A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Abstract Background A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses. Methods To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity. Results It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes. Conclusion Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

Preclinical development of cancer vaccines

Triplex cell vaccine is a cancer immunopreventive cell vaccine that can prevent almost completely mammary tumor onset in HER-2/neu transgenic mice. A future translation of cancer immunoprevention from preclinical to clinical studies should take into account several aspects. The work reported in this thesis deals with the study of three of these aspects: vaccine schedule, activity in a therapeutic set-up and second-generation DNA vaccines. An important element in determining human acceptance and compliance of a treatment protocol is the number of vaccinations. In order to improve the vaccination schedule a minimal protocol was searched, i.e. a schedule consisting of a lower number of administrations than standard protocol but with a similar efficacy. A candidate optimal protocol was identified by the use of an in silico model, SimTriplex simulator. The in vivo test of this schedule in HER-2/neu transgenic mice only partially confirmed in silico predictions. This result shows that in silico models have the potential ability to aid in searching of optimal treatment protocols, provided that they will be further tuned on experimental data. As a further result this preclinical study highlighted that kinetic of antibody response plays a major role in determining cancer prevention, leading to the hypothesis of a threshold that must be reached rapidly and maintained lifetime. Early clinical trials would be performed in a therapeutic, rather than preventive, setting. Thus, the activity of Triplex vaccine was investigated against experimental lung metastases in HER-2/neu transgenic mice in order to evaluate if the immunopreventive Triplex vaccine could be effective also against a pre-existing tumor mass. This preclinical model of aggressive metastatic development showed that the vaccine was an efficient treatment also 4 for the cure of micrometastases. However the immune mechanisms activated against tumor mass were not antibody dependent, i.e. different from those preventing the onset of primary mammary carcinoma. DNA vaccines could be more easily used than cellular ones. A second generation of Triplex vaccine based on DNA plasmids was evaluated in an aggressive preclinical model (BALBp53neu female mice) and compared with the preventive ability of cellular Triplex vaccine. It was observed that Triplex DNA vaccine was as effective as Triplex cell vaccine, exploiting a more restricted immune stimulation.

Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs

Unmethylated CpG dinucleotides in particular base contexts (CpG-S motifs) are relatively common in bacterial DNA but are rare in vertebrate DNA. B cells and monocytes have the ability to detect such CpG-S motifs that trigger innate immune defenses with production of Th1-like cytokines. Despite comparable levels of unmethylated CpG dinucleotides, DNA from serotype 12 adenovirus is immune-stimulatory, but serotype 2 is nonstimulatory and can even inhibit activation by bacterial DNA. In type 12 genomes, the distribution of CpG-flanking bases is similar to that predicted by chance. However, in type 2 adenoviral DNA the immune stimulatory CpG-S motifs are outnumbered by a 15- to 30-fold excess of CpG dinucleotides in clusters of direct repeats or with a C on the 5′ side or a G on the 3′ side. Synthetic oligodeoxynucleotides containing these putative neutralizing (CpG-N) motifs block immune activation by CpG-S motifs in vitro and in vivo. Eliminating 52 of the 134 CpG-N motifs present in a DNA vaccine markedly enhanced its Th1-like function in vivo, which was increased further by the addition of CpG-S motifs. Thus, depending on the CpG motif, prokaryotic DNA can be either immune-stimulatory or neutralizing. These results have important implications for understanding microbial pathogenesis and molecular evolution and for the clinical development of DNA vaccines and gene therapy vectors.

An autologous oral DNA vaccine protects against murine melanoma

We demonstrated that peripheral T cell tolerance toward murine melanoma self-antigens gp100 and TRP-2 can be broken by an autologous oral DNA vaccine containing the murine ubiquitin gene fused to minigenes encoding peptide epitopes gp10025–33 and TRP-2181–188. These epitopes contain dominant anchor residues for MHC class I antigen alleles H-2Db and H-2Kb, respectively. The DNA vaccine was delivered by oral gavage by using an attenuated strain of Salmonella typhimurium as carrier. Tumor-protective immunity was mediated by MHC class I antigen-restricted CD8+ T cells that secreted TH1 cytokine IFN-γ and induced tumor rejection and growth suppression after a lethal challenge with B16G3.26 murine melanoma cells. Importantly, the protective immunity induced by this autologous DNA vaccine against murine melanoma cells was at least equal to that achieved through xenoimmunization with the human gp10025–33 peptide, which differs in its three NH2-terminal amino acid residues from its murine counterpart and was previously reported to be clearly superior to an autologous vaccine in inducing protective immunity. The presence of ubiquitin upstream of the minigene proved to be essential for achieving this tumor-protective immunity, suggesting that effective antigen processing and presentation may make it possible to break peripheral T cell tolerance to a self-antigen. This vaccine design might prove useful for future rational designs of other recombinant DNA vaccines targeting tissue differentiation antigens expressed by tumors.

