Prostaglandin and thromboxane in liposomes: suppression of the primary immune response to liposomal antigens

Liposomes containing lipid A as adjuvant and also containing prostaglandin E2 or thromboxane B2 were examined for the ability to influence induction of humoral immunity against liposomal protein or lipid antigens in rabbits. The protein antigen consisted of cholera toxin that was bound to ganglioside GM1 on the surface of the liposomes. High titers of anti-cholera toxin antibodies were produced and IgM and IgG responses were detected. When the immunizing liposomes contained either prostaglandin E2 or thromboxane B2 as part of the lipid bilayer, the primary immune response, involving both IgM and IgG antibodies, was greatly reduced. The secondary immune response observed after a boosting immunization was not suppressed by liposomal eicosanoids. A similar inhibitory effect on the primary response was observed when liposomal lipid antigens were examined instead of cholera toxin. An inhibitory effect of liposomal prostaglandin E2 on the phagocytic uptake of opsonized liposomes by cultured macrophages was also observed, suggesting that liposomal eicosanoids can have direct local effects on macrophages that might influence the immune response to liposomal antigens.     

Immunoglobulin immobilized liposomal constructs for transmucosal vaccination through nasal route

The aim of the present investigation was to evaluate the prospective of surface-engineered vesicular carriers for mucosal immunization via the nasal route. IgG antibody was immobilized on the surface of hepatitis B surface antigen (HBsAg) antigen–loaded liposomes. The developed formulations were characterized on the basis of physicochemical parameters, such as morphology, particle size, polydispersity index, entrapment efficiency, and zeta potential. Liposomal formulations were then evaluated for in-process antigen stability and storage stability. In vivo studies were conducted to visualize targeting potential, localization pattern, and immunogenicity. In addition, immune response was compared with alum-HBsAg vaccine injected intramuscularly. The serum anti-HBsAg titer, obtained from the postnasal administration of IgG-coupled liposomes, was significantly higher than plain liposomes. Moreover, IgG-coupled liposomes generated both humoral (i.e., systemic and mucosal) and cellular immune responses upon nasal administration, while the alum-adsorbed antigen displayed neither cellular (cytokine level) nor mucosal (IgA) response. The formulation also displayed enhanced transmucosal transport, improved in vitro stability, and effective immunoadjuvant property. To conclude, IgG antibody-coupled liposomes may serve as novel carriers to augment the secretory immune response of antigen encapsulated in the liposomes, apparently by escalating liposome uptake via M cells, thereby rationalizing their use as a carrier adjuvant for nasal subunit vaccines.     

Induction of CD4(+) and CD8(+) Bordetella pertussis toxin subunit S1 specific T cells by immunization with synthetic peptides.

In this study two synthetic peptides from the Bordetella pertussis toxin subunit S1 were conjugated to human anti-idiotypic antibodies and used as an immunogen in cancer patients to induce immunity. The aims of the present report are to explain why no carrier or adjuvant effect of the conjugated pertussis peptides could be established regarding induction of responses against the anti-idiotype and to explore the type and quality of induced anti-pertussis immune responses. The lack of carrier and adjuvant effect of the peptides might be related to the fact that the anti-idiotypic antibodies by themselves include helper epitopes and that none of the patients had a detectable T cell response against any of the selected peptides before immunization, which might be a requirement for an adjuvant effect. However, three of four immunized patients mounted a humoral as well as cellular response against the pertussis peptides used. The induced T cell immunity was restricted to one of the two peptides in responding patients. Established T cell lines and MHC blocking studies indicated that the T cell epitopes of the two peptides had a different MHC restriction. The type of T cell response induced seemed to govern the humoral response. The only durable antibody response was accompanied by the presence of a CD4(+) T cell response against the same peptide. Immunization with an anti-idiotype conjugated to synthetic peptides might thus induce both a B and a T cell response against the peptides and the type of induced T cells (CD4 or CD8) governs the quality of the humoral response. Moreover, the possibility of boosting or inducing a response against the antigen from which the peptide sequences were deduced also seemed feasible.     

