes and downregulation of H-2b molecules were immunized with highly immunogenic E7GGG.GUS DNA vaccine expressing the fused gene of modified HPV16 E7 (E7GGG) with E.coli beta-glucuronidase (GUS). The DNA vaccine was administered by gene gun on days 7 and 14 after s.c. injection of tumor cells. The tumors in situ were injected with recombinant vaccinia virus MVA expressing the gene for murine granulocyte-macrophage colony-stimulating factor (MVA-GM-CSF). Two doses of the DNA vaccine combined with at least two consecutive local treatments with MVA-GM-CSF were able to inhibit significantly the growth of tumors. We have shown by ELISPOT-IFNgamma that in situ expression of the GM-CSF gene did not enhance the E7 specific systemic Tcell response. We found that local injections of MVA-GM-CSF induced an increase of intratumoral CD3+ T cell counts and that the DNA vaccination resulted in up-regulation of MHC type I molecules on tumor cells in vivo. We suppose that i.t. delivery of MVA-GM-CSF changed the local tumor microenvironment and rendered tumors more attractive and better accessible to effector T cells.

[Vaccinia virus as a vector for transduction of dendritic cells]. / Virus vakcinie jako vektor pro transdukci dendritických bunek.

Dendritic cells (DC) are very heterogenous population of professional antigen-presenting cells. Precursor cells migrate from bone marrow to peripheral tissues, where immature DC ingest pathogenic microorganisms and then migrate to secondary lymphoid organs. DC differentiate into mature cells that are capable to prime naive T lymphocytes. DC can be used for immunotherapy of cancer and infectious diseases. Transduction of DC by recombinant viral vectors expressing tumor associated antigens (TAA) can result in efficient antigen presentation to T lymphocytes. DC transduced with recombinant vaccinia virus expressing E7 oncoprotein of human papilloma virus 16 are able to protect mice against the growth of syngeneic papillomavirus transformed tumor cells TC1. Antitumor effect was observed also with nonreplicating viruses.

Effect of IFN-gamma receptor gene deletion on vaccinia virus virulence.

Two vaccina virus (VV) strains, WR and Praha, were selected for a study undertaken to determine whether the virus-encoded interferon-gamma receptor (IFN-gamma R) plays any role in virus virulence. Both of the viruses expressed the B8R gene coding for IFN-gamma R in infected cell cultures. The nucleotide sequence of the Praha virus B8R gene was determined, and, when compared with the published sequence of the WR virus, it only displayed one silent nucleotide substitution. Mutants of the WR and Praha viruses with deleted B8R gene were constructed. In rabbits, skin lesions produced by the WR B8R-deleted mutants were smaller and tended to disappear earlier than those caused by wild-type WR virus. Similar results were obtained with both independently prepared WR B8R-deleted mutants. These data strongly suggested that the product of B8R gene did play a role in virus virulence. A similar comparison of the wild-type Praha virus and its mutant could not be done because of the very low virulence of the parental virus for rabbits.

Immune response to vaccinia virus recombinants expressing glycoproteins gE, gB, gH, and gL of Varicella-zoster virus.

Immunogenicity of Varicella-zoster virus glycoproteins gE, gB, gH, and gL expressed by recombinant vaccinia viruses (VV) separately or simultaneously was determined in mice and guinea pigs by ELISA, Western blotting, radioimmunoprecipitation, plaque reduction assay, and skin test. Single VV-gE and VV-gB recombinants and double VV-gH/gL recombinant elicited specific antibodies with VZV neutralizing activity in mice. Co-expression of gE and gB by one recombinant VV resulted in an increased antibody response in comparison with immunization with single recombinants or their mixtures. Unlike anti-gB and anti-gH/gL antibodies, the gE-specific antibodies had no virus neutralizing activity in absence of complement, and when used alone, they even caused considerable increase of VZV infectious units. Moreover, immune sera containing anti-gE antibodies antagonized complement independent virus-neutralizing activity of anti-gB- and anti-gH/gL-positive sera. The ability to induce delayed hypersensitivity reaction to VZV antigens was observed after immunization of guinea pigs with gE- and/or gB-expressing VVs.

Early gene expression of vaccinia virus strains replicating (Praha) and non-replicating (modified vaccinia virus strain Ankara, MVA) in mammalian cells.

Modified vaccinia virus Ankara strain (MVA) is a safe highly attenuated non-pathogenic virus suitable as a vector for developing various vaccines. Study of expression of a reporter beta-galactosidase gene under the control of an early vaccinia virus (VV) promoter in MVA and non-modified vaccinia virus Praha strain showed that early transcription in MVA is elevated in comparison with non modified VV. This property was demonstrated in various cell cultures including CV1 cells, human lung diploid cells, chicken primary fibroblasts but not in bone marrow-derived mouse dendritic cells. There the relationship between the elevated early transcription and the permisivity of cells for MVA was not observed.

Secondary vaccination with vaccinia virus recombinants: role of residual virulence of recombinants and immunogenicity of extrinsic antigens.