Protective DNA vaccination against organ-specific autoimmunity is highly specific and discriminates between single amino acid substitutions in the peptide autoantigen

DNA vaccines that encode encephalitogenic sequences in tandem can protect from subsequent experimental autoimmune encephalomyelitis induced with the corresponding peptide. The mechanism for this protection and, in particular, if it is specific for the amino acid sequence encoding the vaccine are not known. We show here that a single amino acid exchange in position 79 from serine (nonself) to threonine (self) in myelin basic protein peptide MBP68–85, which is a major encephalitogenic determinant for Lewis rats, dramatically alters the protection. Moreover, vaccines encoding the encephalitogenic sequence MBP68–85 do not protect against the second encephalitogenic sequence MBP89–101 in Lewis rats and vice versa. Thus, protective immunity conferred by DNA vaccination exquisitely discriminates between peptide target autoantigens. No bystander suppression was observed. The exact underlying mechanisms remain elusive because no simple correlation between impact on ex vivo responses and protection against disease were noted.

Prophylactic DNA vaccine for hepatitis C virus (HCV) infection: HCV-specific cytotoxic T lymphocyte induction and protection from HCV-recombinant vaccinia infection in an HLA-A2.1 transgenic mouse model

DNA vaccines express antigens intracellularly and effectively induce cellular immune responses. Because only chimpanzees can be used to model human hepatitis C virus (HCV) infections, we developed a small-animal model using HLA-A2.1-transgenic mice to test induction of HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs) and protection against recombinant vaccinia expressing HCV-core. A plasmid encoding the HCV-core antigen induced CD8+ CTLs specific for three conserved endogenously expressed core peptides presented by human HLA-A2.1. When challenged, DNA-immunized mice showed a substantial (5–12 log10) reduction in vaccinia virus titer compared with mock-immunized controls. This protection, lasting at least 14 mo, was shown to be mediated by CD8+ cells. Thus, a DNA vaccine expressing HCV-core is a potential candidate for a prophylactic vaccine for HLA-A2.1+ humans.

CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice

Successful neonatal immunization of humans has proven difficult. We have evaluated CpG-containing oligonucleotides as an adjuvant for immunization of young mice (1–14 days old) against hepatitis B virus surface antigen. The protein-alum-CpG formulation, like the DNA vaccine, produced seroconversion of the majority of mice immunized at 3 or 7 days of age, compared with 0–10% with the protein-alum or protein-CpG formulations. All animals, from neonates to adults, immunized with the protein-alum vaccine exhibited strong T helper (Th)2-like responses [predominantly IgG1, weak or absent cytotoxic T lymphocytes (CTL)]. Th2-type responses also were induced in young mice with protein-CpG (in 1-, 3-, and 7-day-old mice) and protein-alum-CpG (in 1- and 3-day-old mice) but immunization carried out at older ages gave mixed Th1/Th2 (Th0) responses. DNA vaccines gave Th0-like responses when administered at 1 and 7 days of age and Th1-like (predominantly IgG2a and CTL) responses with 14-day-old or adult mice. Surprisingly, the protein-alum-CpG formulation was better than the DNA vaccine for percentage of seroconversion, speed of appearance, and peak titer of the antibody response, as well as prevalence and strength of CTL. These findings may have important implications for immunization of human infants.

Intralymphatic immunization enhances DNA vaccination

Although DNA vaccines have been shown to elicit potent immune responses in animal models, initial clinical trials in humans have been disappointing, highlighting a need to optimize their immunogenicity. Naked DNA vaccines are usually administered either i.m. or intradermally. The current study shows that immunization with naked DNA by direct injection into a peripheral lymph node enhances immunogenicity by 100- to 1,000-fold, inducing strong and biologically relevant CD8+ cytotoxic T lymphocyte responses. Because injection directly into a lymph node is a rapid and easy procedure in humans, these results have important clinical implications for DNA vaccination.

