The Adjuvant Effect of Liposomes in Eliciting Anti-Galactosyl Antibodies

Negatively-charged, multilamellar liposomes, covalently coupled with p-aminophenyl-β-D-galactopyranoside, elicited in rabbits a nearly equivalent anti-galactosyl immune response in both the presence and the absence of complete Freund's-adjuvant (CFA). The antibody response obtained using liposomes both as the carrier and adjuvant was found to be better than that obtained through the conventional way of using protein carrier in CFA. The results suggest an effective role of liposomes as adjuvant in the production of sugar-specific antibodies.     

Immunochemical determination of gentamicin in serum. II. Preparation of polyclonal gentamicin antibodies]

The author describes the preparation of polyclonal rabbit antibodies against gentamicin. As immunogens gentamicin conjugates with bovine serum albumin were used, or else with thyroglobulin and haemocyanin. The immunization pattern involved combined administration of immunogens by the intravenous route, or by the intramuscular or intradermal route, using liposomes and complete Freund adjuvant. The highest antibody titres against gentamicin found by the ELISA method were obtained when procedures were used where the carrier was serum albumin and thyroglobulin and as adjuvant complete Freund adjuvant was used.     

Liposomes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE) are excellent adjuvants for induction of an immune response to protein and tumor antigens.

Liposomes containing the synthetic lipophilic analog of muramyl dipeptide, muramyl tripeptide phosphatidylethanolamine (MTP-PE), were used as adjuvants for the induction of humoral and cellular immune responses following immunization with protein or tumor antigens. Cellular immune reactions, including delayed-type hypersensitivity and lymphoproliferation in vitro, were observed following immunization of mice with a mixture of antigen and liposome-MTP-PE. Immunization with murine melanoma K1735 cells, admixed with liposomal MTP-PE, induced a protective immune response as demonstrated by the rejection of transplanted tumor cells. Antibody production was also induced following immunization with protein antigens admixed with liposome-MTP-PE. The efficacy of adjuvant activity following immunization with antigens admixed with liposome-MTP-PE was equal to or better than that of complete Freund's adjuvant (CFA). Moreover, liposome-MTP-PE did not have the toxic side effects associated with CFA. These data suggest that phospholipid liposomes containing MTP-PE are superior adjuvants and should receive consideration for vaccine therapy.     

The effect of the physical form of poly(lactic-co-glycolic acid) carriers on the humoral immune response to co-delivered antigen

A model shed antigen, ovalbumin (OVA), was incorporated into polymeric biomaterial carriers made of poly(lactic-co-glycolic acid) (PLGA) in the form of microparticles (MP) or scaffolds (SC). These polymeric biomaterial carrier vehicles with incorporated antigen were then injected or implanted into mice and the resulting time-dependent systemic humoral immune response towards the controlled released OVA was assessed by following the OVA-specific IgG concentration and isotypes using ELISA. To assess the differential level of enhancement of the immune response depending on the form of carrier vehicle (MP vs. SC), the total amount of polymer and OVA delivered was kept constant as well as the release rate of OVA for both carrier vehicles. The level of the humoral immune response was higher and sustained for OVA released from PLGA SC which were implanted with associated tissue damage, and lower and transient when the same amount of polymer and OVA were delivered from PLGA MP, which were minimally invasively delivered by injection. This immune response was primarily Th2 helper T cell-dependent, although for the strong adjuvant, CFA, and PLGA SC carriers there was both a Th2 and Th1 response contribution. These results implicate 'danger signals' associated with the implantation of the scaffolds due to tissue injury which primed the system for an enhanced immune response.     

Humoral and cellular immunity to hepatitis B virus-derived antigens: comparative activity of Freund complete adjuvant alum, and liposomes.