ICR mice were immunized intraperitoneally with two doses (10(6) PFU per dose) of vaccinia virus (VV) recombinants of variable virulence expressing either the strongly immunogenic glycoprotein E (gE) of varicella zoster virus (VZV) or weakly immunogenic hepatitis B virus (HBV) preS2-S (S) antigen. Recombinants expressing gE were able to elicit primary and secondary anti-gE antibody irrespective of their residual virulence; after the second dose they did so even in the presence of VV antibody resulting from primary vaccination dose or under other conditions limiting VV replication. As for the S-recombinants, pronounced anti-S antibody development was only observed in mice which had received the more virulent recombinant virus as the first dose. A repeated dose of S-recombinants was unable to elicit a secondary anti-S antibody response. The present findings do not support the assumption that the poor immunogenicity of some extrinsic antigens could be overcome by administering repeated doses of the particular VV recombinant.

Influence of the parental virus strain on the virulence and immunogenicity of recombinant vaccinia viruses expressing HBV preS2-S protein or VZV glycoprotein I.

Five triple-plaque purified vaccinia virus (VV) lines generated from smallpox Sevac VARIE vaccine (strain Praha) and three VV virus lines similarly derived from Wyeth DRYVAX vaccine were used for preparation of recombinants expressing the hepatitis B virus preS2-S gene. The same five Praha-derived virus lines were used to construct recombinants expressing the varicella-zoster virus (VZV) glycoprotein I (gpI) gene. Recombinants and their parental viruses were tested for the residual neurovirulence in mice. The virus lines and the recombinants derived therefrom differed markedly in this respect. Immunization of mice resulted in high levels of anti-HBsAg antibodies only in the case of recombinants derived from the relatively virulent viruses. In contrast, the levels of VZVgpI antibodies in mice were similar with all VV-VZV recombinants irrespective of the virulence of the parental virus line.

Induction of varicella-zoster virus-neutralizing antibodies in mice by co-infection with recombinant vaccinia viruses expressing the gH or gL gene.

Recombinant vaccinia viruses (VV) expressing the varicella-zoster virus (VZV) glycoprotein H (gH) or glycoprotein L (gL) were constructed. The 94 kDa gH intermediate glycoprotein was synthesized in cell cultures infected with the VV-gH recombinant, but only co-infection with both recombinants resulted in the synthesis of the fully processed 118 kDa gH molecule. The VV-expressed gH and gL formed a complex that displayed the conformational neutralization epitope detectable by means of human VZV gH-specific monoclonal antibody V3. Formation of this epitope was inhibited by tunicamycin but not by monensin. Simultaneous intraperitoneal inoculation of mice with high doses of both VV-gH and VV-gL viruses resulted in the development of VZV-neutralizing, complement-independent antibodies; these antibodies were not detected in mice infected solely with either the VV-gH or the VV-gL recombinant.

Search for optimal parent for recombinant vaccinia virus vaccines. Study of three vaccinia virus vaccinal strains and several virus lines derived from them.

Three vaccinia virus strains (Praha, DD--a DRYVAX Wyeth vaccine-derived virus-and LIVP) were examined for growth in various cell cultures and for virulence and immunogenicity in mice. The viruses did not differ by their growth rates in monkey kidney cells (CV-1), human diploid cells (LEP), rat TK cells (RAT 2) or primary dog kidney cells. The immunogenicity of Praha and DD viruses was similar, the virus LIVP was somewhat more immunogenic. In terms of virulence in 3-day-old mice, the DD virus was the most attenuated. Single-plaque progenies were derived from the original smallpox vaccines VARIE Sevac (strain Praha) and DRYVAX Wyeth and tested for the above markers and DNA restriction patterns. The results obtained demonstrated biological and molecular heterogeneity of the original virus populations. Close linkage was observed between immunogenic activity and virulence in 3-day but not in 3-week mice. The results indicate that smallpox vaccine preparations may serve as an abundant source of virus mutants.

Incidence of apparent restricted diffusion in three different models of cerebral infarction.

High speed magnetic resonance imaging (MRI) and short diffusion times are used to investigate the appearance of restricted diffusion in three different models of cerebral infarction. The models are: the middle cerebral artery occlusion (MCAO) model in the rat, the carotid occlusion model in the gerbil, and the Rose Bengal microvascular occlusion model in the rat. All three were investigated for 16 b-values equally spaced between 10 and 1510 s/mm2 using two distinct experiments. In the ct (constant time) experiment, the diffusion time was held constant at 11.7 ms while the b-value was varied with the gradient strength. In the cg (constant gradient) experiment, the gradient strength was held constant and the b-value increased by varying the diffusion time from 4.4 to 11.7 ms. A monoexponential decay of the signal intensity with b-value in the ct experiment accompanied by nonmonoexponential (NME) decay in the cg experiment is indicative of restricted diffusion. As this phenomenon is detectable only at short diffusion times, it cannot be due to restriction by impermeable membranes, and we have thus termed this apparent restriction. For the MCAO model and the carotid occlusion model, apparent restriction was found both inside the infarct territory and in some regions outside it. No definite evidence for restriction was found for the Rose Bengal model, which was, however, only studied from 24 h post-insult.