Enhancing nicotine vaccine immunogenicity with liposomes

A major liability of existing nicotine vaccine candidates is the wide variation in anti-nicotine immune responses among clinical trial participants. In order to address this liability, significant emphasis has been directed at evaluating adjuvants and delivery systems that confer more robust potentiation of the anti-nicotine immune response. Toward that end, we have initiated work that seeks to exploit the adjuvant effect of liposomes, with or without Toll-like receptor agonist(s). The results of the murine immunization study described herein support the hypothesis that a liposomal nicotine vaccine formulation may provide a means for addressing the immunogenicity challenge.     

Liposome-based cationic adjuvant formulations (CAF): Past,present, and future

The use of liposomes as vaccine adjuvants has been investigated extensively over the last few decades. In particular, cationic liposomal adjuvants have drawn attention, with dimethyldioctadecylammonium (DDA) liposomes as a prominent candidate. However, cationic liposomes are, in general, not sufficiently immunostimulatory, which is why the combination of liposomes with immunostimulators has arisen as a strategy in the development of novel adjuvant systems in recent years. One such adjuvant system is CAF01. In this review, we summarize the immunological properties making CAF01 a promising versatile adjuvant system, which was developed to mediate protection against tuberculosis (TB) but, in addition, has shown promising protective efficacy against other infectious diseases requiring different immunological profiles. Further, we describe the stabilization properties that make CAF01 suitable in vaccine formulation for the developing world, which in addition to vaccine efficacy, are important prerequisites for any novel TB vaccine to reach global implementation. The encouraging nonclinical data led to a preclinical vaccine toxicology study of the TB model vaccine, Ag85B-ESAT-6/CAF01, that concluded that CAF01 has a satisfactory safety profile to advance the vaccine into phase I clinical trials, which are scheduled to start in 2009.     

重组粒细胞-巨噬细胞集落刺激因子长效、缓释制剂的研究进展

重组人粒细胞—巨噬细胞集落刺激因子(GM-CSF)临床上有广泛的应用,然而,由于其体内半衰期很短,治疗周期长、不能通过胃肠道屏障进行口服给药,病人必须接受频繁注射。为了减少其注射频率,延长蛋白作用时间,多年来,科学家们对长效GM-CSF的研发进行了多种尝试。这类研究主要集中于三大类:GM-CSF的PEG化、脂质体包封及基于可降解高分子的缓释剂型。然而,这几类方法具有各自优势的同时,也都存在各种不足,如包封率低、对蛋白活性的保护不足、突释问题等。本文将对这三大类针对GM-CSF的长效制剂研究进展的报道,以及各类方法目前存在的主要优缺点进行综述。     

Analytical Characterization of an Oil-in-Water Adjuvant Emulsion

Adjuvants are typically used in subunit vaccine formulations to enhance immune responses elicited by individual antigens. Physical chemical characterization of novel adjuvants is an important step in ensuring their effective use in vaccine formulations. This paper reports application of a panel of quantitative assays developed to analyze and characterize an oil-in-water adjuvant emulsion, which contains glucopyranosyl lipid A (GLA) and is a squalene-based emulsion. GLA is a fully synthetic analogue of monophosphoryl lipid A, which is a Toll-like receptor type 4 agonist and an FDA-approved adjuvant. The GLA-stable emulsion (GLA-SE) is currently being used for a respiratory syncytial virus vaccine in a phase 2 clinical trial. GLA was quantitated using reverse-phased high-performance liquid chromatography (RP-HPLC) coupled to a mass spectrometric detector, achieving higher assay sensitivity than the charged aerosol detection routinely used. Quantitation of the excipients of GLA-SE, including squalene, egg phosphatidyl choline, and Poloxamer 188, was achieved using a simple and rapid RP-HPLC method with evaporative light scattering detection, eliminating chemical derivatization typically required for these chromophore-lacking compounds. DL-α-tocopherol, the antioxidant of the GLA-SE, was quantitated using a RP-HPLC method with conventional UV detection. The experimental results compared well with values expected for these compounds based on targeted composition of the adjuvant. The assays were applied to identify degradation of individual components in a GLA-SE sample that degraded into distinct aqueous and oil phases. The methods developed and reported here are effective tools in monitoring physicochemical integrity of the adjuvant, as well as in formulation studies.     

Lipid A structures containing novel lipid moieties: synthesis and adjuvant properties

Structurally well-defined immune stimulatory molecules are important components of new generation molecular vaccines. In this paper, the design and synthesis of two lipid A analogues containing an unnatural tri-lipid acyl group are described. In a totally synthetic liposomal vaccine system, these re-designed lipid A analogues demonstrate potent immune stimulatory properties including antigen specific T-cell activation.     