Complete Freud adjuvant, aluminum gel, and liposomes were compared for their ability to enhance the immunogenicity of an intact 22-nm HBsAg particle vaccine and an HBsAg-derived polypeptide vaccine in guinea pigs. Both humoral and cell-mediated immune responses were evaluated. The greatest immune response was obtained with complete Freund adjuvant, regardless of the antigen preparation. Aluminum gel appeared to be a better adjuvant for 22-nm HBsAg particles, but the liposomes rendered polypeptide preparations more immunogenic. The possibility that various proportions were entrapped in aqueous compartments instead of being inserted into the lipid bilayers of liposomes might account for this difference. The development of both humoral and cellular immunity was dependent upon the use of an adjuvant, because aqueous preparations had poor immunogenicity.     

弗氏佐剂与氢氧化铝佐剂对诱导小鼠获得性免疫应答作用的比较

为研究、比较不同佐剂对诱导小鼠产生获得性免疫应答的不同作用,以卵清白蛋白(OVA)为抗原,分别混合完全弗氏佐剂(CFA)或Al(OH)3佐剂,对C57BL/6小鼠进行常规免疫,采用流式细胞技术对细胞内细胞因子IFN-γ和IL-4进行检测;ELISA方法对特异性抗OVA抗体滴度及抗体亚型进行了检测。结果显示在免疫后CFA组产生以IFN-γ为主的细胞因子而Al(OH)3组产生以IL-4为主的细胞因子;两组中均产生特异性抗OVA IgG抗体,但CFA组以IgG2a亚型为主,而Al(OH)3组则以IgG1亚型为主,不产生IgG2a亚型抗体。实验表明,经CFA加抗原免疫后机体产生的免疫应答以Th1型细胞免疫为主,抗体类型为IgG2a;而Al(OH)3佐剂则诱导机体产生Th2型细胞免疫应答,抗体类型为IgG1。     

Liposomes as immunological adjuvants in eliciting antibodies specific to the synthetic polypeptide poly(LTyr,LGlu)-poly(DLAla)–poly(LLys) with high frequency of site-associated idiotypic determinants

The antibody response to the synthetic polypeptide, poly(LTyr, LGlu)-poly(DLAla)–poly(LLys), [(T, G)-A–L], injected entrapped in liposomes which served as adjuvant, has been analyzed. The liposomes used were composed of phosphatidylcholine, cholesterol, dicetylphosphate and DL α-tocopherol (molar ratios as 4:3:0.1:0.5) and therefore, were negatively charged. Since the (T, G)-A–L is also negatively charged, no free complexes were formed. The (T, G)-A–L was found to be entrapped inside the enclosed volume of the liposomes, and no (T, G)-A–L antigenic determinants could be detected on the liposomal membranes. Injection of high-responder C3H.SW (H-2^b) mice with (T, G)-A–L-bearing liposomes demonstrated that the i.p. and the i.v. routes of immunization were efficient in eliciting (T, G)-A–L-specific antibodies, whereas the i.d. injection led to poor antibody responses. The latter route of immunization is the most effective when (T, G)-A–L is injected in complete Freund's adjuvant (CFA). When low doses (0.1 and 1 μg) of (T, G)-A–L were used for immunization, the liposomes were better adjuvants than CFA. The effectiveness of the liposomes as immunological adjuvants was also shown in their ability to induce high-potential, primed memory cells. The pattern of low (H-2^{k, a}) and high (H-2^b) responsiveness to (T, G)-A–L was retained following immunization with (T, G)-A–L entrapped in liposomes, as tested in two pairs of congenic strains. (T, G)-A–L-specific antibodies induced by injection with 1μ antigen entrapped in liposomes bear the (T, G)-A–L site-related idiotypic markers of C3H.SW (Igh-1^a) mice in a significantly higher frequency than the homologous idiotypes, namely the antibodies elicited in this strain against (T, G)-A–L in CFA. Thus, liposomes may serve as adjuvants for the production of relatively restricted (T, G)-A–L-specific antibodies of high qualitiy.     

Parameters affecting the immunogenicity of a liposome-associated synthetic hexapeptide antigen.

