Liposomes based on dimethyldioctadecylammonium promote a depot effect and enhance immunogenicity of soluble antigen

The mechanism behind the immunostimulatory effect obtained with the cationic liposomal vaccine adjuvant DDA:TDB remains unclear. One of the proposed hypotheses is the 'depot effect' in which the liposomal carrier helps to retain the antigen at the injection site thereby increasing the time of vaccine exposure to the immune cells. In the present study we devise a method to quantify the in vivo movement of liposomes and vaccine antigen using the radioisotopes H(3) and I(125) respectively. H(3)-labeled liposomes composed of dimethyldioctadecylammonium bromide (DDA) or an 8:1 molar ratio of DDA and trehalose 6,6-dibehenate (TDB) were administered in combination with I(125)-labeled Ag85B-ESAT-6 antigen, both via intramuscular and subcutaneous injection to mice. Furthermore characterisation of the liposomal system in simulated in vivo conditions was undertaken. Our results show that this dual-labeling technique is functional and reproducible. The administration of Ag85B-ESAT-6 without a liposomal carrier leads to rapid dissemination of the antigen from the site of injection. The administration of Ag85B-ESAT-6 together with either DDA or DDA:TDB liposomes however leads to deposition of the antigen at the injection site with detectable levels still being present 14 days post injection. Neither the incorporation of TDB nor the route of injection had any significant influence on the depot effect of DDA-based liposomes. The presence of TDB in DDA liposomes improves draining of liposomes to the lymph node in addition to increasing monocyte influx to the site of injection as highlighted by the intensive blue colouring of the injection site after pontamine blue staining of phagocytic cells in vivo. Our findings provide conclusive evidence for a cationic liposome-mediated deposition of antigen at the injection site with improved monocyte infiltration.

Liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a CMI response

With respect to liposomes as delivery vehicles and adjuvants for vaccine antigens, the role of vesicle surface charge remains disputed. In the present study we investigate the influence of liposome surface charge and antigen-liposome interaction on the antigen depot effect at the site of injection (SOI). The presence of liposome and antigen in tissue at the SOI as well as the draining lymphatic tissue was quantified to analyse the lymphatic draining of the vaccine components. Furthermore investigations detailing cytokine production and T-cell antigen specificity were undertaken to investigate the relationship between depot effect and the ability of the vaccine to induce an immune response. Our results suggest that cationic charge is an important factor for the retention of the liposomal component at the SOI, and a moderate to high (>50%) level of antigen adsorption to the cationic vesicle surface was required for efficient antigen retention in the same tissue. Furthermore, neutral liposomes expressing poor levels of antigen retention were limited in their ability to mediate long term (14 days) antigen presentation to circulating antigen specific T-cells and to induce the Th1 and Th17 arms of the immune system, as compared to antigen adsorbing cationic liposomes. The neutral liposomes did however induce the production of IL-5 at levels comparable to those induced by cationic liposomes, indicating that neutral liposomes can induce a weak Th2 response.

The SEF14 fimbrial antigen of Salmonella enterica serovar Enteritidis is encoded within a pathogenicity islet

The DNA sequence of the chromosomal gene cluster encoding the SEF14 fimbriae of Salmonella enterica serovar Enteritidis was determined. Five contiguous open reading frames, sefABCDE, were identified. The sefE gene shared significant homology with araC-like positive regulators. Serovar-associated virulence plasmid (SAP) genes orf7,8,9 and pefI were identified immediately adjacent to the sef operon. The pefI gene encoded a putative regulator of the Plasmid-encoded fimbrial antigen (PEF) expression. The entire sef--pef region, flanked by two IS-like elements, was inserted adjacent to leuX that encoded a transfer RNA molecule. The organisation of this region was suggestive of a classic pathogenicity islet. Southern hybridisation confirmed two copies of the SAP derived orf7,8,9 and pefI region in S. Enteritidis, one in the chromosome and one on the SAP. Of other group D Salmonella, only S. Blegdam and S. Moscow harboured both chromosomal and plasmid copies of pefI--orf9 region although polymorphism was evident.