Enhanced potency of individual and bivalent DNA replicon vaccines or conventional DNA vaccines by formulation with aluminum phosphate

DNA vaccines against botulinum neurotoxin (BoNTs) induce protective humoral immune responses in mouse model, but when compared with conventional vaccines such as toxoid and protein vaccines, DNA vaccines often induce lower antibody level and protective efficacy and are still necessary to increase their potency. In this study we evaluated the potency of aluminum phosphate as an adjuvant of DNA vaccines to enhance antibody responses and protective efficacy against botulinum neurotoxin serotypes A and B in Balb/c mice. The administration of these individual and bivalent plasmid DNA replicon vaccines against botulinum neurotoxin serotypes A and B in the presence of aluminum phosphate improved both antibody responses and protective efficacy. Furthermore, formulation of conventional plasmid DNA vaccines encoding the same Hc domains of botulinum neurotoxin serotypes A and B with aluminum phosphate adjuvant increased both antibody responses and protective efficacy. These results indicate aluminum phosphate is an effective adjuvant for these two types of DNA vaccines (i.e., plasmid DNA replicon vaccines and conventional plasmid DNA vaccines), and the vaccine formulation described here may be an excellent candidate for further vaccine development against botulinum neurotoxins.     

Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein

Key Ags of Mycobacterium tuberculosis initially identified in the context of host responses in healthy purified protein derivative-positive donors and infected C57BL/6 mice were prioritized for the development of a subunit vaccine against tuberculosis. Our lead construct, Mtb72F, codes for a 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32(C)-Mtb39-Mtb32(N). Immunization of C57BL/6 mice with Mtb72F DNA resulted in the generation of IFN-gamma responses directed against the first two components of the polyprotein and a strong CD8(+) T cell response directed exclusively against Mtb32(C). In contrast, immunization of mice with Mtb72F protein formulated in the adjuvant AS02A resulted in the elicitation of a moderate IFN-gamma response and a weak CD8(+) T cell response to Mtb32c. However, immunization with a formulation of Mtb72F protein in AS01B adjuvant generated a comprehensive and robust immune response, resulting in the elicitation of strong IFN-gamma and Ab responses encompassing all three components of the polyprotein vaccine and a strong CD8(+) response directed against the same Mtb32(C) epitope identified by DNA immunization. All three forms of Mtb72F immunization resulted in the protection of C57BL/6 mice against aerosol challenge with a virulent strain of M. tuberculosis. Most importantly, immunization of guinea pigs with Mtb72F, delivered either as DNA or as a rAg-based vaccine, resulted in prolonged survival (>1 year) after aerosol challenge with virulent M. tuberculosis comparable to bacillus Calmette-Guérin immunization. Mtb72F in AS02A formulation is currently in phase I clinical trial, making it the first recombinant tuberculosis vaccine to be tested in humans.     

Are Anti-Phosphoupid Antibodies to be Expected after Proteoliposomal Hepatitis A Vaccination?

Abstract

Introduction

The so-called IRIV- (Immunopotentiating Reconstituted Influenza Virosomes) system (1) is a new type of vaccine. During the past five years extensive experience has been accumulated concerning the immunological and side effects in organisms, both animals and humans, in order to draw up an assessment of the new vaccine Although IRIV could be placed in the category of liposomal vaccines this classification is, however, not quite correct. Such a classification could raise certain questions regarding its use in human medicine. These questions are derived from toxicological studies in animals to which liposomes combined with lipid A were administered It would be valuable to clear up the difference between an IRIV and a liposomal vaccine, with and without lipid A The IRIV is made up of a combination of liposomes (without lipid A) and influenza vims envelopes. Above all, the inclusion of liposomes imposes itself since they already receive much consideration as a new immunological adjuvant and will probably, in the near future, find a large place both as vaccines and as therapeutics.     

Comments of R. Glück

Abstract

This paper discusses several liposomal approaches: A T-independent response against hapten or lipopolysaccharide by the addition of a T-epitope (HA2 molecule of influenza); the same response against a synthetic peptide-based vaccine; finally the first commercial liposomal hepatitis A vaccine is discussed.     