West Nile virus vaccines

West Nile virus (WNV) is a mosquito-borne flavivirus that is emerging as a global pathogen. In the last decade, virulent strains of the virus have been associated with significant outbreaks of human and animal disease in Europe, the Middle East and North America. Efforts to develop human and veterinary vaccines have taken both traditional and novel approaches. A formalin-inactivated whole virus vaccine has been approved for use in horses. DNA vaccines coding for the structural WNV proteins have also been assessed for veterinary use and have been found to be protective in mice, horses and birds. Live attenuated yellow fever WNV chimeric vaccines have also been successful in animals and are currently undergoing human trials. Additional studies have shown that immunisation with a relatively benign Australian variant of WNV, the Kunjin virus, also provides protective immunity against the virulent North American strain. Levels of efficacy and safety, as well as logistical, economic and environmental issues, must all be carefully considered before vaccine candidates are approved and selected for large-scale manufacture and distribution.

Liposome-mediated DNA immunisation via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper, we have investigated the application of liposome-entrapped DNA and their cationic lipid composition on such potency after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into liposomes composed of 16 μmol egg phosphatidylcholine (PC), 8 μmoles dioleoyl phosphatidylethanolamine (DOPE) or cholesterol (Chol) and either the cationic lipid 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP) or cholesteryl 3-N-(dimethyl amino ethyl) carbamate (DC-Chol). This method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded incorporation values of 90-94% of the DNA used. Mixing or rehydration of preformed cationic liposomes with 100 μg plasmid DNA also led to similarly high complexation values (92-94%). In an attempt to establish differences in the nature of DNA association with these various liposome preparations their physico-chemical characteristics were investigated. Studies on vesicle size, zeta potential and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, formulation of liposomal DNA by the dehydration-rehydration generated submicron size liposomes incorporating most of the DNA in a manner that prevents DNA displacement through anion competition. The bilayer composition of these dehydration-rehydration vesicles (DRV(DNA)) can also further influence these physicochemical characteristics with the presence of DOPE within the liposome bilayer resulting in a reduced vesicle zeta potential. Subcutaneous liposome-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded NP were substantially higher after dosing twice, 28 days apart with 10 μg liposome-entrapped DNA compared to naked DNA. At all time points measured, mice immunised with naked DNA showed no greater immune response compared to the control, non-immunised group. In contrast, as early as day 49, responses were significantly higher in mice injected with DNA entrapped in DRV liposomes containing DOTAP compared to the control group and mice immunised with naked DNA. By day 56, all total IgG responses from mice immunised with both DRV formulations were significantly higher. Comparison between the DRV formulations revealed no significant difference in immune responses elicited except at day 114, where the humoural responses of the group injected with liposomal formulation containing DC-Chol dropped to significantly lower levels that those measured in mice which received the DOTAP formulation. Similar results were found when the IgG1 and IgG2a subclass responses were determined. These results suggest that, not only can DNA be effectively entrapped within liposomes using the DRV method but that such DRV liposomes containing DNA may be a useful system for subcutaneous delivery of DNA vaccines. © 2003 Taylor & Francis Ltd.