Administration routes affect the quality of immune responses:a cross-sectional evaluation of particulate antigen-delivery systems

Particulate delivery systems such as liposomes and polymeric nano- and microparticles are attracting great interest for developing new vaccines. Materials and formulation properties essential for this purpose have been extensively studied, but relatively little is known about the influence of the administration route of such delivery systems on the type and strength of immune response elicited. Thus, the present study aimed at elucidating the influence on the immune response when of immunising mice by different routes, such as the subcutaneous, intradermal, intramuscular, and intralymphatic routes with ovalbumin-loaded liposomes, N-trimethyl chitosan (TMC) nanoparticles, and poly(lactide-co-glycolide) (PLGA) microparticles, all with and without specifically selected immune-response modifiers. The results showed that the route of administration caused only minor differences in inducing an antibody response of the IgG1 subclass, and any such differences were abolished upon booster immunisation with the various adjuvanted and non-adjuvanted delivery systems. In contrast, the administration route strongly affected both the kinetics and magnitude of the IgG2a response. A single intralymphatic administration of all evaluated delivery systems induced a robust IgG2a response, whereas subcutaneous administration failed to elicit a substantial IgG2a response even after boosting, except with the adjuvanted nanoparticles. The intradermal and intramuscular routes generated intermediate IgG2a titers. The benefit of the intralymphatic administration route for eliciting a Th1-type response was confirmed in terms of IFN-gamma production of isolated and re-stimulated splenocytes from animals previously immunised with adjuvanted and non-adjuvanted liposomes as well as with adjuvanted microparticles. Altogether the results show that the IgG2a associated with Th1-type immune responses are sensitive to the route of administration, whereas IgG1 response associated with Th2-type immune responses were relatively insensitive to the administration route of the particulate delivery systems. The route of administration should therefore be considered when planning and interpreting pre-clinical research or development on vaccine delivery systems.

Cationic liposomes as adjuvants for subunit protein vaccines:their role in the depot effect and antigen processing by APCs

Introduction: The requirement of adjuvants in subunit protein vaccination is well known yet their mechanisms of action remain elusive. Of the numerous mechanisms suggested, cationic liposomes appear to fulfil at least three: the antigen depot effect, the delivery of antigen to antigen presenting cells (APCs) and finally the danger signal. We have investigated the role of antigen depot effect with the use of dual radiolabelling whereby adjuvant and antigen presence in tissues can be quantified. In our studies a range of cationic liposomes and different antigens were studied to determine the importance of physical properties such as liposome surface charge, antigen association and inherent lipid immunogenicity. More recently we have investigated the role of liposome size with the cationic liposome formulation DDA:TDB, composed of the cationic lipid dimethyldioctadecylammonium (DDA) and the synthetic mycobacterial glycolipid trehalose 6,6’-dibehenate (TDB). Vesicle size is a frequently investigated parameter which is known to result in different routes of endocytosis. It has been postulated that targeting different routes leads to different intracellular signaling pathway activation and it is certainly true that numerous studies have shown vesicle size to have an effect on the resulting immune responses (e.g. Th1 vs. Th2). Aim: To determine the effect of cationic liposome size on the biodistribution of adjuvant and antigen, the ensuing humoral and cell-mediated immune responses and the uptake and activation of antigen by APCs including macrophages and dendritic cells. Methods: DDA:TDB liposomes were made to three different sizes (~ 0.2, 0.5 and 2 µm) followed by the addition of tuberculosis antigen Ag85B-ESAT-6 therefore resulting in surface adsorption. Liposome formulations were injected into Balb/c or C57Bl/6 mice via the intramuscular route. The biodistribution of the liposome formulations was followed using dual radiolabelling. Tissues including muscle from the site of injection and local draining lymph nodes were removed and liposome and antigen presence quantified. Mice were also immunized with the different vaccine formulations and cytokine production (from Ag85B-ESAT-6 restimulated splenocytes) and antibody presence in blood assayed. Furthermore, splenocyte proliferation after restimulating with Ag85B-ESAT-6 was measured. Finally, APCs were compared for their ability to endocytose vaccine formulations and the effect this had on the maturation status of the cell populations was compared. Flow cytometry and fluorescence labelling was used to investigate maturation marker up-regulation and efficacy of phagocytosis. Results: Our results show that for an efficient Ag85B-ESAT-6 antigen depot at the injection site, liposomes composed of DDA and TDB are required. There is no significant change in the presence of liposome or antigen at 6hrs or 24hrs p.i, nor does liposome size have an effect. Approximately 0.05% of the injected liposome dose is detected in the local draining lymph node 24hrs p.i however protein presence is low (<0.005% dose). Preliminary in vitro data shows liposome and antigen endocytosis by macrophages; further studies on this will be presented in addition to the results of the immunisation study.