Effect of dehydration and rehydration of the pH-sensitive liposomes containing chimeric gag-V3 virus like particle on their long-term stability

One of the practical limitations with the use of liposomes for delivery of the pharmaceutical substances such as antigens is that liposomes are relatively unstable in storage. In order to extend the stability of liposome in storage without affecting their functional activity, solution-type liposomes were dehydrated to form a structurally intact dry liposomes. Comparative immunological evaluation was carried out for both dry and solution-type liposomes containing gag-V3 chimera, consequently it was found that dry liposomes elicited both humoral and cellular response as efficiently as solution-type liposomes did against the same gag-V3 antigen. Especially, long-term stability of the liposomes was remarkably enhanced by the dehydration made to liposomes without a significant change in its ability to elicit immune responsein vivo. These results indicate that dry pH-sensitive liposome may become an effective delivery and adjuvant system for general vaccine development.     

Preclinical trials of L-lysine-alpha-oxidase, an antitumor enzyme

The effect of L-lysine-alpha-oxidase from a representative of the Trichoderma genus on the humoral immune response to protein antigens was studied. It was shown that repeated fife-fold intravenous administrations of L-lysine-alpha-oxidase in a dose of 35 U/kg had no depressive action on the humoral immunity. The enzyme had no suppressive effect on the delayed type hypersensitivity to xenogenous erythrocytes. The use of L-lysine-alpha-oxidase in a therapeutic dose or in a twice as higher dose had no reliable effect on the leukocyte migration capacity vs. the control.     

Fusogenic potential of prokaryotic membrane lipids. Implication in vaccine development.

Development of protective immunity against many pathogens, particularly viruses, requires fine orchestration of both humoral- and cell mediated-immunity. The immunization of animals with soluble antigens usually leads to the induction of humoral immune responses. In contrast, the activation of a cell-mediated immune response against exogenous antigens has always been a challenge, requiring special strategies to expose them to the proteasome, a multifunctional protease complex in the cytosol of the target cells. The degradation of the protein by the cytosolic proteolytic system forms a cardinal step for the induction of cytotoxic T lymphocytes (CTLs). In the present study, we report that a potent primary CTL response against a soluble protein, ovalbumin, can be induced in mice by encapsulating it in the liposomes comprised of Escherichia coli membrane lipids. These lipids were shown to induce strong membrane-membrane fusion as evident from resonance energy transfer and content mixing assays. Furthermore, the fusion of these liposomes with living cells (J774 A1) was demonstrated to result in effective transfer of a fluorescent lipid probe to the plasma membrane of the cells. Moreover, ricin A, a protein synthesis inhibitor that does not cross plasma membrane, was demonstrated to gain access to the cytosol when it was encapsulated in these liposomes. Finally, the liposomes were demonstrated to behave like efficient vehicles for the in vivo delivery of the antigens to the target cells resulting in the elicitation of antigen reactive CD8+ T cell responses.     

Influence of peptide acylation, liposome incorporation, and synthetic immunomodulators on the immunogenicity of a 1-23 peptide of glycoprotein D of herpes simplex virus: implications for subunit vaccines.

A peptide corresponding to residues 1 to 23 of glycoprotein D of herpes simplex virus type 1 was chemically synthesized and coupled to a fatty acid carrier by standard Merrifield synthesis procedures. The resulting peptide-palmitic acid conjugate (acylpeptide) exhibited enhanced immunogenicity in mice as compared with that exhibited by the free form of the peptide. Incorporation of the acylpeptide into liposomes further increased the immunogenicity of the peptide, while inclusion of the immunomodulators muramyl tripeptide phosphatidylethanolamine and monophosphoryl lipid A into the same liposome stimulated the strongest response. The humoral immune responses induced by the acylpeptide-liposome construct were greater than those induced by peptide in Freund complete adjuvant, and cellular responses were equal. The acylpeptide-immunomodulator-liposome formulation also induced significant levels of protective immunity, although the immunity was less than that induced by herpes simplex virus infection. Acylated peptides, especially in liposomes, were taken up more effectively by draining lymph nodes, which possibly accounts in part for the enhanced immunogenicity of the peptides. Since the acylpeptide-immunoliposome formulation used was nontoxic, it could represent a useful way to enhance immunogenicity of subunit peptides used for vaccine purpose in humans and animals.     

