Review Article Mucosal Vaccine Development Based on...

Review ArticleMucosal Vaccine Development Based on Liposome Technology

Valentina Bernasconi1 Karin Norling2 Marta Bally2 Fredrik Houmloumlk2 and Nils Y Lycke1

1Mucosal Immunobiology and Vaccine Center (MIVAC) Department of Microbiology and Immunology Institute of BiomedicineUniversity of Gothenburg 40530 Gothenburg Sweden2Department of Applied Physics Chalmers University of Technology 41296 Gothenburg Sweden

Correspondence should be addressed to Nils Y Lycke nilslyckemicrobioguse

Received 14 October 2016 Accepted 27 November 2016

Academic Editor Giuseppe A Sautto

Copyright copy 2016 Valentina Bernasconi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Immune protection against infectious diseases is most effective if located at the portal of entry of the pathogen Hence there isan increasing demand for vaccine formulations that can induce strong protective immunity following oral respiratory or genitaltract administration At present only few mucosal vaccines are found on the market but recent technological advancements anda better understanding of the principles that govern priming of mucosal immune responses have contributed to a more optimisticview on the future of mucosal vaccines Compared to live attenuated vaccines subcomponent vaccines most often protein-basedare considered safer more stable and less complicated to manufacture but they require the addition of nontoxic and clinically safeadjuvants to be effective In addition another limiting factor is the large antigen dose that usually is required for mucosal vaccinesTherefore the combination ofmucosal adjuvants with the recent progress in nanoparticle technology provides an attractive solutionto these problems In particular the liposome technology is ideal for combining protein antigen and adjuvant into an effectivemucosal vaccine Here we describe and discuss recent progress in nanoparticle formulations using various types of liposomes thatconvey strong promise for the successful development of the next generation of mucosal vaccines

1 Introduction

Most pathogens enter the body through mucosal surfacesand therefore vaccines that target the respiratory gastroin-testinal or urogenital tracts are attractive as they stimulatelocal protection against infections However because ofthe requirements for strong mucosal adjuvants and usuallyrelatively large amounts of antigen only few such vaccineshave been developed and most of these are live atten-uated vaccines Whereas live attenuated vaccines can beeffective subcomponent vaccines are usually safer and withless manufacturing and regulatory complications There-fore efforts are focused on developing mucosal vaccinesbased on subcomponents but this also requires identifyingappropriate and effective mucosal adjuvants to enhance theimmune response Subcomponent vaccines can consist ofbacterial whole cell components virus-like particles or otherparticles polysaccharides complete protein structures orpeptides that delivered at mucosal membranes together withan adjuvant can stimulate strong immune responses and

protection against infection Suchmucosal vaccines aremuchwarranted as they carry several advantages over injectablevaccines In particular mucosal vaccines can elicit both localand systemic immune responses and they are safer as theydo not require needles and may allow for mass vaccina-tion when pandemic spread of infection is a threat [1 2]Mucosal vaccination could also lead to increased complianceand reduce the risk of spreading transmissible diseases ashas been experienced with spread of hepatitis C and HIVinfections following the use of injectable vaccines [3] Mostimportantly mucosal immunization elicits antigen-specificlocal IgA and systemic IgG antibodies as well as strongsystemic and tissue resident CD4+ and CD8+ T cell immu-nity (Figure 1) Despite these advantages only few mucosalvaccines are commercially available The reason for this isthe need for safe and effective mucosal adjuvants and thefact that many vaccine formulations require protection fromdegradation of the antigens as seen for example after oraladministration [4] Consequently the development of novelcombinations of antigen and adjuvant into nanoparticles for

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2016 Article ID 5482087 16 pageshttpdxdoiorg10115520165482087