Subunit vaccines distearoylphosphatidylcholine-based liposomes entrapping antigen offer a neutral alternative to dimethyldioctadecylammonium-based cationic liposomes as an adjuvant delivery system

The adjuvanticity of liposomes can be directed through formulation to develop a safe yet potent vaccine candidate. With the addition of the cationic lipid dimethyldioctadecylammonium bromide (DDA) to stable neutral distearoylphosphatidylcholine (DSPC):cholesterol (Chol) liposomes, vesicle size reduces while protein entrapment increases. The addition of the immunomodulator, trehalose 6,6-dibehenate (TDB) to either the neutral or cationic liposomes did not affect the physiochemical characteristics of these liposome vesicles. However, the protective immune response, as indicated by the amount of IFN-? production, increases considerably when TDB is present. High levels of IFN-? were observed for cationic liposomes; however, there was a marked reduction in IFN-? release over time. Conversely, for neutral liposomes containing TDB, although the initial amount of IFN-? was slightly lower than the cationic equivalent, the overall protective immune responses of these neutral liposomes were effectively maintained over time, generating good levels of protection. To that end, although the addition of DSPC and Chol reduced the protective immunity of DDA:TDB liposomes, relatively high protection was observed for the neutral counterpart, DSPC:Chol:TDB, which may offer an effective neutral alternative to the DDA:TDB cationic system, especially for the delivery of either zwitterionic (neutral) or cationic molecules or antigens.

Formulation and characterisation of an effective particulate delivery vehicle for the novel sub-unit vaccine antigen, Ag85B-ESAT-6

This research focused on the formation of particulate delivery systems for the sub-unit fusion protein, Ag85B-ESAT-6, a promising tuberculosis (TB) vaccine candidate. Initial work concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyl dioctadecyl ammonium (DDA). These studies demonstrated that addition of the immunomodulatory trehalose dibehenate (TDB) enhanced the physical stability of the system whilst also adding further adjuvanticity. Indeed, this formulation was effective in stimulating both a cell mediated and humoural immune response. In order to investigate an alternative to the DDA-TDB system, microspheres based on poly(DL-lactide-co-glycolide) (PLGA) incorporating the adjuvants DDA and TDB, either alone or in combination, were first optimised in terms of physico-chemical characteristics, followed by immunological analysis. The formulation incorporating PLGA and DDA emerged as the lead candidate, with promising protection data against TB. Subsequent optimisation of the lead microsphere formulation investigated the effect of several variables involved in the formulation process on physico-chemical and immunological characteristics of the particles produced. Further, freeze-drying studies were carried out with both sugar-based and amino acid-based cryoprotectants, in order to formulate a stable freexe-dried product. Finally, environmental scanning electron microscopy (ESEM) was investigated as a potential alternative to conventional SEM for the morphological investigation of microsphere formulations. Results revealed that the DDA-TDB liposome system proved to be the most immunologically efficient delivery vehicle studied, with high levels of antibody and cytokine production, particularly gamma-interferon (IFN-ϒ), considered the key cytokine marker for anti-mycobacterial immunity. Of the microsphere systems investigated, PLGA in combination with DDA showed the most promise, with an ability to initiate a broad spectrum of cytokine production, as well as antigen specific spleen cell proliferation comparable to that of the DDA-TDB formulation.

