Xiao, Qi Fu; (1993) Liposomes in immunomodulation and vaccines: Immunopotentiation, co-adjuvants and effects of liposomal characteristics. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

There is a need for a safe and effective immunological adjuvant in human immunization programs. One such adjuvant may be liposomes which have the major advantages of being biodegradable and non- immunogenic. The wide variety of lipid compositions of liposomes renders them highly versatile and able to meet specific needs for immunopotentiation. In particular for synthetic peptide vaccines, because of their lack of immunogenicity, liposomes as immunological adjuvants are becoming more and more attractive. Numerous independent studies on the effectiveness of liposomes in potentiating the immune response of a variety of antigens have been documented. However, few reports on the application of liposomes as an immunopotentiating carrier for synthetic peptides, have been published. In addition, there exists considerable controversy with regard to the liposomal characteristics deemed optimal for such action, and questions as to the mode(s) of liposomal immunoadjuvant action remain largely unanswered. In this thesis there will be discussions of the following points. 1. The possibility of using liposomes as immunological adjuvants for synthetic poliovirus peptides has been investigated. Polio types 3-VP2 (W1) and 1-VP2 (W2) peptides have been successfully entrapped in liposomes made by the Dehydration-Rehydration Vesicle (DRV) procedure with up to 87% of the peptides entrapped, depending on the liposomal composition. In work designed to study immunoadjuvant action of liposomes for such peptides, distearoyl phosphatidylcholine (DSPC) DRV liposomes were found superior to all lipid compositions tested in terms of adjuvant effect. A secondary immune response was also obtained with dimyristoyl phosphatidylcholine (DMPC), egg phosphatidylcholine (PC) or dilauroyl phosphatidylcholine (DLPC) liposomes but not with dipalmitoyl phosphatidylcholine (DPPC) liposomes or the free peptides. More specifically, DSPC liposomes produced a secondary immune response (all IgG subclasses tested) which was significantly higher than that produced by DMPC DRV liposomes (P<0.01). DMPC liposomes also acted as immunoadjuvants to the peptide in inducing a secondary response (with an observation period of 118 days). In dose-response studies a 20 μg dose of liposomal W1 or W2 was found superior to other small dosages (i.e. 5, 1.25 and 0.31 µg) in terms of immunoadjuvant action of liposomes. On the other hand, targeted adjuvanticity of liposomes was not found to further improve immunoadjuvant action of liposomes for such peptides. But interleukin-2 (IL-2) as a co-adjuvant co-entrapped in the same liposomes was found to further improve immunoadjuvant action of liposomes to W1 peptide which is, by itself, inactive. It can be concluded that liposomes could be used as immunological adjuvants and carriers in peptide vaccines. 2. Role of IL-2 or the novel positively charged lipids in co-adjuvant action of liposomes has been investigated in this thesis. When the use of IL-2 either passively incorporated into DRV liposomes or covalently coupled it to the surface of liposomes, immunoadjuvant action of liposomes was further improved for hepatitis B surface antigen. There was no significant difference between the entrapped and linked modes. In addition, the role of stearylamine (SA) and [N(1-(2,3-dioleyloxy)-N,N,N-triethyl-ammonium] (DOTMA) which are positively charged lipids has been investigated in co-adjuvant action of liposomes. It was found that when DSPC liposomes formulated with 20% SA (wt/wt) but not with DOTMA or PC liposomes formulated with 20% DOTMA (wt/wt) but not with SA, immunoadjuvant action of liposomes was further improved using tetanus toxoid as antigen model. Such results suggest that (1) The positively charged liposomes can act as immunoadjuvants; (2) Immunoadjuvant action of liposomes could be further improved when the choice of novel positively charged lipids to form liposomal antigen formulation. 3. Finally, the possible advantage of presenting two antigens (entrapped in the same liposomes) to the host (Balb/c mice) simultaneously has been also investigated. Bovine serum albumin (BSA) and tetanus toxoid were co-entrapped into the same liposomes composed of 33 ?mol DSPC or PC and equimolar cholesterol. Results indicate that secondary immune response (all subclasses of IgG tested) for both antigens entrapped in the same liposomes was significantly higher than that produced by the antigens alone in similar DRV liposomes. The antigens were also separately incorporated into liposomes which were then mixed and used to immunize the animals. Similar results to those for co-entrapped antigens were obtained. These are the first findings to suggest that multiple antigen entrapment in the same liposomes may have great advantages in using such liposomal formulations for polyvalent vaccines. There is a need to further investigate this observation.

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