The effect of liposome association on the immunogenicity of the hexapeptide IRGERA was investigated. When administered in the absence of a carrier and adjuvant this peptide, which corresponds to a linear epitope located at the C-terminus of histone H3, was not immunogenic. When mice were immunized with the peptide covalently linked to the surface of small unilamellar vesicles containing monophosphoryl lipid A as adjuvant, a relatively long-lasting response with memory cell induction was observed. The anti-peptide antibodies raised in this way reacted with the cognate sequence in the native histone. In contrast, coupling of the peptide to the surface of large vesicles yielded both an IgM and IgG response of short duration whereas encapsulation of the free peptide in large vesicles was ineffective. These results indicate that with short synthetic peptides, liposomes provide a substitute for a carrier protein. However, an adjuvant has to be incorporated in the vesicles in order to obtain an efficient immune response. Such an approach may be useful for designing synthetic vaccines.     

A comparison of commercially available adjuvants for use in research.

We evaluated an adjuvant, TiterMax, as an alternative to complete Freund's adjuvant (CFA) for producing antisera in animals. TiterMax, consists of a microparticulate stabilized water-in-oil emulsion of a metabolizable oil, squalene, with the adjuvant block copolymer CRL89-41. This paper reports two evaluations of TiterMax versus CFA and other commercially available adjuvants. In the first study, mice were immunized with a hapten, trinitrophenol, conjugated to hen egg albumin (TNP-HEA) in one of several adjuvants: TiterMax, CFA, Adjuvax, Ribi adjuvant system (RAS), Alhydrogel or Lipovant. TiterMax induced higher longer lasting titers with fewer injections than any of the other adjuvants. The magnitude of the response to TNP varied with species and route of immunization. In the second study, CFA, TiterMax, Adjuvax and RAS were compared in rabbits, mice and goats. Animals were immunized with luteinizing hormone-releasing hormone (LHRH) conjugated to BSA in each adjuvant using comparable protocols. TiterMax induced titers against the peptide equivalent to CFA in all three species. The inflammatory responses induced by TiterMax were mild and transient compared with those induced by CFA. These data suggest that TiterMax is an effective alternative to CFA in many situations.     

The effect of surface-coupled antigen of liposomes in immunopotentiation

The effect of surface coupled antigens of liposomes on the immunological response has been investigated. Lysozyme was covalently coupled to neutral and positively charged liposomes using glutaraldehyde. Subcutaneous administration of these preparations stimulated a significant antibody response higher than that elicited by the antigen entrapped in neutral liposomes. Immunization by liposomal antigens together with complete Freund's adjuvant resulted in strong immune responses, highest with the antigen coupled to neutral and positively charged liposomes followed by the antigen entrapped in neutral liposomes. Primary and secondary immunization with lysozyme, both entrapped and coupled to liposomes, evoked an IgG response.     

Suppression of myosin-induced and adoptively transferred myocarditis by prior treatment with complete Freund's adjuvant.

BACKGROUND: We evaluated the effect of pretreatment with complete Freund's adjuvant (CFA) on experimentally induced myocarditis concomitant with the assessment of antigen-specific properties of disease-triggering lymphocytes. METHODS AND RESULTS: Rats pretreated with CFA a week prior to myosin immunization developed a significantly attenuated myocardial inflammation as compared to control-treated animals. Furthermore, prior administration of CFA virtually abolished histological evidence of myocarditis induced by transfer of antimyosin lymphocytes. CFA administered subcutaneously prior to myosin immunization resulted in a significant reduction in lymph node cell reactivity to myosin. Assessment of cultured medium from lymphocytes obtained from CFA-pretreated myosin-immunized rats revealed reduced levels of interferon gamma but an increased production of IL-10, suggesting an induction of a Thl to Th2 switch. CONCLUSIONS: Thus, CFA treatment suppressed both myosin-induced as well as adoptively transferred myocarditis concomitant with induction of antigen-specific unresponsiveness to myosin and skewing of the immune response in favor of a Th2-dominated profile.     

