key: cord-0034906-61cyofjx authors: Turánek, Jaroslav; Mašek, Josef; Křupka, Michal; Raška, Milan title: Functionalised Nanoliposomes for Construction of Recombinant Vaccines: Lyme Disease as an Example date: 2013-06-13 journal: Molecular Vaccines DOI: 10.1007/978-3-319-00978-0_10 sha: 93a1731e7eb0079dbff0cb5a3baa8cd6ff459250 doc_id: 34906 cord_uid: 61cyofjx Liposomes (phospholipid bilayer vesicles) represent an almost ideal carrier system for the preparation of synthetic vaccines due to their biodegradability and capacity to protect and transport molecules of different physicochemical properties (including size, hydrophilicity, hydrophobicity, and charge). Liposomal carriers can be applied by invasive (e.g. i.m., s.c., i.d.) as well as non-invasive (transdermal and mucosal) routes. In the last 15 years, liposome vaccine technology has matured and several vaccines containing liposome-based adjuvants have been approved for human and veterinary use or have reached late stages of clinical evaluation. Given the intensifying interest in liposome-based vaccines, it is important to understand precisely how liposomes interact with the immune system and how they stimulate immunity. It has become clear that the physicochemical properties of liposomal vaccines – method of antigen attachment, lipid composition, bilayer fluidity, particle charge, and other properties – exert strong effects on the resulting immune response. In this chapter we will discuss some aspects of liposomal vaccines including the effect of novel and emerging immunomodulator incorporation. The application of metallochelating nanoliposomes for development of recombinant vaccine against Lyme disease will be presented as a suitable example. we will discuss some aspects of liposomal vaccines including the effect of novel and emerging immunomodulator incorporation. The application of metallochelating nanoliposomes for development of recombinant vaccine against Lyme disease will be presented as a suitable example. Vaccinology as a scientifi c fi eld is undergoing dramatic development. Sophisticated techniques and rapidly growing in-depth knowledge of immunological mechanisms are at hand to exploit fully the potential for protecting from, as well as curing of diseases through vaccination. In spite of great successes like eradication of smallpox in the 1970s and in a lesser extent poliomyelitis (two important milestones in medical history), new challenges have arisen to be faced. Rapidly changing ecosystems and human behaviour, an ever-increasing density of human and farmed animal populations, a high degree of mobility resulting in rapid spreading of pathogens in infected people and animals, poverty and war confl icts in the third world, and many other factors contribute to the more frequent occurrence and rapid dissemination of new as well as some old infectious disease. The three infections that most heavily affl ict global health are AIDS, tuberculosis, and malaria. As an example of new viral pathogens, Ebola virus, SARS coronavirus, or new strains of infl uenza virus can be mentioned [ 1 ] . Rapid sequencing of the genome of pathogens leads to development of sensitive molecular diagnostic tools and augments identifi cation and expression of recombinant antigenic targets for future vaccines [ 2 ] . Special fi eld represents immunotherapy of cancer, where anticancer vaccines could be a powerful weapon for long-term effective treatment. The progress in vaccine development is tightly connected not only with new fi ndings in immunology but also in molecular biology and biotechnology. The new term "reverse vaccinology" was proposed by Rappuoli to describe a complex genome-based approach toward vaccine design [ 3 ] . In comparison with conventional approaches which require a laborious process of attenuation or inactivation of pathogens, or selection of individual components important for induction of immune response, reverse vaccinology offers the possibility of using genomic information derived from in silico analyses for direct design and production of protective antigen using recombinant technology. This approach can signifi cantly reduce the time necessary for the identifi cation of antigens for development of candidate vaccine and enables systematic identifi cation of all potential antigens even from pathogens which are diffi cult or currently impossible to culture. Of course, this approach is limited to identifi cation of protein or glycoprotein antigens, omitting such important vaccine components such as polysaccharides and glycolipids. The principal question for reverse vaccinology consists in identifi cation of protective antigen, which presents the main hurdle of this approach. Nevertheless, once the protective antigen is identifi ed it enables scientists to systemically classify such antigens, and develop efficient preparations virtually against any pathogen that has had its genome sequence determined. Subunit vaccines offer superior safety profi les and can be manufactured with minimal risk of contamination [ 4 , 5 ] . When coupled with appropriate adjuvants, they can also focus the immune response on protective or highly conserved antigenic determinants that may not elicit a potent response during natural infection or after vaccination with an inactivated or attenuated pathogen [ 6 , 7 ] . A common observation from the process of elicitation of the adaptive immune response is that the antigen by itself is not a stimulating agent. In other words, administration of absolutely pure recombinant protein antigens and synthetic peptide antigens generally does not induce specifi c immune response. Therefore