Influence of microbial antigen formulation and delivery route on the immune response

Recent technological advances have resulted in the production of safe subunit and synthetic small peptide vaccines. Unfortunately, these vaccines are weakly or non-immunogenic in the absence of an immunological adjuvant (agents that can induce strong immunity to antigens). In addition, in order to prevent and/or control infection at the mucosal surface, stimulation of the mucosal immune system is essential. This may be achieved via the common mucosal immune system by exposure to antigen at a mucosal surface remote from the area of infection. Initial studies investigated the potential of multiple emulsions in effecting oral absorption and the subsequent immune responses to a lipopolysaccharide vaccine (LPS) after immunisation. Nasal delivery of LPS was carried out in parallel work using either aqueous solution or gel formulations. Tetanus toxoid vaccine in simple solution was delivered to guinea pigs as free antigen or entrapped in DSPC liposomes. In addition, adsorbed tetanus toxoid vaccine was delivered nasally free or in an aerosil gel formulation. This work was extended to investigate guinea pigs immunised by various mucosal routes with a herpes simplex virus subunit vaccine prepared from virus infected cells and delivered in gels, multiple emulsions and liposomes. Comparable serum antibody responses resulted but failed to produce enhanced protection against vaginal challenge when compared to subcutaneous immunisation with alhydrogel adjuvanted vaccine. Thus, immunisation of the mucosal surface by these methods may have been inadequate. These studies were extended in an attempt to protect against HSV genital challenge by construction of an attenuated Salmonella typhimurium HWSH aroA mutant expressing a cloned glycoprotein D-l gene fused to the Es-cherichia coli lac z promoter. Preliminary work on the colonisation of guinea pigs with S. typhimurium HWSH aroA mutants were carried out, with the aim of using the guinea pig HSV vaginal model to investigate protection.

Novel formulations for antigen delivery using biodegradable polymers: new approaches for the use of new and established adjuvants

The use of immunological adjuvants has been established since 1924 and ever since many candidates have been extensively researched in vaccine development. The controlled release of vaccine is another area of biotechnology research, which is advancing rapidly with great potential and success. Encapsulation of peptide and protein drugs within biodegradable microspheres has been amongst the most successful of approaches within the past decade. The present studies have focused on combining the advantages of microsphere delivery systems composed of biodegradable polylactide (PLLA) and polylactide-co-glycolide (PLGA) polymers with that of safe and effective adjuvants. The research efforts were directed to the development of single-dose delivery vehicles which, can be manufactured easily, safely, under mild and favourable conditions to the encapsulated antigens. In pursuing this objective non ionic block copolymers (NIBCs) (Pluronics@ LI01 and L121) were incorporated within poly-dl-lactide (PDLA) micorospheres prepared with emulsification-diffusion method. LI0I and L121 served both as adjuvants and stabilising agents within these vaccine delivery vehicles. These formulations encapsulating the model antigens lysozyme, ovalbumin (OVA) and diphtheria toxoid (DT) resulted in high entrapment efficiency (99%), yield (96.7%) and elicited high and sustained immune response (IgG titres up to 9427) after one single administration over nine months. The structural integrity of the antigens was preserved within these formulations. In evaluating new approaches for the use of well-established adjuvants such as alum, these particles were incorporated within PLLA and PLGA microspheres at much lesser quantities (5-10 times lower) than those contained within conventional alum-adsorbed vaccines. These studies focused on the incorporation of the clinically relevant tetanus toxoid (TT) antigen within biodegradable microspheres. The encapsulation of both alum particles and TT antigen within these micropheres resulted in preparations with high encapsulation efficiency (95%) and yield (91.2%). The immune response to these particles was also investigated to evaluate the secretion of serum IgG, IgG1, IgG2a and IgG2b after a single administration of these vaccines. The Splenic cells proliferation was also investigated as an indication for the induction of cell mediated immunity. These particles resulted in high and sustained immune response over a period of 14 months. The stability of TT within particles was also investigated under dry storage over a period of several months. NIBC microspheres were also investigated as potential DNA vaccine delivery systems using hepatitis B plasmid. These particles resulted in micro spheres of 3-5 μm diameter and were shown to preserve the integrity of the encapsulated (27.7% entrapment efficiency) hepatitis B plasmid.