Liposomes as carriers for allergy immunotherapy

With the aim of obtaining an efficient but safer vaccine for allergy immunotherapy, the possibility of using liposomes as adjuvants has been considered given their proven low toxicity, adjuvant properties and ability to maintain the encapsulated substance in their interior for some time in vivo. Different methods of encapsulating allergenic extracts into neutral, positively, and negatively charged DPPC: cholesterol liposomes have been investigated and the immune response provoked by these in mice was studied and compared to the immune response to free allergen or other adjuvants such as aluminium hydroxide. The results obtained show that allergen encapsulated in all three types of liposomes elicit an increase in specific IgG levels higher than that provoked by free allergen, however, both encapsulation efficiency and specific IgG titre were higher when positively charged (DPPC: cholesterol stearylamine) liposomes were used. Specific IgE levels to allergen in positive and neutral liposomes was lower than to allergen in negative liposomes or adsorbed to Al(OH)3. No difference were found in the behaviour of liposomes prepared by different methods (the results were obtained from pooled sera from different groups of mice so there is no statistical data). These results confirm the immunoadjuvant effect of liposomes for allergy immunotherapy. Further studies to determine their lack of toxicity, pharmacokinetic studies and human clinical studies are necessary to confirm their adequacy for human use and advantage over current immunotherapy methods.     

Induction of humoral manifestations of autoimmunity following intraperitoneal injection of complete Freund's adjuvant in mice

Several animal models of arthritis are produced using complete Freund's adjuvant (CFA) alone or with collagen as an arthritogen. Successful induction of arthritis is reported to require that the adjuvant mixture be administered by intradermal or subcutaneous routes. The resulting arthritis is caused by primarily cellular immune responses. Data presented in this paper show that giving CFA by intraperitoneal (I.P.) inoculation results in a humoral autoimmune response, with no obvious signs of arthritis. This humoral autoimmune response is characterized by production of autoantibodies to nuclear and cytoplasmic antigens, elevated levels of circulating immune complexes, and in approximately 25% of mice, rheumatoid factor.     

The acquired immune deficiency syndrome.

Liposomes prepared with human LS174T colon tumour cell membranes induce specific primary xenogeneic immune responses in BALB/c splenocytes in vitro. Characterization of the adjuvant role of these liposomes was accomplished by determining the effect on immune induction of several modifications on the liposomal carrier. The results showed that the carrier effect of liposomes was mediated primarily by tumour antigens exposed on the outer surface. Trypsin treatment of the liposomes eliminated 95% of the surface protein and significantly (P less than 0.05) reduced the ability of liposomes to induce cytotoxic splenocytes. The generation of cytolytic activity with liposomes was dose-dependent, with a 10 micrograms protein threshold and a maximal response at 100 micrograms. 'Rigid' liposomes were shown to be significantly (P less than 0.05) more efficacious than fluid liposomes in inducing cytotoxicity. In addition, the data indicate that the xenogeneic cell-mediated immunity exhibits identical classes of effector cells as found in murine-murine reactions. Lymphocytes bearing the THY-1, Lyt-1 and Lyt-2 surface markers were necessary for immune induction. The role of Lyt-123 subpopulation was suggested by the inability to achieve normal cytolytic levels by reconstitution with Lyt-1 plus Lyt-2 cells. Adherent cells were, as expected, necessary for the generation of primary immunity. Indeed, the interaction of I-A+ adherent cells with liposomes for at least 8 h was required to generate subsequent maximal T cell cytotoxic activity. The phenotype of the cytotoxic effector cell was Thy-1+, Lyt-2+, and I-Ad-. If this were an allo-or syngeneic, and not a xenogeneic system, this study would be of less interest. However, when coupled with the known molecular homologies between murine and human lymphocyte antigens, these results suggest that the concept of cross species major histocompatibility complex (MHC) restriction is tenable. Thus the liposome is not only an effective antigen carrier, but also a functional adjuvant for in vitro induced cell-mediated immunity.     