2 Journal of Immunology Research

Epithelial cells

Dendritic cell

MHC IIpeptide

TCR

B cell

Mucosal surface

Inductive site Effector site

Secretory IgA

NALT

T cell zone

Follicular DC

B cell follicle

TFH cell

p p

TFH cell

B cell

Germinal centre

Lymph node

Plasma cell

IgA B cell

Blood stream

Vaccine

FAE cells

CD8+ T cell

CD4+ T cell

CD4+ and CD8

+ effectorand memory T cells

Figure 1 Principles for induction of mucosal immune responses after intranasal vaccination The respiratory mucosal immune systemconsists of clusters of lymphoid cells beneath the mucosal epithelium hosting both innate and adaptive immune cells [29] There is a cleardistinction between inductive and effector sites and these are also physically separated Inductive sites are organized lymphoid tissues whereantigen is taken up by DCs and other APCs The effector sites on the other hand are tissues that provide protection against infection wherespecific antibodies and CD4+ and CD8+ effector and memory T cells reside [30] The main inductive sites for mucosal immune responsesafter intranasal vaccination are known as nasopharynx-associated lymphoid tissue (NALT) which harbors B cell follicles and T cell zones inwell demarked microanatomical areas [31] Antigens are taken up by DCs that get access to the luminal content either through direct uptakethrough the epitheliumor via the follicle associated epithelium (FAE) that overlay theNALT After antigen uptake the immatureDCs undergomaturation and subsequently leave themucosal tissue for the draining lymph nodes alternatively if already in the NALT the DCs will directlyprime naive CD4+ or CD8+ T cells Activated CD4+ T cells differentiate into various subsets T helper 1 (Th1)Th2 orTh17 cells regulatory Tcells (Tregs) or follicular helper T cells (TFH) The latter are critically needed for the expansion and differentiation of the activated B cells inthe germinal center (GC) which is formed in the B cell follicle in the lymph node after vaccination TFH cells are involved in the developmentof long-lived plasma cells and memory B cells in the GC

the next generation of effective mucosal vaccines is muchneeded

Liposomes have been extensively used as delivery vehi-cles for vaccine antigens some of the advantages of these

formulations are (a) protection against antigen degradation(b) tissue depot effects or slow release of antigen and (c)facilitated uptake of antigen by antigen presenting cells (APC)[5 6] Phosphatidylcholines are the most common lipids

Journal of Immunology Research 3

employed for liposome manufacturing However nanoparti-cles can be developed fromawide range of lipids andproteinswhich have been found to also alter their physicochemicaland biological properties Classical liposomes are now alsogradually being replaced by more advanced technologieswith the new generation of lipid-based nanovesicles (L-NVs)which have more elaborate functions and less weaknessesNiosomes transfersomes sphingosomes and other nonli-posomal lipid-based nanoparticles are excellently reviewedby Grimaldi et al [7] For example the virus-like particle(VLP) or virosome L-NV incorporates virus-derived orrecombinant proteins that in this way are effectively deliveredto the immune system [8] This technology is used in twocommercial vaccines Inflexal (against influenza) and Epaxal(against hepatitis A) [9 10] Currently several liposome-based vaccine delivery systems against infectious diseases areundergoing clinical testing (Table 1)

Mucosal vaccines were initially designed to be adminis-tered orally Later also intranasal vaccines were developedand today several different routes of administration are beingexplored formucosal vaccination including pulmonary gen-ital tract rectal and sublingual routes Whereas preclinicalexamples in animal models have shown that in principleall of these routes work well only the oral and intranasalroutes have been used for licensed human vaccines [11] Thereason for this may simply be attributed to that a majority ofthese vaccines are against gastroenteric infections (requiringoral vaccines) Importantly the adjuvant choice is criticalbecause it enhances and modulates the immune response tothe vaccine For example the breadth the quality and thelong-term protective effect of the vaccine may be directlydependent on the adjuvant [12] Liposomes can also functionas adjuvants in their own right as they have been shown toenhance immune responses even after oral administration[13] A particular type of liposomes that is layersomeswhich are liposomes coated with single or multiple layers ofbiocompatible polyelectrolytes has been found to stimulatesignificant serum IgG and mucosal IgA antibodies and Tcell responses producing IL-2 and IFN-120574 [14] Noteworthythough the oral route most often requires high amountsof antigen and protection against enzymatic degradationMoreover an effective oral liposome vaccine should beeffective at breaching the mucus barrier to facilitate uptakeof antigen by gut mucosal antigen presenting cells (APCs)

Whereas oral vaccination provides a real challenge tovaccine developers the intranasal (in) route is more permis-sive In fact in vaccination has several advantages comparedto oral vaccination These include the need for less antigenand a substantially reduced risk of antigen degradation[15] Interestingly IgG-coupled liposomes with an enhancedtransmucosal transport were more immunogenic than plainliposomes given in [16] Because of the compartmentaliza-tion of the mucosal immune response due to the acquisitionof tissue-specific homing receptors on activated lymphocytesnasal immunization also promotes a much stronger specificimmune response in the respiratory tract compared to oralimmunization This also results in that excellent genital tractimmunity can be achieved after in immunizations while thisis not the case after oral vaccinationThus local secretory IgA

(sIgA) antibodies and genital tract cytotoxic T cells weremoreeffectively stimulated after in immunization than throughoral immunizations [17ndash20]

In this review we will describe and discuss liposomes asvaccine delivery vehicles for efficient mucosal immunizationIn the first section the impact of liposome composition andstructure for vaccine efficacy will be discussed and in the sec-ond section the nature and qualities of the resulting immuneresponses following liposome vaccination will be describedFinally in the last section we have attempted to summarizethe current standing of the field of liposome-based vaccinesand future perspectives towards the development of the nextgeneration of effective mucosal vaccines