TLR7 imidazoquinoline ligand 3M-019 is a potent adjuvant for pure protein prototype vaccines

Cancer vaccines, while theoretically attractive, present difficult challenges that must be overcome to be effective. Cancer vaccines are often poorly immunogenic and may require augmentation of immunogenicity through the use of adjuvants and/or immune response modifiers. Toll-like receptor (TLR) ligands are a relatively new class of immune response modifiers that may have great potential in inducing and augmenting both cellular and humoral immunity to vaccines. TLR7 ligands produce strong cellular responses and specific IgG2a and IgG2b antibody responses to protein immunogens. This study shows that a new TLR7 ligand, 3M-019, in combination with liposomes produces very strong immune responses to a pure protein prototype vaccine in mice. Female C57BL/6 mice were immunized subcutaneously with ovalbumin (OVA, 0.1 mg/dose) weekly 4x. Some groups were immunized to OVA plus 3M-019 or to OVA plus 3M-019 encapsulated in liposomes. Both antibody and cellular immune responses against OVA were measured after either two or four immunizations. Anti-OVA IgG antibody responses were significantly increased after two immunizations and were substantially higher after four immunizations in mice immunized with OVA combined with 3M-019. Encapsulation in liposomes further augmented antibody responses. IgM responses, on the other hand, were lowered by 3M-019. OVA-specific IgG2a levels were increased 625-fold by 3M-019 in liposomes compared to OVA alone, while anti-OVA IgG2b levels were over 3,000 times higher. In both cases encapsulation of 3M-019 in liposomes was stronger than either liposomes alone or 3M-019 without liposomes. Cellular immune responses were likewise increased by 3M-019 but further enhanced when it was encapsulated in liposomes. The lack of toxicity also indicates that this combination may by safe, effective method to boost immune response to cancer vaccines.     

Intranasal Immunization with DOTAP Cationic Liposomes Combined with DC-Cholesterol Induces Potent Antigen-Specific Mucosal and Systemic Immune Responses in Mice

Despite the progress made by modern medicine, infectious diseases remain one of the most important threats to human health. Vaccination against pathogens is one of the primary methods used to prevent and treat infectious diseases that cause illness and death. Vaccines administered by the mucosal route are potentially a promising strategy to combat infectious diseases since mucosal surfaces are a major route of entry for most pathogens. However, this route of vaccination is not widely used in the clinic due to the lack of a safe and effective mucosal adjuvant. Therefore, the development of safe and effective mucosal adjuvants is key to preventing infectious diseases by enabling the use of mucosal vaccines in the clinic. In this study, we show that intranasal administration of a cationic liposome composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N'',N''-dimethylaminoethane)-carbamoyl] (DC-chol) (DOTAP/DC-chol liposome) has a potent mucosal adjuvant effect in mice. Intranasal vaccination with ovalbumin (OVA) in combination with DOTAP/DC-chol liposomes induced the production of OVA-specific IgA in nasal tissues and increased serum IgG1 levels, suggesting that the cationic DOTAP/DC-chol liposome leads to the induction of a Th2 immune response. Additionally, nasal-associated lymphoid tissue and splenocytes from mice treated with OVA plus DOTAP/DC-chol liposome showed high levels of IL–4 expression. DOTAP/DC-chol liposomes also enhanced OVA uptake by CD11c⁺ dendritic cells in nasal-associated lymphoid tissue. These data demonstrate that DOTAP/DC-chol liposomes elicit immune responses via an antigen-specific Th2 reaction. These results suggest that cationic liposomes merit further development as a mucosal adjuvant for vaccination against infectious diseases.     

