Roberta L. Richards

Interactions of plant lectins with glycolipids in liposomes

Abstract A panel of five plant lectins with different binding specificities was used to determine if plant lectins could bind specifically to membrane-associated glycolipids. Ricinis communis and wheat germ agglutinins both bound specifically to mixed brain gangliosides and globoside I from human erythrocytes. Wheat germ agglutinin also bound to ganglioside GM1 and human erythrocyte ceramide trihexoside, but not to ceramide dihexoside, mono-, or digalactosyl diglycerides. Concanavalin A bound to liposomes with or without glycolipid substituents, and this binding was partially inhibited by α-methyl mannoside. This study indicates that lectins can specifically recognize and bind to certain glycolipids in membranes.

Immunization against the murine malaria parasite Plasmodium yoelii using a recombinant protein with adjuvants developed for clinical use.

Mice vaccinated with a recombinant protein containing the two EGF-like modules of Plasmodium yoelii merozoite surface protein-1 in liposomes or combined with the formulations SBAS2.1 and SBAS2, were protected against a lethal malaria infection. The protection achieved with these adjuvants developed for clinical use was as good as or better than that achieved with Freunds adjuvant. A parasite-specific response was needed for protection. Analysis of the immunoglobulin sub-class response showed that MSP-1-specific IgG1, and to a lesser extent IgG2a and IgG2b, were induced, suggesting that these antibodies were important for protection. Mice passively immunized with serum or purified IgG from vaccinated mice had delayed onset of parasitemia and were able to control the infection.

Immunogenicity of liposomal malaria sporozoite antigen in monkeys: adjuvant effects of aluminium hydroxide and non-pyrogenic liposomal lipid A

The immunogenicity of a recombinant protein (R32tet32) containing sequences from the tetrapeptide repeat region of the circumsporozoite protein of Plasmodium falciparum was enhanced by encapsulation in liposomes containing lipid A and adsorption of the liposomes with alum. The toxicities and efficacies of preparations containing different types and doses of lipid A were assessed by studying pyrogenicity in rabbits and adjuvanticity in monkeys. In each case liposomal lipid A was 25-fold to 200-fold less pyrogenic than free lipid A. Monophosphoryl lipid A, whether free or in liposomes, was the least pyrogenic of the three lipid A preparations tested. High antibody levels were obtained after immunization of rhesus monkeys with a formulation consisting of alum-adsorbed liposomes in which the liposomes contained R32tet32 and a strongly pyrogenic dose of native lipid A. Excellent antibody levels were also observed in monkeys immunized with a combination of R32tet32 encapsulated in alum-adsorbed liposomes containing non-pyrogenic doses of monophosphoryl lipid A and alum. The adjuvant effect was related to the dose of the lipid A in the liposomes, and the adjuvant effect was still strongly expressed despite suppression of the pyrogenic effect of lipid A. Antibody levels were considerably lower in monkeys immunized with liposomes lacking lipid A. It was concluded that a non-pyrogenic formulation of alum-adsorbed liposomes, in which the liposomes contained both lipid A and an encapsulated synthetic sporozoite antigen, shows considerable promise for inducing high titres of antibodies to sporozoites.

Immune reactivities of antibodies against glycolipids—I: Properties of anti-galactocerebroside antibodies purified by a novel technique of affinity binding to liposomes

Abstract A new, simple technique is described for purifying specific anti-glycolipid (galactocerebroside) antibodies from antiserum. The antibodies first were adsorbed to galactocerebroside-containing liposomes and then were recovered, after washing the liposomes, by elution with 1 M NaI, or by extraction with a saline-chloroform mixture. Depending on the antiserum used, this procedure resulted in approximately a 175-fold to 3800-fold purification of antibodies from whole serum when calculated on an activity/protein basis. The antibodies were tested for activity by means of complement-dependent glucose release from separate galactocerebroside-containing indicator liposomes. It was found that the hydrophobic portions both of the galactocerebroside and the surrounding phospholipids had an influence on antibody binding. The relative anti-galactocerebroside activity, as determined by liposomal glucose release, was directly related to the galactocerebroside fatty acyl chain length. This suggested that the presence of a longer fatty acyl chain in the glycolipid molecule made the carbohydrate more accessible to binding with specific antibody. Furthermore, the degree of adsorption of antibody activity from the antiserum was progressively decreased when the hydrophilic region of the phospholipid membrane was increased due to the presence of phospholipids containing progressively longer fatty acids. These results were consistent with the hypothesis that under certain conditions the immunologically-active group on galactocerebroside may be partially ‘buried’ in the phospholipid bilayer and may be less available for antibody binding. With certain antisera, the antibodies eluted from liposomes having longer phospholid fatty acids (distearoyllecithin) had higher avidity for galactocerebroside than did those from liposomes having shorter phospholipid fatty acids (dimyristoyllecithin). In contrast, a long fatty acyl chain of galactocerebroside (lignoceroyl galactocerebroside) resulted in eluted antibodies having a lower avidity than when a shorter glycolipid (stearoyl or palmitoyl galactocerebroside) was used. It was thought that these results were caused by preferential binding of high affinity antibodies under conditions in which binding was partially hindered due to steric conditions.