Liposomes as a Mucosal Adjuvant System: An Intranasal Liposomal Influenza Subunit Vaccine and the Role of IgA in Nasal Anti-Influenza Immunity

Abstract

Liposomes exhibit potent immunoadjuvant activity in a variety of experimental vaccine formulations. We have investigated the mucosal adjuvant activity of liposomes in an influenza subunit vaccine. Mice were immunized intranasally (I.N.) with the major surface antigen of influenza virus, hemagglutinin (HA), mixed with negatively charged liposomes. Inclusion of the liposomes in the vaccine resulted in a marked stimulation of the serum IgG response against the antigen. In addition, the liposomal preparation, but not the antigen alone, induced a significant secretory IgA (s-IgA) response, not only in the lungs and nasal cavity, but also at the mucosa of the urogenital tract. The adjuvant activity of the liposomes appeared to be independent of a physical association of the antigen with the liposomes: Stimulation of antibody responses was observed even when liposomes and antigen were administered separately in time. Serum IgG and local s-IgA responses to I.N. immunization with the liposomal vaccine were comparable to the corresponding responses induced by an influenza infection. Mice immunized with the liposomal vaccine or mice recovered from an influenza infection were completely protected from (re)infection. Protection from nasal infection was abrogated by treatment of the mice with an anti-IgA antiserum, while anti-IgG had no effect, indicating that s-IgA plays an essential role in nasal anti-influenza immunity.     

Liposomes containing glucosyl ceramide specifically bind T4 bacteriophage: a self-assembling nanocarrier formulation

A unique formulation is described comprising liposomes containing glucosyl ceramide (GluCer) in the lipid bilayer to which bacteriophage T4 was attached. Binding of the phage T4 did not occur to glycolipids, such as galactosyl ceramide, containing an aldose in which the C-2 or C-4 conformations were not identical to glucose. These results strongly support previous proposals that glucose is a major receptor moiety for T4 binding to Escherichia coli. By using the binding of T4 to liposomal GluCer, we further describe a formulation that can be used as a self-assembling combined antigen and adjuvant carrier. A peptide antigen derived from C-trimer (Ct) of HIV-1 gp41 was fused to the highly antigenic outer capsid protein (Hoc), a nonessential protein of T4 that spontaneously binds to the T4 capsid. This resulted in display of the Ct-Hoc construct on the T4 capsid, and specific binding of a human monoclonal antibody that recognizes a peptide sequence of Ct was demonstrated. Liposomes containing monophosphoryl lipid A (MPLA) have been demonstrated to have potent adjuvant activities for experimental vaccines both in humans and animals, and because of this, mice were immunized with the Ct-Hoc-T4 construct that was bound to liposomes containing both GluCer and MPLA, resulting in the induction of high titers of Ct-specific antibodies. We conclude that liposomes containing both GluCer and MPLA can spontaneously bind to a construct of T4 that displays antigens that spontaneously binds to the capsid of T4 bacteriophage. This formulation could be utilized as an easily manufactured self-assembling antigen and adjuvant carrier.     

Saponin-adjuvanted particulate vaccines for clinical use

Saponins are well recognised as potent immune stimulators, but their applicability as vaccine adjuvants have been limited due to associated toxicity. Formulation of saponin adjuvant with cholesterol and phospholipid produces the particulate ISCOMATRIX adjuvant, and when antigen is also contained within the particle, an ISCOM vaccine is produced. These particulate vaccines retain the adjuvant activity of the saponin component but without toxicity. Saponin-adjuvanted particulate vaccines have significant potential as a novel strategy in vaccine development. This review discusses (i) recent methodologies which have attempted to increase the flexibility and applicability of this technology by modifying either the vaccine composition or the mode of formulation; (ii) recent evaluations of these technologies for inducing protection against infectious diseases and as cancer immunotherapeutics.     

Effectiveness of liposomal paclitaxel against MCF-7 breast cancer cells

Paclitaxel is an effective chemotherapeutic agent that is widely used for the treatment of several cancers, including breast, ovarian, and non-small-cell lung cancer. Due to its high lipophilicity, paclitaxel is difficult to administer and requires solubilization with Cremophor EL (polyethoxylated castor oil) and ethanol, which often lead to adverse side effects, including life-threatening anaphylaxis. Incorporation of paclitaxel in dimyristoylphosphatidylcholine:dimyristoylphosphatidylglycerol (DPPC:DMPG) liposomes can facilitate its delivery to cancer cells and eliminate the adverse reactions associated with the Cremophor EL vehicle. Accordingly, the effectiveness of liposomal paclitaxel on MCF-7 breast cancer cells was examined. The results from this study showed that (i) the lipid components of the liposomal formulation were nontoxic, (ii) the cytotoxic effects of liposomal paclitaxel were improved when compared with those seen with conventional paclitaxel, and (iii) the intracellular paclitaxel levels were higher in MCF-7 cells treated with the liposomal paclitaxel formulation. The results of these studies showed that delivery of paclitaxel as a liposomal formulation could be a promising strategy for enhancing its chemotherapeutic effects.     