Immunogenicity and safety of liposome-vaccine encapsulating hepatitis B surface antigen and phosphodiester CpG oligodeoxynucleotides

CpG oligodeoxynucleotides (CpG ODN) as adjuvant have been extensively studied in recent years. Phosphodiester CpG ODN (PO CpG ODN) can perfectly mimic bacterial DNA in enhancing immune response but are vulnerable to nucleases in vivo . This study aimed to evaluate the immunostimulatory potential and safety of phosphodiester CpG ODN encapsulated in nonphospholipid liposomes. BALB/c mice were immunized intramuscularly with different formulations of liposomes,CpG ODN and hepatitis B surface antigen (HBsAg). The results demonstrated that the encapsulated PO CpG ODN were protected against rapid degradation in vivo and retained their adjuvant activity. PO CpG ODN encapsulated with HBsAg in liposomes induced strong Th1-biased or Th1/Th2 mixed humoral immune response in mice with the magnitude similar to their phosphothioate equivalent in the same formulation. High IFN-gamma production induced by this formulation confirmed the generation of strong cellular immune response. Additionally, co-delivery of HBsAg and PO CpG ODN improved the immune response over that obtained with separate delivery. Safety experiment showed that liposome-encapsulaed PO CpG ODN and HBsAg caused mild systemic and moderate local adverse reaction. In conclusion, our data shows that PO CpG ODN encapsulated in liposomes fully exhibit their Th1-type adjuvant activity and act as a potential adjuvant for vaccines.     

Improvement of long-lasting response and antibody affinity by the complexation of antigen with complement C3b

Complement protein C3 plays a major role in cell regulation and immune response. This last point is mainly due to C3's capacity to act as a bifunctional link between antigen and immunocompetent cells. In a previous work, we have reported the adjuvant effect produced by linking C3 fragments to antigen. In this paper, we characterize the long-term secondary antibody response induced by C3b-antigen complexes. Mice were immunized using either a protein (hen egg lysozyme) or an ovalbumin-derived peptide as antigen. We compared the secondary response elicited by these antigens covalently linked to C3b or emulsified in complete Freund's adjuvant (CFA). Our results provide evidence that C3b linkage induces better long-term adjuvant effect than CFA, resulting in the production of a higher specific IgG titer with a better affinity.     

Saponin adjuvant primes for a dominant interleukin-10 production to ovalbumin and to Trypanosoma cruzi antigen.

The adjuvant activity of saponin for T-cell responses was evaluated and compared with that of complete Freund's adjuvant (CFA) in two antigen systems: a lysate of the protozoa Trypanosoma cruzi and ovalbumin (OA). Strong delayed-type hypersensitivity and T-cell proliferate responses, comparable with those stimulated by CFA, were observed for both antigens following immunization with saponin as adjuvant. Upon in vitro secondary antigen stimulation, high interleukin-10 (IL-10) and low interferon-gamma (IFN-gamma) levels were observed in lymph node (LN) cell cultures from saponin-immunized mice in contrast with the high IFN-gamma and decreased IL-10 production by LN cells from CFA-immunized mice. Production of IL-10 and IFN-gamma in these conditions was CD4-activation dependent. Concanavalin A (Con A)-stimulated interleukin-4 (IL-4) production was higher in saponin-immunized mice than in CFA-immunized mice. IL-10 produced by LN cells from saponin-immunized mice suppressed IFN-gamma production and Con A-induced proliferation. Taken together, these data are consistent with in vivo stimulation of both T-helper (Th)1 and Th2-type cells by immunization with saponin; in vitro a Th2-type cytokine response with high IL-10 production predominates, indicating preferential priming towards a Th2-type response. Immunization with CFA induced a Th1-type cytokine response. To our knowledge this is the first report in which an adjuvant is shown to prime for a dominant IL-10 production.     

Liposome-entrapped T-cell peptide provides help for a co-entrapped B-cell peptide to overcome genetic restriction in mice and induce immunological memory.