Enhancement of immune responses by DNA vaccination through targeted gene delivery using mannosylated cationic liposome formulations following intravenous administration in mice

The present study investigated the potency of the mannosylated cationic liposomes (Man liposomes) that we have developed in novel DNA vaccine carrier. Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. The potency of the Man liposome/pCMV-OVA complex was compared with naked pCMV-OVA and that complexed with DC-Chol liposomes. In cultured mouse peritoneal macrophages, MHC class I-restricted antigen presentation of the Man liposome/pCMV-OVA complex was significantly higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. After intravenous administration, OVA mRNA expression and MHC class I-restricted antigen presentation on CD11c+ cells and inflammatory cytokines, such as TNF-alpha, IL-12, and IFN-gamma, that can enhance the Th1 response of the Man liposome/pCMV-OVA complex were higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. Also, the spleen cells from mice immunized by intravenous administration of the Man liposome/pCMV-OVA complex showed the highest proliferation response and IFN-gamma secretion. These findings suggest that the targeted delivery of DNA vaccine by Man liposomes is a potent vaccination method for DNA vaccine therapy.     

脂质体、免疫刺激复合物和壳聚糖作为黏膜免疫载体的研究现状

查阅国内外文献,采用整理归纳、引用和分析的方法,概述了鼻黏膜免疫载体材料脂质体、免疫刺激复合物和壳聚糖的生物学特性及其在气溶胶疫苗领域的应用。脂质体、免疫刺激复合物和壳聚糖作为气溶胶疫苗鼻黏膜免疫的载体,能够增强疫苗的免疫原性,提高机体的体液免疫和细胞免疫水平,因此,可作为疫苗抗原激发特异免疫应答的生物材料。     

The Use of Transmembrane pH Gradient-Driven Drug Encapsulation in the Pharmacodynamic Evaluation of Liposomal Doxorubicin

Abstract

The toxicity and efficacy properties of doxorubicin entrapped inside liposomes are sensitive to the physical characteristics of the vesicle carrier system. Studies addressing such relationships must use preparation procedures with the ability to independently vary vesicle size, lipid composition and drug to lipid ratio while maintaining high trapping efficiencies. The transmembrane pH gradient-driven encapsulation technique allows such liposomal doxorubicin formulations to be prepared. Pharmacokinetic, toxicology and antitumour studies with these systems have revealed several important relationships between liposome physical properties and biological activity. The acute toxicity of liposomal doxorubicin is related primarily to the ability of the liposomes to retain doxorubicin after administration. Including cholesterol and increasing the degree of acyl chain saturation of the phospholipid component in the liposomes significantly decreases drug leakage in the blood, reduces cardiac tissue accumulation of doxorubicin and results in increased LD₅₀ values. In contrast, the efficacy of liposomal doxorubicin is most influenced by liposome size. Specifically, liposomes with a diameter of approximately 100 nm or less exhibit enhanced circulation lifetimes and antitumour activity. While these relationships appear to be rather straightforward, there exist anomalies which suggest that a more thorough evaluation of liposomal doxorubicin pharmacokinetics may be required in order to fully understand its mechanism of action. A key feature in this regard is the ability to differentiate between non-encapsulated and liposome encapsulated doxorubicin pools in the circulation as well as in tumours and normal tissues. This represents a major challenge that must be addressed if significant advances in the design of more effective liposomal doxorubicin formulations are to be achieved.     

Oral delivery of antigens in liposomes with some lipid compositions modulates oral tolerance to the antigens

Several liposomes containing ovalbumin (OVA), a model antigen, with different lipid compositions were prepared in order to evaluate their ability to induce oral tolerance. Oral administration of these liposomal OVAs induced suppression of the proliferative responses of popliteal lymph node cells from the treated mice to OVA, suggesting that these treated mice were tolerized. The efficiency of the induction of oral tolerance was affected by the liposome composition. OVA entrapment in these liposomes could modulate the tolerizing dose of OVA itself. These results suggest that some liposomes can be suitable antigen-delivery systems for modulated and/or effective induction of oral tolerance.     