Liposomes containing lipid A : a potent nontoxic adjuvant for a human malaria sporozoite vaccine

Liposomes containing lipid A have been developed as adjuvants for inducing humoral immunity to synthetic antigens containing repeat sequence epitopes from the circumsporozoite protein of Plasmodium falciparum. Preclinical studies demonstrated that liposomes containing lipid A and encapsulated antigen could overcome immunosuppression observed with antigen alone. When liposomes containing lipid A were adsorbed with aluminum hydroxide (alum), further stimulation of humoral immunity against encapsulated antigen was observed in animals. In the presence of huge doses of liposomal lipid A pyrogenicity was not observed and adjuvant activity was enhanced. A phase I human clinical trial has been initiated utilizing a vaccine containing a synthetic recombinant antigen and monophosphoryl lipid A in liposomes and nonliposomal alum as a further adjuvant. Preliminary results confirm that the vaccine lacks significant acute toxicity in humans and causes very strong specific humoral immunity against the appropriate epitopes of the target antigen.

Induction of cytolytic and antibody responses using Plasmodium falciparum repeatless circumsporozoite protein encapsulated in liposomes

Plasmodium circumsporozoite (CS) protein-induced antibody and T-cell responses are considered to be important in protective immunity. Since the key repeat determinant of the CS protein may actually restrict the recognition of other potential T- and B-cell sites, a modified Plasmodium falciparum CS protein lacking the central repeat region, RLF, was expressed in Escherichia coli. On purification, RLF was encapsulated into liposomes [L(RLF)] and used for the in vivo induction of cytolytic T lymphocytes (CTL) and antibodies. Immunization of B10.Br (H-2k) mice with L(RLF), but not with RLF, induced CD8+ CTL specific for the P. falciparum CS protein CTL epitope, amino acid residues 368-390. Anti-L(RLF) serum reacted with antigens on intact sporozoites and inhibited sporozoite invasion of hepatoma cells. Antibody specificity studies in New Zealand White rabbits revealed new B-cell sites localized in amino acid residues 84-94, 91-99, 97-106 and 367-375. Although the mechanisms by which liposomes enhance cellular and humoral immune responses remain unknown, liposome-formulated vaccines have been well tolerated in humans; hence, their use in vaccines, when efficacy depends on antibody and CTL responses, may be broadly applicable.

Influence of temperature on complement-dependent immune damage to liposomes.

Maximal release of trapped liposomal glucose, in the presence of saturating amounts of liposomal antigen (galactocerebroside), antiserum (anti-galactocerebroside), and complement, was dependent on temperature. At lower temperatures (20--25 degrees C), maximal glucose release was inversely related to liposomal phospholipid fatty acyl chain length (dimyristoyl phosphatidylcholine > dipalmitoyl phosphatidylcholine > distearoyl phosphatidylcholine > sphingomyelin). At higher temperatures (32--35 degrees C) a limiting plateau of glucose release, at approx. 60%, was reached, or approached, by all preparations. Sphingomyelin liposomes still released less glucose than those prepared from other phospholipids, even at 35 degrees C. The titers of antiserum and complement (ABL50/ml and CL50/ml) were dependent on temperature, and differences based on liposomal phospholipid fatty acyl chain length were observed. Analysis of antiserum and complement-dependence on temperature, and on phospholipid type, revealed that although antibody binding to galactocerebroside undoubtedly was subject to steric hindrance due to interference by surrounding phospholipids at 20--25 degrees C, steric hindrance did not play a major role in blocking antibody binding above 32 degrees C.

Selection of an adjuvant for vaccination with the malaria antigen, MSA-2

Various formulations of the Plasmodium falciparum merozoite surface antigen, MSA-2, were made and tested in animals in order to select one for use in human vaccine trials. Recombinant constructs representing both major allelic forms of MSA-2 were formulated with a range of adjuvants and used to immunize rabbits, mice and sheep. After immunization, antibody responses obtained with the most potent adjuvants were at least tenfold greater than responses obtained with the least potent adjuvant Alhydrogel, which was used as the reference standard, although its lower potency indicated against its further use in clinical trials. Based on broadly similar results obtained with the three animal species, several adjuvants, including the water-in-oil adjuvant Montanide ISA 720, the oil-in-water adjuvant SAF-1, and liposomes containing lipid A formulated with Alhydrogel were demonstrated to be potent and potentially suitable for the clinical evaluation of MSA-2 as a candidate malaria vaccine antigen. Of these, ISA 720 was selected for further trial.

Liposome spin immunoassay: A new sensitive method for detecting lipid substances in aqueous media

A new sensitive immunoassay procedure is described for quantitative detection of glycolipids and other lipids in aqueous media. As with other immunoassays specific antiserum is first reacted with the free lipid hapten. The amount of antibody activity remaining is measured by assaying the release, in the presence of complement, of spin label marker from liposomes containing the same lipid hapten. Using this method, 2.6 pmol of aqueous Forssman hapten was detected, and the sensitivity could be increased further.

Novel adjuvant strategies for experimental malaria and AIDS vaccines

Adjuvant research has improved the ability of biotechnology to generate novel vaccines. Numerous strategies for enhancing the immunogenicity of synthetic peptides and proteins have been identified. This overview focuses on adjuvant development and vaccine delivery systems that provide new tools for amplifying the effectiveness of ongoing malaria and AIDS vaccine development programs. In addition, some of the complex challenges and issues that have become associated with the delivery of modern vaccines in man are outlined. As adjuvant research continues to open new opportunities in vaccine development, there is renewed expectation that further generations of safe and potent vaccines will be possible against a broad spectrum of infectious agents and cancer.