there is a need for potent co-stimulation by coadministration of appropriate adjuvants, biocompatible carrier systems and application devices for vaccines consisting of highly purifi ed antigens. These particulate systems are supposed to mediate effi cient delivery to antigen-presenting cells and may induce infl ammation through activation of innate immunity [ 8 -10 ] . Lyme disease is the most frequent zoonosis both in Europe and the United States. Disease may progress into a chronic form and cause damage of the nervous or cardiovascular system, joints, skin, or eyes. Infected patients can be affected by prolong work disability or even permanent invalidity. Prevention and treatment of disease thus becomes a long-term priority for medical research. Some symptoms typical for Lyme disease have been known since the beginning of the twentieth century -acrodermatitis chronica atrophicans, erythema migrans, lymphocytoma, and meningopolyradiculoneuritis [ 11 ] . Nevertheless, the aetiological agent Borrelia burgdorferi was described quite recently on the basis of endemic juvenile arthritis accompanied with erythema migrans [ 12 , 13 ] . Later it was shown that B. burgdorferi sensu lato is a complex of several sibling species. Currently, 12 species are distinguished and new variants are identifi ed continuously, so the number of Borrelia species is probably not fi nal. At least three species are known to be pathogenic for human -B. burgdorferi sensu stricto, B. afzelii and B. garinii . The number of annually reported Lyme disease cases continually increases in many geographical areas. This may be due to the actual spread of the disease or alternatively, to improved diagnostic methods [ 14 ] . The genus Borrelia Swellengrebel 1907 is a member of the family Spirochaetaceae together with genera Leptospira and Treponema . In the USA, the only one causative agent -B. burgdorferi s.s. -was described. In Europe, two species -B. afzelii and B. garinii -are the most common. Borreliae are typical for their spiral-shaped cells, 10-30 μm in length and 0.2-0.3 μm in diameter. This shape is caused by periplasmatic fl agella responsible for the high motility in viscous environment like connective tissue. Borrelia lacks the rigid cell wall and the surface is composed mainly of lipoproteins with strictly controlled expression pattern, essential for adaptation for external conditions [ 15 ] . The disease has a vector character. Most of the time, it is transmitted to humans by infected ticks of the genus Ixodes , but Borrelia has also been found in the midgut of the mosquitoes and other blood sucking insect. The role of haematophagous insect in disease transmission is still unclear [ 16 , 17 ] . Reservoir competence has been described in the broad spectrum of wildlife animals, e.g. rodents (genera Apodemus, Clethrionomys, Microtus, Rattus , etc.), squirrels ( Sciurus sp. ), hares ( Lepus sp. ), and birds (genera Turdus, Phasianus, Carduelis and Fringilla , etc.). Human is a terminal host, incapable of further spreading of infection [ 18 ] . Lyme disease can have three stages -early localised infection, early disseminated infection, and late persistent infection, but not all stages develop in every infected individual. It appears that a signifi cant portion of infections has asymptomatic course without clinical symptoms, but with elevated levels of anti-Borrelia-specifi c antibodies. The fi rst stage is characterized by nonspecifi c symptoms including fever, chills, headache, lethargy, and/or muscle and joints pain, about 70 % is accompanied by erythema migrans at the site of bacterial entry. If the infection is not eliminated by the host immune system or antibiotics treatment, it may disseminate and affect central nervous system (meningitis, radiculopathy, seventh cranial nerve palsy) or cardiovascular system (atrioventricular heart block, myopericarditis). The late persistent infection may develop months to years after the transmission and commonly affects the nervous system, skin, joints, or less frequently heart, eyes, or other organs [ 15 ] . Treatment of Lyme disease is based on the application of antibiotics. For adults administration of doxycycline is recommended. For children mainly beta-lactam antibiotics are prescribed. For the treatment of advanced forms, cephalosporins or penicillin G are recommended. Long-term usage of antibiotics in persistent forms of Lyme disease has only limited effect [ 19 ] . The fi rst effort to design vaccine against Lyme disease followed shortly after the discovery of Borrelia as the aetiological agent causing this disease. First experiments demonstrated the immunogenicity of bacterial whole cell lysates. Later, specifi c proteins were identifi ed to be recognised by the host immune system [ 20 ] . Expression of surface antigens by Borrelia is highly variable. In the tick host, Borrelia expresses outer surface proteins A (OspA) and B (OspB). Genes coding both proteins are located within one operon and expression of these proteins is dependent on housekeeping sigma factor RpoD (σ 70 ). Presumed function of OspA and OspB is the adhesion to tick gut epithelium. When tick starts blood feeding on the vertebrate host, both proteins are downregulated. At the same time the expression of proteins dependent on alternative sigma factor RpoS (σ 38 ) is induced, e.g. (OspC) and OspF and decorin-binding proteins DbpA and DbpB. These proteins are required for Borrelia transmission to vertebrate and for initial stages of vertebrate infection. In later stages, OspC is downregulated and the expression of VlsE protein (Vmp-like sequence, expressed) is induced. It