阿霉素脂质体的制备及其体外抗肿瘤活性的初步研究

目的:应用超声波分散法制备脂质体阿霉素,并比较脂质体阿霉素与游离性阿霉素抗肿瘤活性。方法:以卵磷脂和胆固醇为原料,将阿霉素包封于脂质体中,采用超声分散法制备脂质体阿霉素,对其在290-700nm范围内进行紫外扫描,用SephedexG-50柱分离脂质体阿霉素并计算其包封率。以昆明种小鼠为载体建立肿瘤模型(S180型肉瘤)和细胞荧光染色法研究脂质体阿霉素的抗肿瘤活性,以ZITA SIZER3000型表面电位与粒度测定仪测定其粒径分布。结果:脂质体阿霉素在480nm处有最大吸收峰值,包封率达91.3%,细胞荧光染色显示,脂质体及游离型阿霉素均对S180细胞有明显的抑制作用。结论:此法制备的脂质体阿霉素包封率高,粒径分布集中,脂质体阿霉素较游离型阿霉素有较强的抗肿瘤活性剂及较低的细胞毒作用,对阿霉素的临床应用有一定的参考价值。     

The influence of repeated injections on pharmacokinetics and biodistribution of different types of sterically stabilized immunoliposomes

Sterically stabilized immunoliposomes (IL) with diameters of about 135 nm carrying mouse IgG, either coupled directly to the liposome surface, or linked to the terminal ends of grafted poly(ethylene glycol) (PEG) chains by a recently described conjugation procedure (Cyanur-PEG-PE), were intravenously injected into rats and the elimination kinetics and biodistribution were determined and compared with control liposomes. The amounts of conjugated antibodies were about 30 μg/μmol total lipid for all IL. In naive rats, plain pegylated liposomes displayed the longest blood circulation time, whereas the terminal-coupled IL exhibited the fastest elimination. Liposomes containing the underivatized anchor molecules circulate nearly as long as plain pegylated liposomes, indicating that the fast elimination of the IL can be attributed to the presence of antibodies.A second injection of identical liposomes 14 days after the first injection had a considerable influence on the pharmacokinetic parameters of the liposomes. The circulation time of plain pegylated liposomes drastically dropped by half and their uptake by the liver increased concomitantly, indicating that the PEG, upon repeated injection, ceases to function as an efficient barrier reducing opsonization and/or immune reactions. The circulation time of conventional IL was moderately reduced upon a second injection, whereas that of the terminally coupled IL was nearly unaffected. These differences among the IL demonstrate that the pharmacokinetic behavior of IL is strongly dependent on the antibody conjugation site on the liposome. The observed effects of repeated injections were similar for liposomes of 90-nm diameter. The phenomena described may have important implications for the repeated application of IL as drug carriers.     

Targeting Stealth liposomes in a murine model of human small cell lung cancer.

Tumor accumulation and therapeutic activity of Stealth liposomes loaded with doxorubicin (DXR) were examined in Balb/c nude mice xenografts inoculated subcutaneously with the human small cell lung cancer (SCLC) cell line, H69. Mice were treated with non-targeted liposomes (SL) or liposomes targeted with antagonist G coupled to the liposome surface (SLG). SLG showed 30-44-fold higher binding to H69 cells harvested from H69 xenografts than SL. At 48 and 72 h post injection, tumor accumulation of [(125)I]tyraminylinulin-containing liposomes was shown to be dependent on liposome size but independent of the presence of the targeting ligand. Maximum tumor uptake of either SLG or SL ranged from 2 to 4% of injected dose/g of tissue. In therapeutic studies, mice received three weekly injections of 3 or 6 mg free DXR/kg or 3 or 10 mg liposomal DXR/kg at initial tumor volumes of either 7 or 33 mm(3). The therapeutic efficacy of DXR-containing SL or SLG was significantly improved over free DXR, but SLG did not improve anti-tumor efficacy relative to SL. Stealth liposomes containing DXR have potential as a therapy against human SCLC tumors.     

The influence of repeated injections on pharmacokinetics and biodistribution of different types of sterically stabilized immunoliposomes

