Antigens reconstitution

Schroit, A. J., and Key, M. E. (1983). Induction of syngeneic tumor-specific immunity by liposomes reconstituted with L2C tumor-cell antigens, Immunology. 49. 431-438. 

Figure 1 Continued on next page) Immunopotentiating reconstituted influenza virosomes (IRIV) induced antigen specific proliferation of CD4+CD45RO+ cells. (A) Peripheral blood mononuclear cells (PBMQ from healthy donors n=3) were cultured in the absence of stimuli (Neg), in the presence of IRIV (V), and in the presence of control liposomes (L) at the indicated dilutions. Proliferation was measured on day 6 of culture by H-thymidine incorporation. (B) Cord blood mononuclear cells from two donors were cultured in the absence of stimuli (Neg) or in the presence of phytohaemag-glutinin (PHA), concanavalin A (ConA), IRIV (V) or L at the indicated concentrations. Proliferation was measured on day 3 of culture for PHA and ConA cultures and on day 6 for IRIV and L stimulated cultures. (Q Purified CD4+ or CD8+ cells were cocultured with autologous irradiated PBMC in the absence of stimuli (Neg) and in the presence of IRIV (V) at the indicated concentrations. Proliferation was measured on day 6 of culture by H-thymidine incorporation. (D) Purified CD4/CD45RA+ cells and CD4/CD45RO-I-cells were isolated from PBMC of one healthy donor and cocultured with autologous irradiated PBMC in the presence of IRIV (V) or L at the indicated concentration. Proliferation was measured on day 6 of culture by H-thymidine incorporation. Source From Ref 6.

Figure 6 Immunopotentiating reconstituted influenza virosomes (IRIV) adjuvant effects in the induction of tumor associated antigen-specific cytotoxic T cell. CD14-negative cells from a healthy donor peripheral blood mononuclear cells were cocultured with autologous immature dendritic cells (iDC) in the presence of Melan-A/Mart-l27-35, alone (A) or supplemented with either control liposomes (B) or IRIV (1 50, C). On day 7, culture cells were restimulated with Melan-A/MART-127-35 pulsed iDC and cultured for six further days [see Materials and Methods ]. On day 7 after restimulation cells were stained with fluorescein isothiocyanate-conjugated anti-CD8 and phosphatidylethanolamine-conjugated HL A-A0201 /Melan-A/MART -127-3 5 tetramers. Source From Ref. 6.

Studies performed at RCRM have shown that hematopoietic and immune systems reconstitution after irradiation depends greatly on the functional abilities of the stem cells. Subset analysis and expression of CD34-i- antigens on bone marrow and peripheral blood cells were studied in Chernobyl accident clean-up workers including patients with leukemia and myelodysplasia and patients exposed to the natural levels of irradiation (table 2). 

Field, H., G.T. Yarranton, and A.R. Rees, Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity. Protein Eng, 1990. 3(7) 641-7. 

Epitope excision was performed by application of 2-5 xg antigen to the antibody microcolumn (Tian et al. 2005). Binding was performed for 60 min at 20°C. After washing with binding buffer, proteolytic digestion was carried out on the column with 0.2 xg protease in 200 xl PBS for 2 h at 37°C. Supernatant unbound peptides were removed using washing buffer and the epitope dissociated by addition of 500 pi 0.1 % trifluoroacetic acid (TFA). After incubation for 15 min at 20 C, the epitope eluate was lyophilised and reconstituted in 10 pi NaAc buffer. 

Two commercial vaccines based on virosome technology are currendy on the market. Epaxal (Berna Biotech Ltd, Bern, Switzerland), a hepatitis A vaccine, has inactivated hepatitis A virus particles adsorbed on the surface of the immunopotentiating reconstituted influenza virosomes (IRIV). In Inflexal V (Berna Biotech Ltd) the virosome components themselves are the vaccine protective antigens (185). Recently, in phase I study liposome-encapsulated malaria vaccine (containing monophosphoryl lipid A as adjuvant in the bilayer), the formulation showed induction of higher level of anti-malaria antibody in human volunteers (186). Some liposomal formulations are under investigation in preclinical studies against Yersina pestis, ricin toxin and Ebola Zaire virus (77, 187). 

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