enables Borrelia escape from specifi c humoral response due to high VlsE variability. Early humoral response to Borrelia is characterised by production of IgM antibodies specifi c to OspC, fl agellins (p39 and p41), and BmpA (p39). Later IgG antibodies against p39, p41, p83/100, DbpA and VlsE arise. In some cases, antibodies against OspA and OspB can be detected in later stages of the disease [ 15 ] . Subunit vaccines of fi rst generation were based on OspA antigen, which is highly expressed during cultivation of Lyme disease spirochetes in vitro. Preclinical trials with vaccines based on OspA antigen demonstrated protection in animal models. OspA antigen was produced either as a recombinant protein or lipoprotein, or it was expressed on the surface of Escherichia coli , Salmonella typhimurium , or Mycobacterium bovis . Two OspA-based vaccine candidates were developed and tested. The ImuLyme vaccine (Pasteur Merieux-Connaught) based on purifi ed recombinant OspA antigen expressed in E. coli and LYMErix (GlaxoSmithKline) containing purifi ed recombinant OspA from B. burgdorferi s.s. [ 21 ] . In both vaccines protein was adsorbed on aluminium hydroxide. In clinical trials involving more than 10,000 people, LYMErix vaccine was found to confer protective immunity to Borrelia in 76 % of adults and 100 % of children with only mild or moderate and transient adverse effects [ 20 ] . Based on these results, LYMErix was approved by the Food and Drug Administration (FDA) on December 21, 1998. Subsequently, hundreds of vaccinee reported the development of autoimmune side effects. Supported by some patient advocacy groups, a number of class-action lawsuits were fi led against GlaxoSmithKline, alleging the vaccine had caused these health problems. These claims were investigated by the FDA and the US Centers for Disease Control (CDC), who found no connection between the vaccine and the autoimmune complaints [ 22 , 23 ] . Despite the lack of evidence, sales plummeted and LYMErix was withdrawn from the US market by GlaxoSmithKline in February 2002 in the setting of negative media coverage and fears of vaccine side effects. There are a number of reasons for the slow development of a Lyme disease vaccine, including reluctance by pharmaceutical companies to get burned in a similar way to LYMErix manufacturers. Others argue that it is simply more profi table for drug manufacturers to 'treat' the symptoms of Lyme palliatively, giving antiinfl ammatory medications, pain relievers, and other medications where antibiotics fail to address a patient's ailments. The continued availability of a Lyme disease vaccine for dogs has made many people question the motives of drug companies and the psychological component of many symptoms often blamed on Lyme disease. Unlike humane medicine, several licensed veterinary Lyme disease vaccines are available for companion pets. Three are formulations of B. burgdorferi bacterin (Merilym, Merial, Germany; Galaxy Lyme, Schering Plough, USA; LymeVax, Fort Dodge, USA). A European vaccine based on combination of bacterins from B. garinii and B. afzelii is produced for dogs (Biocan, Bioveta, Czech Republic) [ 24 ] . There are also available veterinary subunit vaccines based on recombinant OspC antigen (ProLyme, Intervet, USA; Recombitek Lyme, Merial, USA). All of the veterinary vaccines are applied in two-dose immunisation scheme with boosters recommended yearly. Safety data are limited and minor crossreactivity between heterologous Borrelia species has been reported [ 25 ] . Thus, the poor crossprotective coverage is a problem of currently available vaccines. Second generation of OspA subunit vaccines is based on recombinant OspA with genetically removed potentially cross-reactive T-cell epitopes, formerly suspected for the induction of autoimmunity [ 26 ] . Development of broadly cross-protective rOspA vaccine must address issues pertaining to sequence variation, because at least seven serotypes of OspA do exist. Multivalent-chimeric OspA proteins have been developed by molecular cloning incorporating the protective epitopes from several OspA serotypes [ 27 ] . Nevertheless, OspA antigen-based vaccines require repeated vaccination to keep high titre of specifi c antibodies in vaccinee serum to prevent transfer of bacteria from tick into human host. New interesting antigen for vaccine development is tick ferritin 2, which is responsible for maintaining iron homeostasis. Iron is an essential but potentially toxic element; therefore, during feeding, ticks must deal with the challenge of an enormous iron supply in the blood meal. Ferritins, the iron storage proteins, play a pivotal role in this process. It was shown that vaccination with recombinant FER2 signifi cantly reduces tick infestations in vaccinated rabbits infected with Ixodes ricinus and in cattle infected with Rhipicephalus microplus and Rhipicephalus annulatus . These results support the inclusion of FER2 as a promising candidate antigen for development of new anti-tick vaccines [ 28 ] . In spite of the fact that ferritin 2 is not the antigen derived from Borrelia , the vaccination with this antigen prevents long-term feeding of tick and transmission of Borrelia as well as other tick-transmitted pathogens into host. In this aspect ferritin 2 could be superior to OspA and is of interest to include it in development of combined multiantigen vaccine. As mentioned above, several new antigens were discovered and studied as possible targets for new vaccines. We focus on OspC antigen, which received considerable attention in the effort to develop a broadly protective Lyme disease vaccine. OspC is an