Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines.

CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract.

Impact of Poloxamer 188 Material Attributes on Proteinaceous Visible Particle Formation in Liquid Monoclonal Antibody Formulations.

Surfactants such as Poloxamer 188 (PX188) play an important role in controlling particle formation in biotherapeutic formulations due to interfacial stresses. This study demonstrates for the first time that hydrophobicity of PX188 is a potential critical material attribute (CMA) as far as control of visible particle (VP) formation is concerned. We have found that within PX188 lots satisfying pharmacopeial specifications, there is variability in material attributes such as hydrophobicity, as determined from their reversed-phase high-performance liquid chromatography profiles. However, it currently remains unknown how such variability in hydrophobicity of PX188 affects surfactant function and VP formation. Here, we compared the effect of seven PX188 lots in two monoclonal antibody drug product formulations under various stress conditions. Notably, proteinaceous VP formation was reduced while using a PX188 lot with higher hydrophobicity. Our findings emphasize the importance of monitoring lot-to-lot variability of PX188 and provide insight into potential CMA for improving and controlling material attributes of PX188 for use in liquid biotherapeutic formulations.

Detection of material-derived differences in the stiffness of egg yolk phosphatidylcholine-containing liposomes using atomic force microscopy.

Naturally sourced phospholipids are used in many liposomal pharmaceuticals. The present report describes a method to detect the effects of different egg yolk phosphatidylcholines (EPCs) on liposomal physicochemical properties. Five EPC-containing liposomes were prepared using five different EPCs obtained from different suppliers. There was no significant difference in purity between each EPC. The stiffness of the liposomes was examined via atomic force microscopy (AFM) in relation to the liposomal membrane permeability coefficient of encapsulated calcein after gel filtration, which is indicative of liposomal stability including the release of a hydrophilic drug from a liposome. Although the size of the liposome and the encapsulation efficiency of calcein did not significantly change with the type of EPC used, the liposome stiffness was found to vary depending on the EPC used, and liposomes with a similar stiffness were found to show a similar membrane permeability to calcein. Our results indicate the usefulness of stiffness measurement, using AFM as the analytical method, to detect material-derived differences in EPC-containing liposomes that affect drug release from the liposomes. Because drug release is one of the most important liposomal functions, combining this method with other analytical methods could be useful in selecting material for the development and quality control of EPC-containing liposomes.

Protection from inactivation of the adenine nucleotide translocator during hypoglycaemia-induced apoptosis by mitochondrial phospholipid hydroperoxide glutathione peroxidase.

We demonstrated that mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx) first suppressed the dissociation of cytochrome c (cyt c) from cardiolipin (CL) in mitochondrial inner membranes and then apoptosis caused by the hypoglycaemia by the prevention of peroxidation of CL [Nomura, Imai, Koumura, Arai and Nakagawa (1999) J. Biol. Chem. 274, 29294-29302; Nomura, Imai, Koumura, Kobayashi and Nakagawa (2000) Biochem. J. 351, 183-193]. The present study shows the involvement of peroxidation of CL in the inactivation of adenine nucleotide translocator (ANT) and the opening of permeability transition pores by using the system of ANT-reconstituted liposome and isolated mitochondria. ANT activity appeared in dioleoyl phosphatidylcholine proteoliposome containing 10% (mol/mol) CL or phosphatidylglycerol (PG), but not other classes of phospholipids. ANT activity was competitively inhibited by the addition of cardiolipin hydroperoxide (CLOOH) in reconstituted liposomes containing CL. However, phosphatidylcholine hydroperoxide failed to inactivate the activity of ANT. The activity of ANT in reconstituted liposomes, including CLOOH, recovered when CLOOH in reconstituted liposome was reduced to hydroxycardiolipin by incubation with PHGPx. The activity of ANT was determined in rat basophil leukaemia RBL2H3 cells after their exposure to 2-deoxyglucose. ANT activity decreased to 50% of the control level by 4 h in response to apoptosis. In parallel, cyt c and apoptosis-inducing factor (AIF) were released from mitochondria. Suppression of the accumulation of CLOOH by overexpression of PHGPx in mitochondria effectively prevented the inactivation of ANT, the opening of permeability transition pores and the release of cyt c and AIF from mitochondria in hypoglycaemia-induced apoptotic cells. These findings suggest that mitochondrial PHGPx might be involved in the modulation of the activity of ANT and the opening of pores for the release of cyt c via the modulation of levels of CLOOH in the mitochondria.