Novel bioadhesive formulations for mucosal and parenteral delivery of vaccines

In this work we have established the efficient mucosal delivery of vaccines using absorption enhancers and chitosan. In addition, the use of chitosan was shown to enhance the action of other known adjuvants, such as CTB or Quil-A. Collectively, the results presented herein indicate that chitosan has excellent potential as a mucosal adjuvant. We have evaluated a number of absorption enhancers for their adjuvant activity in vivo. Polyornithine was shown to engender high scrum immune reasons to nasally delivered antigens, with higher molecular weight polyornithine facilitating the best results. We have demonstrated for the first time that vitamin E TPGS can act as mucosal adjuvant. Deoxycholic acid, cyclodextrins and acylcarnitines were also identified as effective mucosal adjuvants and showed enhanced immune responses to nasally delivered TT, DT and Yersinia pestis V and F1 antigens. Previously, none of these agents, common in their action as absorption enhancing agents, have been shown to have immunopotentiating activity for mucosal immunisation. We have successfully developed novel surface modified microspheres using chitosan as an emulsion stabiliser during the preparation of PLA microspheres. It was found that immune responses could be substantially increased, effectively exploiting the immunopenetrating characteristics of both chitosan and PLA microspheres in the same delivery vehicle. In the same study, comparison of intranasal and intramuscular routes of administration showed that with these formulations, the nasal route could be as effective as intramuscular delivery, highlighting the potential of mucosal administration for these particulate delivery systems. Chitosan was co-administered with polymer microspheres. It was demonstrated that this strategy facilitates markedly enhanced immune responses in both magnitude and duration following intramuscular administration. We conclude that this combination shows potential for single dose administration of vaccines. In another study, we have shown that the addition of chitosan to alum adsorbed TT was able to enhance immune responses. PLA micro/nanospheres were prepared and characterised with discreet particle size ranges. A smaller particle size was shown to facilitate higher scrum IgG responses following nasal administration. A lower antigen loading was additionally identified as being preferential for the induction of immune responses in combination with the smaller particle size. This may be due to the fact that the number of particles will be increased when antigen loading is low, which may in turn facilitate a more widespread uptake of particles. PLA lamellar particles were prepared and characterised. Adsorbed TT was evaluated for the potential to engender immune responses in vivo. These formulations were shown to generate effective immune responses following intramuscular administration. Positively charged polyethylcyanoacrylate and PLA nanoparticies were designed and characterised and their potential as delivery vehicles for DNA vaccines was investigated. Successful preparation of particles with narrow size distribution and positive surface charge (imparted by the inclusion of chitosan) was achieved. In the evaluation of antibody responses to DNA encoded antigen in the presence of alum administered intranasally, discrimination between the groups was only seen following intramuscular boosting with the corresponding protein. Our study showed that DNA vaccines in the presence of either alum or Quil-A may advantageously influence priming of the immune system by a mucosal route. The potential for the combination of adjuvants, Quil-A and chitosan, to enhance antibody responses to plasmid encoded antigen co-administered with the corresponding protein antigen was shown and this is worthy of further investigation. The findings here have identified novel adjuvants and approaches to vaccine delivery. In particular, chitosan or vitamin E TPGS are shown here to have considerable promise as non-toxic, safe mucosal adjuvants. In addition, biodegradable mucoadhesive delivery systems, surface modified with chitosan in a single step process, may have application for other uses such as drug and gene delivery.

Liposome (Lipodine™)-mediated DNA vaccination by the oral route

Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen (HBsAg) was incorporated by the dehydration-rehydration method into Lipodine™ liposomes composed of 16 μmoles phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC), 8 μmoles of (dioleoyl phosphatidylethanolamine (DOPE) or cholesterol and 4 μmoles of the cationic lipid 1,2-dioleoyl-3-(trimethylammonium propane (DOTAP) (molar ratios 1:0.5:0.25). Incorporation efficiency was high (89-93% of the amount of DNA used) in all four formulations tested and incorporated DNA was shown to be resistant to displacement in the presence of the competing anionic sodium dodecyl sulphate molecules. This is consistent with the notion that most of the DNA is incorporated within the multilamellar vesicles structure rather than being vesicle surface-complexed. Stability studies performed in simulated intestinal media also demonstrated that dehydration-rehydration vesicles (DRV) incorporating DNA (DRV(DNA)) were able to retain significantly more of their DNA content compared to DNA complexed with preformed small unilamellar vesicles (SUV-DNA) of the same composition. Moreover, after 4h incubation in the media, DNA loss for DSPC DRV(DNA) was only minimal, suggesting this to be the most stable formulation. Oral (intragastric) liposome-mediated DNA immunisation studies employing a variety of DRV(DNA) formulations as well as naked DNA revealed that secreted IgA responses against the encoded HBsAg were (as early as three weeks after the first dose) substantially higher after dosing with 100 μg liposome-entrapped DNA compared to naked DNA. Throughout the fourteen week investigation, IgA responses in mice were consistently higher with the DSPC DRV(DNA) liposomes compared to naked DNA and correlated well with their improved DNA retention when exposed to model intestinal fluids. To investigate gene expression after oral (intragastric) administration, mice were given 100 μg of naked or DSPC DRV liposome-entrapped plasmid DNA expressing the enhanced green fluorescent protein (pCMV.EGFP). Expression of the gene, in terms of fluorescence intensity in the draining mesenteric lymph nodes, was much greater in mice dosed with liposomal DNA than in animals dosed with the naked DNA. These results suggest that DSPC DRV liposomes containing DNA (Lipodine™) may be a useful system for the oral delivery of DNA vaccines.