essential virulence factor that is critical for the establishment of early infection in mammals [ 29 ] and the sequence of the protein is not undergoing mutation during infection [ 30 ] . OspC is a 22 kDa immunodominant lipoprotein that is anchored in the spirochete outer membrane by an N -terminal tripalmitoyl-S -glycerylcysteine [ 31 ] . The factor complicating the exploitation of OspC as a vaccine candidate is the existence of OspC variation. Molecular phylogenetic analyses have revealed that OspC sequences form at least 38 distinct OspC types. This OspC variation arises primarily through genetic exchange and recombination and not by hypermutation with concomitant immune selection [ 32 ] . Owing to this sequence variation the protective range of single OspC-based vaccine is narrow. A potential approach to solve the high variability of the OspC is to prepare (a) polyvalent vaccine containing several OspC variants [ 33 ] ; (b) generation of recombinant chimeric protein that consists of protective epitopes from those OspC types associated with disease [ 34 ] ; and (c) combination of two or more different recombinant immunogens like OspC, DbpA, and fi bronectin-binding protein (BBK32) [ 35 ] . From the biotechnological point of view the full-length OspC is diffi cult to express in a high yield and purity as a recombinant protein [ 36 ] . The removal of the lipidisation signal substantially increases both the yield (28 mg/1 of the bacterial culture) and purity (93 %) of the recombinant OspC protein [ 37 ] . On the other side delipidised rOspC exerted very low immunogenicity [ 38 , 39 ] and strong adjuvanticity of vaccine formulation is necessary to induce specifi c antibody response especially in IgG2 subclass (in experimental mice) important for effective complement activation and opsonisation. In effort to adhere to all above requirements for OspC vaccine, we developed experimental vaccine based on functionalised metallochelating nanoliposomes and synthetic non-pyrogenic adjuvants derived from muramyl dipeptide. This formulation induced strong immune response in experimental mice, which was superior to aluminium hydroxide formulation. Owing to the versatility and safety as an adjuvant, liposomes thus represent promising platform for developing the clinically acceptable vaccine against Lyme disease. Next part of the chapter is focused on liposomal-based vaccines. Of the numerous particulate delivery systems that have been developed until now, phospholipid bilayer vesicles (liposomes) are among the most promising. Gregoriadis and Allison fi rst reported the use of liposomes as immunological adjuvants in 1974 [ 40 , 41 ] . Since that time, liposomes and related vesicular carriers have been established as robust systems for induction of humoral and cellmediated immunity to a broad spectrum of infectious diseases and cancers [ 42 ] . Liposomes represent the oldest nanoparticle systems described for applications in biological studies as model membranes and in medicine. Over 44 years, liposomes have been shown to be suitable drug delivery systems for applications ranging from cosmetics and dermatology to medical applications such as therapy of infections, anticancer therapy, and veterinary vaccination [ 43 ] . Liposome-based vaccines have been around for approximately 30 years, and numerous liposome variants have been developed, some with evident immune-stimulatory properties and attractive safety profi les, resulting in registration of several products on the market and progressing of others to advanced stage of clinical testing. Epaxal ® (Crucell, hepatitis A, formalin-inactivated hepatitis A virus adsorbed to virosomes) and Infl exal ® V (Crucell, infl uenza, virosomes -reconstituted infl uenza viral membranes) are two examples of marketed liposome-based vaccines for human application. Infl exal ® V is licensed in 43 countries with over 60 million doses applied [ 44 , 45 ] . Furthermore, at least eight liposome-based adjuvant systems are currently approved for human use or undergoing clinical evaluation [ 46 ] . Liposomes represent almost ideal carrier/ delivery systems for the components of synthetic vaccines due to their biodegradability and their ability to retain/incorporate a variety of essential vaccine components simultaneously, even components possessing quite different physicochemical properties (different size, hydrophobicity, charge, etc.). Different synthetic vaccine components can be encapsulated within the aqueous cavities of liposomes (if hydrophilic) or associated with liposome bilayers (if at least partially hydrophobic in character). Furthermore, essential components can be attached to either internal or external outer leafl et membrane by electrostatic, covalent, or metallochelation interactions. Biochemically the most diverse vaccine components are adjuvants required for effi cacious activation of innate immunity cells including antigen-presenting cells (APC) (e.g. monophosphoryl lipid A, CpG oligonucleotides, muramyl dipeptide, and analogues). In addition, adjuvants can be combined with antigens such as soluble or membrane proteins to provoke strong specifi c immune response. Finally, the liposomes may present carrier for targeting of antigens and adjuvants to antigen-presenting cells [ 47 ] . The laboratory and industrial procedures for the liposome preparation have been established, and liposomes have been approved by the US Food and Drug Administration for biomedical applications. The potential for the participation of liposome-based recombinant vaccines in the human and veterinary vaccine market is very promising [ 48 ] . The benefi cial