Preparation of nanoliposomes linked to HER2/neu-derived (P5) peptide containing MPL adjuvant as vaccine against breast cancer

The study was aimed at evaluating antitumor and immunomodulatory effects of liposomal vaccine composed of P5 human epidermal growth factor receptor 2 (HER2)/neu-derived peptide coupled to the surface of high-temperature nanoliposomes containing distearoylphosphocholine:distearoylphosphoglycerol:Chol:dioleoylphosphatidylethanolamine (DOPE) comprising monophosphoryl lipid A (MPL) adjuvant in HER2/neu overexpressing the breast cancer model. BALB/c mice bearing TUBO carcinoma were subcutaneously immunized with formulations containing 10 µg P5 peptide and 25 µg MPL three times with 2-week intervals. To determine immuno responses in immunized mice, the amount of released interferon-γ and IL-4 were measured by the enzyme-linked immunospot method and the flow cytometric analysis on the isolated splenocytes. The results demonstrated that tumor-bearing mice immunized with Lip/DOPE/MPL/P5 formulation had the most released interferon-γ and the highest cytotoxic T lymphocyte responses that led to the lowest tumor size and the longest survival time than those of other formulations. The results achieved by Lip/DOPE/MPL/P5 formulation could make it a suitable candidate to induce effective antigen-specific tumor immunity against breast cancer.     

Pros and cons of the liposome platform in cancer drug targeting

Coating of liposomes with polyethylene-glycol (PEG) by incorporation in the liposome bilayer of PEG-derivatized lipids results in inhibition of liposome uptake by the reticulo-endothelial system and significant prolongation of liposome residence time in the blood stream. Parallel developments in drug loading technology have improved the efficiency and stability of drug entrapment in liposomes, particularly with regard to cationic amphiphiles such as anthracyclines. An example of this new generation of liposomes is a formulation of pegylated liposomal doxorubicin known as Doxil or Caelyx, whose clinical pharmacokinetic profile is characterized by slow plasma clearance and small volume of distribution. A hallmark of these long-circulating liposomal drug carriers is their enhanced accumulation in tumors. The mechanism underlying this passive targeting effect is the phenomenon known as enhanced permeability and retention (EPR) which has been described in a broad variety of experimental tumor types. Further to the passive targeting effect, the liposome drug delivery platform offers the possibility of grafting tumor-specific ligands on the liposome membrane for active targeting to tumor cells, and potentially intracellular drug delivery. The pros and cons of the liposome platform in cancer targeting are discussed vis-à-vis nontargeted drugs, using as an example a liposome drug delivery system targeted to the folate receptor.     

Pros and Cons of the Liposome Platform in Cancer Drug Targeting

Coating of liposomes with polyethylene-glycol (PEG) by incorporation in the liposome bilayer of PEG-derivatized lipids results in inhibition of liposome uptake by the reticulo-endothelial system and significant prolongation of liposome residence time in the blood stream. Parallel developments in drug loading technology have improved the efficiency and stability of drug entrapment in liposomes, particularly with regard to cationic amphiphiles such as anthracyclines. An example of this new generation of liposomes is a formulation of pegylated liposomal doxorubicin known as Doxil® or Caelyx®, whose clinical pharmacokinetic profile is characterized by slow plasma clearance and small volume of distribution. A hallmark of these long-circulating liposomal drug carriers is their enhanced accumulation in tumors. The mechanism underlying this passive targeting effect is the phenomenon known as enhanced permeability and retention (EPR) which has been described in a broad variety of experimental tumor types. Further to the passive targeting effect, the liposome drug delivery platform offers the possibility of grafting tumor-specific ligands on the liposome membrane for active targeting to tumor cells, and potentially intracellular drug delivery. The pros and cons of the liposome platform in cancer targeting are discussed vis-à-vis nontargeted drugs, using as an example a liposome drug delivery system targeted to the folate receptor.     

Induction of high-titer neutralizing antibodies, using hybrid human immunodeficiency virus V3-Ty viruslike particles in a clinically relevant adjuvant.