Sterically stabilized immunoliposomes (IL) with diameters of about 135 nm carrying mouse IgG, either coupled directly to the liposome surface, or linked to the terminal ends of grafted poly(ethylene glycol) (PEG) chains by a recently described conjugation procedure (Cyanur-PEG-PE), were intravenously injected into rats and the elimination kinetics and biodistribution were determined and compared with control liposomes. The amounts of conjugated antibodies were about 30 microg/micromol total lipid for all IL. In naive rats, plain pegylated liposomes displayed the longest blood circulation time, whereas the terminal-coupled IL exhibited the fastest elimination. Liposomes containing the underivatized anchor molecules circulate nearly as long as plain pegylated liposomes, indicating that the fast elimination of the IL can be attributed to the presence of antibodies.A second injection of identical liposomes 14 days after the first injection had a considerable influence on the pharmacokinetic parameters of the liposomes. The circulation time of plain pegylated liposomes drastically dropped by half and their uptake by the liver increased concomitantly, indicating that the PEG, upon repeated injection, ceases to function as an efficient barrier reducing opsonization and/or immune reactions. The circulation time of conventional IL was moderately reduced upon a second injection, whereas that of the terminally coupled IL was nearly unaffected. These differences among the IL demonstrate that the pharmacokinetic behavior of IL is strongly dependent on the antibody conjugation site on the liposome. The observed effects of repeated injections were similar for liposomes of 90-nm diameter. The phenomena described may have important implications for the repeated application of IL as drug carriers.     

Enhanced Gene Delivery and Expression in Human Hepatocellular Carcinoma Cells by Cationic Immunoliposomes

Abstract

A targeted vector allowing enhanced gene transfer to human hepatocellular carcinoma (HCC¹) cells in vitro was developed using cationic liposomes covalently conjugated with the mAb AF-20. This high affinity antibody recognizes a rapidly internalized 180 kDa cell surface glycoprotein which is abundantly expressed on the surface of human HCC and other cancer cells. Quantitative binding analysis of liposomes with target cells by flow cytometry showed specific association of mAb-targeted liposomes with human HCC cells. Using mAb-targeted cationic liposomes containing 20% DOTAP, in the presence or absence of serum, gene expression in HuH-7 cells was enhanced up to 40-fold as compared to liposomes conjugated with an isotype-matched non-relevant control antibody. Transfection specificity was not observed in a control cell line that does not express the antigen recognized by mAb AF-20. This study demonstrates that cationic liposome formulations can be targeted with monoclonal antibodies (mAbs) to enhance specific in vitro gene delivery and expression in the presence or absence of serum.     

Immune response to orally consumed antigens and probiotic bacteria

The gut mucosal system must fulfil conflicting roles in suppressing immune responses against orally fed antigens (tolerance) while still retaining the ability to respond to potential enteric pathogens. It must also, to a large degree, not mount an immune response against commensal enteric bacteria and the administration of large numbers of probiotic bacteria formulated as dietary supplements in food products. Contrary to this dogma, it has been found that feeding ovalbumin as a marker antigen, in association with selected probiotic bacteria, appears to prime for an intestinal immune response that is further augmented by skin vaccination. Skin immunization is known to stimulate a strong innate, humoral and cellular immune response. Such dominant immunogenic signals appear to override tolerogenic signals engendered by oral feeding of antigen. High-dose antigen feeding stimulated a strong Th2-dependent antibody response to skin vaccination but completely suppressed cytotoxic T cell responses. This was true even when ovalbumin was administered in conjunction with various selected probiotic bacteria. However, while yeast appeared to be better at priming for an enhanced humoral response, Lactobacillus fermentum and Staphylococcus carnosus were more effective in enhancing the postvaccinal lymphoproliferative response against ovalbumin.     

Improvement of adaptive immunity by antigen-carrying biodegradable nanoparticles

One of the most important aspects in vaccine development is to induce potent antigen-specific immune responses. In this study, we examined the immunological activities of antigen-carrying biodegradable poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) in mice. The immunization with ovalbumin (OVA)-carrying γ-PGA NPs (OVA-NPs) could induce significant expansion of antigen-specific CD8⁺ T cells. Unlike complete Freund’s adjuvant, subcutaneous (s.c.) inoculation of OVA-NPs to footpad did not generate injection site swelling. Although OVA-NPs could induce both antigen-specific cellular and humoral immune responses, the dominant induction of either cellular or humoral immunity was found to depend on their administration routes. Strong antibody production was observed by s.c. immunization, yet no antibody was identified by intranasal immunization. Thus, γ-PGA NPs are a safe and efficient antigen carrier with unique immunological properties.