effect of liposomal carriers consists in their ability to ensure (1) protection and stabilisation of the antigen, including reconstitution of its native conformation, (2) enhanced uptake by APC by passive or active targeting and (3) enhanced or controlled antigen processing after uptake. Interaction of liposomal vaccine and liposomal immunomodulators with immune cells and cooperation between innate and adaptive branches of immune system is schematically represented in Fig. 36.1 . Liposome-based nanoparticle formulations used for vaccination can be broadly grouped into several classes: Conventional liposomes composed of neutral lipids and cholesterol have been the most widely studied due to their greatest versatility -desired formulation parameters can be achieved through modifi cation of the lipid composition or vesicle preparation method. These liposomes were used for steric entrapment of protein antigens in early stage of liposome-based vaccine research. These classical liposomes are relatively ineffective at enhancing the immunogenicity of antigens because the phospholipids do not act as adjuvants. Adjuvant effect of these liposomes is based on multimeric presentation of antigen, and their adjuvant effect can be optimised by variation of lipid composition, co-incorporation of various adjuvant molecules (e.g. MLP-A, MDP, CpG oligonucleotides, trehalose dibehenate), and attachment of antigen. Transferosomes are ultradeformable liposomes with enhanced skin penetration properties. They are composed of phosphatidylcholine and cholate (9:2 M ratio). Presence of cholate endowed these vesicles with high elasticity enables them to squeeze through pores in the stratum corneum. As a result of high elasticity of the bilayer, vesicles of the size of 200-300 nm can pass the highly compact stratum corneum and deliver their content into deep parts of the skin. Transferosomes are utilised as carrier system for topical application of vaccines [ 49 ] . Cationic transferosomes were also tested for noninvasive topical vaccination with pDNA vaccine against hepatitis B [ 50 ] . Surface adsorption on the cationic liposomes, via electrostatic interactions, is another useful method for attachment of antigens (primarily proteins) on liposomes. The recent work of Christensen, Perrie, and others with protein antigens adsorbed to cationic DDA/TDB (dimethyldioctadecylammonium/ trehalose-6,6-dibehenate) liposomes has demonstrated that formulations with surface-adsorbed antigens can be highly stable and elicit robust antibody and cell-mediated immune responses in mice and ferrets [ 42 , 51 -53 ] . Incorporation of synthetic immunostimulatory molecules like lipophilic TDB into cationic liposomes is able to potentiate signifi cantly their adjuvant effect [ 52 ] . It was also observed that certain cationic lipids, originally synthesised as transfection reagents, induce robust IFN-γ and TNF-α response in serum and antitumour immunity in murine models of pulmonary metastasis and fi brosarcoma [ 54 ] . DOTAP is an example of such a cationic lipid used for many years as component of liposomal gene delivery system exerted an enantiospecifi c adjuvant effect in dendritic cells [ 55 ] . Virosomes are a special class proteoliposomes prepared from reconstituted infl uenza virus membranes supplemented with PC [ 56 ] . The physicochemical features of virosomes are constrained by their well-defi ned composition and method of preparation, but these vesicles benefi t greatly from the inherent delivery properties (effi cient cell binding, internalisation, and cytosolic release) and immunogenicity of the infl uenza virus. Archaeosomes are liposomes prepared from special lipids isolated from archaebacteria, which are extremophiles, and their cell membranes contain lipids and proteins resistant to extreme temperatures, pH, and salt concentration. These lipids are less sensitive to oxidation and the membrane of archaeosomes is resistant to action of bile acid salts. Therefore they are suitable for enteral application. Immune responses induced by archaeosomes are comparable to those induced by complete Freund's adjuvants [ 57 ] . Functionalised liposomes in general represent various liposomal structures having their surface modifi ed by various ligands (biotin, oligosaccharides, peptides), lipids with reactive head groups, pH or temperatures sensitive polymers, monoclonal antibodies or their fragments, lectins, etc. to tailor their functions with respect to selective interaction with targeted extra or intracellular structures (e.g. pathogenic microorganisms, tumour cells, immune cells, blood clothes). Targeting of antigen to antigen-presenting cells is of great importance for enhancement of effi cacy of subunit recombinant vaccines. In this chapter we will introduce a new system for construction of liposomal-based vaccines. This system employs metallochelating lipids for binding of HIS-tagged recombinant antigens onto the surface of liposomes. One of the most critical parameters infl uencing the immunogenicity of liposomal vaccines is the method by which the antigen is physically or chemically associated with the formulation. The most common modes of association include covalent lipid conjugation (either preor post-vesicle formation), non-covalent surface attachment (via biotin, NTA-Ni-His, or antibody-epitope interactions), encapsulation, and surface adsorption (Fig. 36.2 ) . Many of the early studies of liposomal peptide and protein antigenicity in mice compared encapsulated antigens to those conjugated to the surface of preformed liposomes. Early studies by Alving, Gregoriadis, Therien, and others confi rmed