Discriminating antigen and non-antigen using proteome dissimilarity:bacterial antigens

It has been postulated that immunogenicity results from the overall dissimilarity of pathogenic proteins versus the host proteome. We have sought to use this concept to discriminate between antigens and non-antigens of bacterial origin. Sets of 100 known antigenic and nonantigenic peptide sequences from bacteria were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we were unable to determine a threshold able to separate meaningfully antigen from non-antigen. Thus, antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to the human genome.

Discriminating antigen and non-antigen using proteome dissimilarity III:tumour and parasite antigens

Computational genome analysis enables systematic identification of potential immunogenic proteins within a pathogen. Immunogenicity is a system property that arises through the interaction of host and pathogen as mediated through the medium of a immunogenic protein. The overt dissimilarity of pathogenic proteins when compared to the host proteome is conjectured by some to be the determining principal of immunogenicity. Previously, we explored this idea in the context of Bacterial, Viral, and Fungal antigen. In this paper, we broaden and extend our analysis to include complex antigens of eukaryotic origin, arising from tumours and from parasite pathogens. For both types of antigen, known antigenic and non-antigenic protein sequences were compared to human and mouse proteomes. In contrast to our previous results, both visual inspection and statistical evaluation indicate a much wider range of homologues and a significant level of discrimination; but, as before, we could not determine a viable threshold capable of properly separating non-antigen from antigen. In concert with our previous work, we conclude that global proteome dissimilarity is not a useful metric for immunogenicity for presently available antigens arising from Bacteria, viruses, fungi, parasites, and tumours. While we see some signal for certain antigen types, using dissimilarity is not a useful approach to identifying antigenic molecules within pathogen genomes.

Discriminating antigen and non-antigen using proteome dissimilarity II:viral and fungal antigens

Immunogenicity arises via many synergistic mechanisms, yet the overall dissimilarity of pathogenic proteins versus the host proteome has been proposed as a key arbiter. We have previously explored this concept in relation to Bacterial antigens; here we extend our analysis to antigens of viral and fungal origin. Sets of known viral and fungal antigenic and non-antigenic protein sequences were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we could not determine a threshold able meaningfully to separate non-antigen from antigen. We conclude that viral and fungal antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to mammalian genomes.

Comparison of vesicle based antigen delivery systems for delivery of hepatitis B surface antigen

There is a clinical need for a more effective vaccine against hepatitis B, and in particular vaccines that may be suitable for therapeutic administration. This study assesses the potential of cationic surfactant vesicle based formulations using two agents; the cationic amine containing [N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) or dimethyl dioctadecylammonium bromide (DDA) with hepatitis B surface antigen (HBsAg). Synthetic mycobacterial cord factor, trehalose 6,6′-dibehenate (TDB) has been used as an adjuvant and the addition of 1-monopalmitoyl glycerol (C16:0) (MP) and cholesterol (Chol) to DDA-TDB is assessed for its potential to facilitate formation of dehydration-rehydration vesicles (DRV) at room temperature, and the effect of this on immune responses. A DRV formulation is directly compared to an adsorbed formulation of the same composition and preparation protocol (MP:dioleoyl phosphoethanolamine (DOPE):Chol:DC-Chol) and the direct substitution of MP with phosphatidylcholine (PC) is also compared in DRV antigen-entrapped formulations. MP and Chol were shown to facilitate the use of DDA-TDB in DRV formulations prepared at room temperature, whilst there was marginal alteration of immunogenicity (a reduction in HBsAg-specific IL-2). The HBsAg adsorbed DRV formulation was not significantly different from the HBsAg entrapped DRV formulation. Overall, DDA formulations incorporating TDB showed markedly increased antigen specific splenocyte proliferation and elicited cytokine production concomitant with a strong T cell driven response, delineating formulations that may be useful for further evaluation of their clinical potential. © 2007 Elsevier B.V. All rights reserved.

Developing solid particulate vaccine adjuvants - surface bound antigen favouring a humoural response, whereas entrapped antigen shows a tendency for cell mediated immunity

This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.