Particulate delivery systems for vaccines

This review focuses on the use of particulate delivery systems for the purposes of immunization. This includes poly(lactide-co-glycolide) (PLGA), ISCOMs, liposomes, niosomes, virosomes, chitosan, and other biodegradable polymers. These systems are evaluated in terms of their use as carriers for protein subunit and DNA vaccines. There is an extensive focus on recent literature, the understanding of biological interactions, and relation of this to our present understanding of immunological mechanisms of action. In addition, there is consideration of formulation techniques including emulsification, solvent diffusion, DNA complexation, and entrapment. The diversity of formulation strategies presented is a testament to the exponential growth and interest in the area of vaccine delivery systems. A case study for the application of particulate vaccine carriers is assessed in terms of vaccine development and recent insights into the possible design and application of vaccines against two of the most important pathogens that threaten mankind and for which there is a significant need: Mycobacterium tuberculosis and human immunodeficiency virus. This review addresses the rationale for the use of particulate delivery systems in vaccine design in the context of the diversity of carriers for DNA- and protein-based vaccines and their potential for application in terms of the critical need for effective vaccines. © 2005 by Begell House, Inc.

Lyophilisation and sterilisation of liposomal vaccines to produce stable and sterile products

The advantages of liposomes as delivery systems for peptide, protein and DNA vaccines is well-recognised, unfortunately their application has been stinted by their instability during storage and their limited shelf-life. Further, sterilisation of these systems has been problematic, with degradation of the liposomes being reported after sterilisation using the various techniques available. Work form our laboratory has investigated techniques that can be applied to particulate liposomal vaccines such that they can be prepared in a freeze-dried and sterile format. In this article, we describe techniques for the lyophilisation, cryoprotection and sterilisation of liposomal vaccines. Applying these methods allows for the retention of both the chemical integrity of the lipids and the key physico-chemical characteristics of the liposomes (e.g., particle size, zeta potential, and dynamic viscosity), thus supporting the enhanced transition of liposomal vaccines from the bench to the clinic. © 2006 Elsevier Inc. All rights reserved.

Design and development of cationic liposomes as DNA vaccine adjuvants

Cationic liposomes have been extensively explored for their efficacy in delivering nucleic acids, by offering the ability to protect plasmid DNA against degradation, promote gene expression and, in the case of DNA vaccines, induce both humoural and cellular immune responses. DNA vaccines may also offer advantages in terms of safety, but they are less effective and need an adjuvant to enhance their immunogenicity. Therefore, cationic liposomes can be utilised as delivery systems and/or adjuvants for DNA vaccines to stimulate stronger immune responses. To explore the role of liposomal systems within plasmid DNA delivery, parameters such as the effect of lipid composition, method of liposome preparation and presence of electrolytes in the formulation were investigated in characterisation studies, in vitro transfection studies and in vivo biodistribution and immunisation studies. Liposomes composed of 1,2-dioleoyl-sn-glycero 3-phosphoethanolamine (DOPE) in combination with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-stearoyl-3- trimethylammonium-propane (DSTAP) were prepared by the lipid hydration method and hydrated in aqueous media with or without presence of electrolytes. Whilst the in vitro transfection efficiency of all liposomes resulted to be higher than Lipofectin, DSTAP-based liposomes showed significantly higher transfection efficiency than DOTAP-based formulations. Furthermore, upon intramuscular injection of liposomal DNA vaccines, DSTAP-based liposomes showed a significantly stronger depot effect at the injection site. This could explain the result of heterologous immunisation studies, which revealed DSTAP-based liposomal vaccines induce stronger immune responses compared to DOTAP-based formulations. Previous studies have shown that having more liposomally associated antigen at the injection site would lead to more drainage of them into the local lymph nodes. Consequently, this would lead to more antigens being presented to antigen presenting cells, which are circulating in lymph nodes, and this would initiate a stronger immune response. Finally, in a comparative study, liposomes composed of dimethyldioctadecylammonium bromide (DDA) in combination with DOPE or immunostimulatory molecule of trehalose 6,6-dibehenate (TDB) were prepared and investigated in vitro and in vivo. Results showed that although DDA:TDB is not able to transfect the cells efficiently in vitro, this formulation induces stronger immunity compared to DDA:DOPE due to the immunostimulatory effects of TDB. This study demonstrated, while the presence of electrolytes did not improve immune responses, small unilamellar vesicle (SUV) liposomes induced stronger humoural immune responses compared to dehydration rehydration vesicle (DRV) liposomes. Moreover, lipid composition was shown to play a key role in in vitro and in vivo behaviour of the formulations, as saturated cationic lipids provided stronger immune responses compared to unsaturated lipids. Finally, heterologous prime/boost immunisation promoted significantly stronger immune responses compared to homologous vaccination of DNA vaccines, however, a single immunisation of subunit vaccine provoked comparable levels of immune response to the heterologous regimen, suggesting more immune efficiency for subunit vaccines compared to DNA vaccines.