that both methods are generally effective for inducing antibody and T-cell responses to associated protein antigens such as albumin and tetanus toxoid [ 58 -60 ] . In some cases, covalent antigen conjugation results in superior antibody induction, which is not surprising because B-cell receptors can recognise intact antigen on the liposome surface [ 61 ] . As the size and complexity of the antigen decreases, the benefi t of their surface conjugation for antibody induction becomes more pronounced, as found for the synthetic peptides for which surface conjugation provides immune responses superior to encapsulation [ 61 , 62 ] . With respect to potential application of metallochelating liposomes for the construction of vaccines, the question of in vitro and especially in vivo stability is of great importance. This problem could be divided into two fi elds: fi rst, the stability of the liposomes themselves and second, the effect of the components presented in biological fl uids (e.g. proteins and ions) on the stability of the metallochelating bond. In the example of OspC proteoliposomes, the gel permeation chromatography data indicated a good in vitro stability of the OspC proteoliposomes during the chromatographic process, within which they experience a shear stress and dilution. Also after incubating OspC proteoliposomes in serum at 37 °C, we demonstrated the stability of the metallochelating bond linking the protein to the liposomal surface [ 37 ] . In fact, in vivo fate of liposomes after the intradermal application is different from the situation after intravenous injection. First, dilution of proteoliposomes after intradermal application is not so rapid and second, the ratio of tissue fl uid proteins to proteoliposomes is more favourable to proteoliposomes owing to their relatively high concentration at the site of application. In the case of another route of application -intradermal -the fl ow rate of tissue fl uid within intradermal extracellular matrix is even lower in comparison with muscle tissue or blood vessels. This fact is often overlooked. The stability of metallochelating bond probably depends also on the character of the particular protein. It was shown that Ni-NTA3-DTDA liposomes (containing three functional chelating lipid) with single-chain Fv fragments (anti CD11c) bound onto the liposomal surface were able to target dendritic cells in vitro as well as in vivo. The application of Ni-NTA3-DTDA probably endows the metallochelating bond with a higher in vivo stability [ 63 ] , but this improved stability did not infl uence higher immunogenicity [ 64 ] . Generally, metal ions, physicochemical character of the metallochelating lipids, and their surface density belong to the factors that could be optimised to get a required in vivo stability and therefore strong immune response. Binding of recombinant OspC (rOspC) onto metallochelating liposomes was confi rmed by TEM, GPC, SDS PAGE and dynamic light scattering methods. Stability of the metallochelating bond in model biological fl uids was studied by the incubation of rOspC liposomes with human serum. This study showed that after 1 h incubation at 37 °C, more than 60 % of rOspC was still associated with liposomes. Based on this data, therefore, the half-life of rOspC proteoliposomes in serum was estimated to be at least 1 h. Increase in stability of surface-exposed antigens on the liposomes can be achieved by chemical binding onto the outer liposomal surface that is appropriately functionalised. With respect to a potential application for the construction of vaccines, the question of in vitro and especially in vivo stability is of great importance. There are only few references reporting the metallochelating bond implemented in the construction of supramolecular structures as vaccine carriers [ 65 -69 ] . Various ways for association of protein antigen with liposome is depicted in Fig 36. 2a . Real structures revealed by various microscopy methods are presented in Fig. 36 .3 . The interaction between the innate and the adaptive immune responses is paramount in generating an antigen-specifi c immune response. The initiation of innate immune responses begins with the interaction of pathogen-associated molecular patterns (PAMPs) on the pathogen side with pattern-recognising receptors (PRR) such as Toll-like receptors (TLRs) on the host cells involved in the innate immunity (e.g. dendritic cells). A major functional criterion commonly used for the evaluation of various new adjuvants involves their ability to stimulate the innate immunity cells. This would include engaging other PRRs and the co-receptors and intracellular adaptor signalling proteins with which they are associated. PAMPs and their derivatives are utilised by adjuvant developers to harness the power of innate immunity to channel the immune response in a desired direction. Based on the identifi cation of several TLRs and PAMPs recognised by them, various PAMP agonists were tested as adjuvants. Examples of TLR-PAMP-specifi c interaction include bacterial or viral unmethylated immunostimulating CpG oligonucleotides interacting with TLR9 and liposaccharide and its component monophosphoryl lipid A (MPLA) interacting with TRL4. These two types of adjuvants are in advanced stage of testing in clinical trials and some already licensed vaccines contain MPLA in liposomal form [ 70 , 71 ] . New lipophilic adjuvant (see Fig. 36 .4 ) suitable for construction of liposome-based vaccines is the mycobacterial cord factor trehalose-6,6-dimycolate (TDM), and its synthetic analogue trehalose-6,6-dibehenate (TDB) are potent adjuvants for Th1/Th17 vaccination