The localization of neutralization determinants within the envelope glycoproteins of human immunodeficiency virus (HIV) has been largely achieved by immunizing small animals in conjunction with Freund's adjuvant. However, for eventual use in humans, candidate HIV vaccine components must also be efficacious in a nontoxic formulation. We describe here the production of hybrid Ty viruslike particles carrying the major neutralizing domain of HIV and demonstrate the induction of high-titer virus-neutralizing antibodies and an HIV-specific T-cell proliferative response after immunization in conjunction with aluminum hydroxide. As aluminum hydroxide and aluminum phosphate are the only adjuvants currently licensed for use in humans, these observations have implications for the development of an effective vaccine against HIV.     

Immunogenicity of an inactivated adjuvanted whole‐virion influenza A (H5N1, NIBRG‐14) vaccine administered by intramuscular or subcutaneous injection

The immunogenicity and safety profile of an inactivated whole‐virion influenza A (H5N1, NIBRG‐14) vaccine with alum adjuvant that was administered by IM or SC injection in a phase I clinical study involving 120 healthy Japanese men aged 20–40 years is described. The serological response of the IM group was stronger than that of the SC group. Local adverse events were less severe with IM injection than with SC injection, while similar systemic adverse events were seen in both groups. These results indicate that, when administering an inactivated whole virion vaccine with alum adjuvant for pandemic influenza, IM injection may achieve better immunogenicity and safety than SC injection.     

Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems: Effect of charge,fluidity and antigen-to-lipid ratio

The reverse vaccinology approach has recently resulted in the identification of promising protein antigens, which in combination with appropriate adjuvants can stimulate customized, protective immune responses. Although antigen adsorption to adjuvants influences vaccine efficacy and safety, little is generally known about how antigens and adjuvants interact at the molecular level. The aim of this study was to elucidate the mechanisms of interactions between the equally sized, but oppositely charged model protein antigens α-lactalbumin and lysozyme, and i) the clinically tested cationic liposomal adjuvant CAF01 composed of cationic dimethyldioctadecylammonium (DDA) bromide and trehalose-6,6′-dibehenate (TDB) or ii) the neutral adjuvant formulation NAF01, where DDA was replaced with zwitterionic distearoylphosphatidylcholine (DSPC). The effect of liposome charge, bilayer rigidity, isoelectric point and antigen-to-lipid ratio was investigated using dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, intrinsic fluorescence and Langmuir monolayers. The net anionic α-lactalbumin adsorbed onto the cationic liposomes, while there was no measureable attractive interaction with the zwitterionic liposomes. In contrast, the net cationic lysozyme showed very little interaction with either types of liposome. Adsorption of α-lactalbumin altered its tertiary structure, affected lipid membrane packing below and above the phase transition temperature, and neutralized the liposomal surface charge, resulting in reduced colloidal stability and liposome aggregation. Langmuir studies revealed that α-lactalbumin was not squeezed out of DDA monolayers upon compression, which suggests additional hydrophobic interactions.     

Potentiating effects of MPL on DSPC bearing cationic liposomes promote recombinant GP63 vaccine efficacy: high immunogenicity and protection

Background

Vaccines that activate strong specific Th1-predominant immune responses are critically needed for many intracellular pathogens, including Leishmania. The requirement for sustained and efficient vaccination against leishmaniasis is to formulate the best combination of immunopotentiating adjuvant with the stable antigen (Ag) delivery system. The aim of the present study is to evaluate the effectiveness of an immunomodulator on liposomal Ag through subcutaneous (s.c.) route of immunization, and its usefulness during prime/boost against visceral leishmaniasis (VL) in BALB/c mice.

Methodology/Principal Findings

Towards this goal, we formulated recombinant GP63 (rGP63)-based vaccines either with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) or entrapped within cationic liposomes or both. Combinatorial administration of liposomes with MPL-TDM during prime confers activation of dendritic cells, and induces an early robust T cell response. To investigate whether the combined formulation is required for optimum immune response during boost as well, we chose to evaluate the vaccine efficacy in mice primed with combined adjuvant system followed by boosting with either rGP63 alone, in association with MPL-TDM, liposomes or both. We provide evidences that the presence of either liposomal rGP63 or combined formulations during boost is necessary for effective Th1 immune responses (IFN-γ, IL-12, NO) before challenge infection. However, boosting with MPL-TDM in conjugation with liposomal rGP63 resulted in a greater number of IFN-γ producing effector T cells, significantly higher levels of splenocyte proliferation, and Th1 responses compared to mice boosted with liposomal rGP63, after virulent Leishmania donovani (L. donovani) challenge. Moreover, combined formulations offered superior protection against intracellular amastigote replication in macrophages in vitro, and hepatic and splenic parasite load in vivo.

Conclusion

Our results define the immunopotentiating effect of MPL-TDM on protein Ag encapsulated in a controlled release system against experimental VL.