We have investigated the possibility of a T-cell epitope peptide providing help for a B-cell epitope peptide when both peptides are co-entrapped in the same liposomes. Epitope models used were a 28 amino acid peptide from the S region of the hepatitis B surface antigen (HBsAg) (subtype adw) containing an H-2s Th-cell epitope, and a 33 amino acid peptide from the pre-S1 region of the HBsAg (subtype adw) designed to exclude an adjacent H-2s T-cell epitope, the latter (pre-S1) peptide being recognized by SJL (H-2s) mice as a B-cell epitope. SJL(H-2s) mice were immunized twice intramuscularly with S or pre-S1 peptide alone, co-entrapped in the same liposomes or entrapped in separate liposomes which were mixed before injection. Analysis of sera for anti-peptide IgG1 antibodies revealed that the Th-cell peptide provided help for the pre-S1 peptide only when the two peptides were co-entrapped in the same vesicles. This helper effect was found to correlate with the ability of S peptide (co-entrapped with the pre-S1) to stimulate T-cell proliferation in vitro. There was no IgG1 response against pre-S1 peptide in mice immunized with a mixture of the free peptides or a mixture of separately entrapped peptides. A helper effect, albeit much weaker, was also observed in mice immunized with the two peptides emulsified in incomplete Freund's adjuvant. Antisera from mice immunized with both peptides co-entrapped in liposomes were found to bind to full length (pre-S1 containing) recombinant HBsAg. Moreover, binding values were much higher than those seen with antisera from animals immunized with the liposomal S peptide above, presumably because of full access of anti-pre-S1 antibodies to the pre-S1 region of the rHBsAg. It is concluded that liposomes could serve not only as an immunological adjuvant for peptides but also as a carrier for Th- and B-cell epitopes thus eliminating the need for covalent linkage to a carrier protein.     

Immunoadjuvant action of liposomes: comparison with other adjuvants

Dehydration-rehydration vesicles (DRV liposomes) composed of equimolar phospholipid and cholesterol and containing bovine serum albumin (BSA) were used together with free BSA to immunize Balb/C mice. Primary and secondary immune responses (IgG1) to the liposomal antigen, as measured by ELISA in mouse sera, were similar for egg phosphatidylcholine (PC) and distearoyl phosphatidylcholine (DSPC) DRV, and much greater than those elicited by free BSA. The adjuvanticity of PC DRV was compared with that of aluminium salts (alum), complete Freund's adjuvant (CFA) and N-acetyl muramyl-L-threonyl-D-isoglutamine ([Thr1]MDP), the latter used as such or in a liposome form co-entrapped with the antigen. DRV (with or without co-entrapped [Thr1]MDP), and alum were equally strong in producing primary and secondary immune responses (IgG1) to BSA. Such responses were significantly higher than those achieved with CFA and [Thr1]MDP alone. The implications of these results for the potential role of liposomes as immunological adjuvants in vaccines are discussed.     

The pan HLA DR-binding epitope improves adjuvant-assisted immunization with a recombinant protein containing a malaria vaccine candidate

The pan HLA DR-binding epitope (PADRE) has been proposed as a simple carrier epitope suitable for use in the development of synthetic and recombinant vaccines. Using the mouse model, we evaluated whether PADRE could improve adjuvant-assisted immunizations with a recombinant malarial protein containing the 19kDa C-terminal region of merozoite surface protein 1 (MSP1(19)) that is a Plasmodium vivax vaccine candidate. Initially, the antibody immune response was evaluated in C57BL/6 mice, a mouse strain which develops a strong T cell immune response to PADRE. When administered in distinct adjuvant formulations, antibody titers induced by the recombinant protein His(6)MSP1(19)-PADRE were not significantly different to those generated by complete/incomplete Freund's adjuvant (CFA/IFA) in terms of magnitude, affinity, IgG subclasses and longevity. However, in C57BL/6 mice immunized with the recombinant protein His(6)MSP1(19), strong antibody responses could be generated in the presence of CFA/IFA but not other classes of adjuvants such as CpG ODN 1826 or MPL/TDM. Similarly, in BALB/c mice that do not develop T cells specific for PADRE, the recombinant protein His(6)MSP1(19)-PADRE failed to induce high antibody titers in the presence of adjuvants other than CFA/IFA. Our results indicated that when adjuvants that are not as strong as CFA/IFA are employed, the presence of PADRE greatly improved adjuvant-assisted antibody immune responses induced by a malarial recombinant antigen. Considering the great limitations of adjuvants for human use, our observation may improve the rational design of new vaccine formulations.