that activate Syk-Card9 signalling in APCs [ 72 ] . The last group of potent synthetic or semisynthetic adjuvants is derived from muramyl dipeptide (MDP). Muramyl dipeptide and other muropeptides are derived from very specifi c group of PAMPs represented by peptidoglycans (PGN). Both Grampositive and Gram-negative bacteria contain PGN which consists of numerous glycan chains that are cross-linked by oligopeptides. These glycan chains are composed of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) with the amino acids coupled to the muramic acid. Muropeptides are breakdown products of PGN that bear at least the MurNAc moiety and one amino acid [ 73 ] . One of the prominent muropeptides is muramyl dipeptide (MDP), which is known since the 1970s. We will deal with this compound in more detail. Recently, the molecular bases for MDP recognition and subsequent stimulation of the host immune system have been uncovered. Myeloid immune cells (monocytes, granulocytes, neutrophils, and also DCs) possess two types of intracellular receptor for MDP/MDP analogues, namely, NOD2 and cryopyrin (infl ammasome-NALP-3 complex) [ 74 -76 ] . These two receptors recognise MDP/MDP analogues minimal recognition motifs for bacterial cell wall peptidoglycans [ 77 ] . Another recently reported sensor of MDP is cryopyrin (also known as CIAS1 and NALP3), which is a member of the NOD-LRR family [ 74 ] . Cryopyrin is a part of the infl ammasome complex that is responsible for processing caspase-1 to its active form. Caspase-1 cleaves the precursors of interleukin IL-1β and IL-18, thereby activating these proinfl ammatory cytokines and promoting their secretion. IL-1β is known to be a strong endogenous pyrogen induced by MDP. We showed that norAbu-MDP and that norAbu-GMDP analogues were not pyrogenic even at a high concentration, much higher than the concentrations used for vaccination [ 15 , 65 , 66 ] . The expression of NOD2 in dendritic cells is of importance with respect to the application of MDP analogues as adjuvants. Nanoparticles like liposomes are able to provide a direct codelivery of a danger signal (e.g., MDP) together with the recombinant antigen and therefore to induce an immune response instead of an immune tolerance. This is especially important for weak recombinant antigens or peptide antigens. Clearly, the recognition of MDP by DCs is crucial for the application of MDP analogues as adjuvants. Within the cell, MDP/MDP analogues trigger intracellular signalling cascades that culminate in the transcriptional activation of inflammatory mediators such as the nuclear transcription factor NF-κB. Liposomes probably play the role of efficient carriers for MDP and its analogues on the pathway from extracellular milieu into the cytosol, where they trigger intracellular signalling cascades that culminate in transcriptional activation of inflammatory mediators such as the nuclear transcription factor NF-κB pathway. Since the discovery and first synthesis of MDP, about one thousand various derivatives of MDP have been designed, synthesised, and tested to develop an appropriate drug for an immunotherapeutic application that would be free of the side effect exerted by MDP. The main side effects of MDP are pyrogenicity, rigor, headache, flue-like symptoms, hypertension, etc. Only several preparations reached the stage of clinical testing and only mifamurtide was approved for the treatment of osteosarcoma [ 77 ] . Vaccinology at the beginning of the 21st century SARS -beginning to understand a new virus Reverse vaccinology The global impact of vaccination against hepatitis B: a historical overview Safety, immunogenicity, and efficacy of quadrivalent human papillomavirus (types 6, 11, 16, 18) recombinant vaccine in women aged 24-45 years: a randomised, double-blind trial Recent advances in the discovery and delivery of vaccine adjuvants Vaccine adjuvant systems: enhancing the effi cacy of sub-unit protein antigens Immunity in response to particulate antigen-delivery systems The role of infl ammasomes in the immunostimulatory effects of particulate vaccine adjuvants Towards an understanding of the adjuvant action of aluminium History and characteristics of Lyme borreliosis Erythema chronicum migrans and Lyme arthritis. The enlarging clinical spectrum Lyme disease-a tick-borne spirochetosis? Biological aspects of Lyme disease spirochetes: Unique bacteria of the Borrelia burgdorferi species group Prevention of lyme disease: promising research or sisyphean task? Positive fi ndings of Borrelia burgdorferi in Culex (Culex) pipiens pipiens larvae in the surrounding of Brno city determined by the PCR method What is the percentage of pathogenic borreliae in spirochaetal fi ndings of mosquito larvae? Ecology of Borrelia burgdorferi sensu lato in Europe Two controlled trials of antibiotic treatment in patients with persistent symptoms and a history of Lyme disease The prevention of Lyme disease with vaccine A vaccine consisting of recombinant Borrelia burgdorferi outer-surface protein A to prevent Lyme disease. Recombinant Outer-Surface Protein A Lyme Disease Vaccine Study Consortium Lyme disease: uphill struggle The Lyme vaccine: a cautionary tale Testing of the Biocan B inj. ad us. vet. vaccine and development of the new recombinant vaccine against canine borreliosis Characterization of the humoral immune response in dogs after vaccination against the Lyme borreliosis agent A study with fi ve commercial vaccines using two different vaccination schedules Molecular mimicry in Lyme arthritis demonstrated at the single cell level: LFA-1 alpha L is a partial agonist for outer surface protein A-reactive T cells Immunization with a polyvalent OspA vaccine protects mice against Ixodes ricinus tick bites infected by Borrelia burgdorferi ss, Borrelia garinii and Borrelia afzelii Characterization of ferritin 2 for the control of tick infestations Delineating the requirement for the Borrelia burgdorferi virulence factor OspC in the mammalian host Stability of Borrelia burgdorferi outer surface protein C under immune selection pressure Identifi cation of Borrelia burgdorferi outer surface proteins OspC phylogenetic analyses support the feasibility of a broadly protective polyvalent chimeric Lyme disease vaccine An octavalent lyme disease vaccine induces antibodies that recognize all incorporated OspC type-specifi c sequences Analysis of antibody response in humans to the type A OspC loop 5 domain and assessment of the potential utility of the loop 5 epitope in Lyme disease vaccine development Multicomponent Lyme vaccine: three is not a crowd Isolation and purifi cation of recombinant outer surface protein C (rOspC) of Borrelia burgdorferi sensu lato Enhancement of immune response towards non-lipidized Borrelia burgdorferi recombinant OspC antigen by binding onto the surface of metallochelating nanoliposomes with entrapped lipophilic derivatives of norAbuMDP Biological activities of native and recombinant Borrelia burgdorferi outer surface protein A: dependence on lipid modifi cation Inability of outer-surface protein C (OspC)-primed mice to elicit a protective anamnestic immune response to a tick-transmitted challenge of Borrelia burgdorferi Liposomes as immunological adjuvants Entrapment of proteins in liposomes prevents allergic reactions in pre-immunised mice Liposomal vaccine delivery systems Engineering liposomes for drug delivery: progress and problems Epaxal: a virosomal vaccine to prevent hepatitis A infection Eleven years of Infl exal V-a virosomal adjuvanted infl uenza vaccine Design considerations for liposomal vaccines: infl uence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens Liposomal vaccines -targeting the delivery of antigen Two years into reverse vaccinology Non-invasive vaccine delivery in transfersomes, niosomes and liposomes: a comparative study Cationic transfersomes based topical genetic vaccine against hepatitis B N′-Dimethylaminoethane)carbomyl] cholesterol (DC-Chol), and 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP): prolonged liposome retention mediates stronger Th1 responses The vesicle size of DDA:TDB liposomal adjuvants plays a role in the cell-mediated immune response but has no signifi cant effect on antibody production Liposomal cationic charge and antigen adsorption are important properties for the effi cient deposition of antigen at the injection site and ability of the vaccine to induce a CMI response LPD lipopolyplex initiates a potent cytokine response and inhibits tumor growth Enantiospecifi c adjuvant activity of cationic lipid DOTAP in cancer vaccine Virosomes for antigen and DNA delivery Immunization of mice with lipopeptide antigens encapsulated in novel liposomes prepared from the polar lipids of various Archaeobacteria elicits rapid and prolonged specifi c protective immunity against infection with the facultative intracellular pathogen. Listeria monocytogenes Liposomes as adjuvants with immunopurifi ed tetanus toxoid: infl uence of liposomal characteristics Liposomal adjuvanticity: effect of encapsulation and surface-linkage on antibody production and proliferative response Immunopotentiation of the humoral response by liposomes: encapsulation versus covalent linkage Antibody and cytotoxic T-lymphocyte responses to a single liposome-associated peptide antigen Parameters affecting the immunogenicity of a liposome-associated synthetic hexapeptide antigen Targeting dendritic cells with antigen-containing liposomes: a highly effective procedure for induction of antitumor immunity and for tumor immunotherapy Antibody response to polyhistidinetagged peptide and protein antigens attached to liposomes via lipid-linked nitrilotriacetic acid in mice Metallochelating liposomes with associated lipophilised norAbuMDP as biocompatible platform for construction of vaccines with recombinant His-tagged antigens: preparation, structural study and immune response towards rHsp90 Immobilization of histidine-tagged proteins on monodisperse metallochelation liposomes: preparation and study of their structure Attaching histidinetagged peptides and proteins to lipid-based carriers through use of metal-ion-chelating lipids Association of antigens to ISCOMATRIX adjuvant using metal chelation leads to improved CTL responses Preparation and characterization of nickel nanoparticles for binding to his-tag proteins and antigens Liposomes containing lipid A: an effective, safe, generic adjuvant system for synthetic vaccines Activation of Toll-like receptor 9 and production of epitope specifi c antibody by liposome-encapsulated CpG-DNA Cutting edge: Mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analog trehalosedibehenate MDP and other muropeptides -direct and synergistic effects on the immune system NALP3 forms an IL-1beta-processing infl ammasome with increased activity in Muckle-Wells autoinfl ammatory disorder Mini-review: the role of peptidoglycan recognition in innate immunity Peptido glycan signaling in innate immunity and infl am matory disease Mifamurtide for the treatment of nonmetastatic osteosarcoma Acknowledgements This work was supported by grants: GAČR P304/10/1951 to J.T. and M.R. GAP503/12/G147 and the Ministry of Education, Youth and Sports of the Czech Republic (CZ.1.07/2.3.00/20.0164) to J.T. and M.R.