2 Liposomes as Vaccine Delivery Vehicles

Liposomes are spherical lipid bilayer structures with anaqueous core ranging in size from tens of nanometers toseveral micrometers in diameter Liposome technology wasfirst explored in the 1960s by Bangham et al as a modelsystem for diffusion of ions across biological membranesand already in the 1970s there was an interest in using themfor drug delivery [39 40] At that time some researchersalso tested them for adjuvant functions and ever sincethey have been used in various vaccine formulations owingto their inherent structural and chemical properties [41]Phospholipids are most commonly the main constituents ofthe shell that delimit the aqueous core of the liposomeThesemolecules are amphiphilic and contain a hydrophobic tailconsisting of two fatty acids linked by a glycerol backboneto a hydrophilic headgroup made up of phosphate andpotentially another organicmolecule that together determinetheir chemical properties as shown in Figure 2 In an aqueousenvironment this polarized structure facilitates self-assemblyinto arrangements with the fatty acids facing each otherand forming an oil-like compartment between the outwards-facing phosphate groups In liposomes the arrangementis a hollow sphere a vesicle consisting of either a singleor multiple phospholipid bilayers forming unilamellar ormultilamellar particles Other types of lipids all with theamphiphilic structure in common may be incorporated inliposomes Examples of other interesting lipid categories inthe context of vaccine carrier formulations are sterols mostnotably cholesterol (Chol) sphingolipids with a sphingosinebackbone linked to a headgroup and a single acyl chain tailand fat-soluble vitamins such as tocopherols (vitamin E)

The fact that liposomes have both a hydrophobic anda hydrophilic region makes them versatile and useful ascarriers for antigens hydrophobic peptides or proteins canbe inserted into the inner hydrophobic center of the bilayerwhile hydrophilic molecules can be either encapsulated inthe core of vesicles or bound to their surface (Figure 3(c))Surface binding of the antigen can be achieved by covalentattachment or it can occur through adsorption or electrostaticinteractions Alternatively an antigen-bound hydrophobicanchor can be inserted into the bilayer Another advantage ofliposomes in vaccine formulations is that their physicochem-ical properties are highly adaptable and their size chargeand lamellarity can be tailored to meet the requirements


Table 1 Examples of liposome adjuvant vaccines against infectious diseases tested in clinical trialsThis compilation of completed or ongoingstudies involving liposomes for vaccination of humans was generated with data from ClinicalTrialsgov Here we have indicated the targetdisease the vaccine composition the route of administration the clinical testing stage and the reference number

Name Disease Description Route ofadministration Sponsor Status ClinicalTrialsgov

Identifier

FMP012 withAS01B Malaria

Falciparum malaria protein (FMP012)in a formulation based on liposomesmixed with the immunostimulantsmonophosphoryl lipid (MPL) and

Quillaja saponariaMolina fraction 21

Intramuscularinjection

US Army MedicalResearch and

GlaxoSmithKlinePhase 1 NCT02174978

TVDV withVaxfectin [21] Dengue fever

Tetravalent dengue vaccine (TVDV)with Vaxfectin cationic lipid-based

adjuvant


US Army MedicalResearch andMaterielCommand

Phase 1 NCT01502358

Ag85B-ESAT-6 withCAF01 [22]

Tuberculosis

Subunit protein antigenAg85B-ESAT-6 with two-componentliposomal adjuvant system composed

of a cationic liposome vehicle(dimethyldioctadecylammonium(DDA) stabilized with a glycolipidimmunomodulator (trehalose

66-dibehenate (TDB))


Statens SerumInstitut Phase 1 NCT00922363

Biocine withlipid A [23] HIV

Recombinant envelope proteinrgp120HIV-1SF2 combined with lipid

AIntradermal

National Instituteof Allergy and

Infectious DiseasesPhase 1 NCT00001042

PAMVACwith GLA-SEor GLA-LSQ

Malaria

Placental malaria vaccine candidateadjuvant with alhydrogel

glucopyranosyl lipid adjuvant-stableemulsion (GLA-SE) or glucopyranosyl

lipid adjuvant-liposome-QS-21formulation (GLA-LSQ)


TuebingenUniversity Hospital Phase 1 NCT02647489

ID93 withGLA-SE [24] Tuberculosis

Recombinant fusion proteinincorporating fourM tuberculosisantigens (ID93) formulated with

glucopyranosyl lipid adjuvant- (GLA-)stable emulsion (SE)




Novelliposomalbasedintranasalinfluenzavaccine

Influenza Liposomal-based influenza vaccine Intranasal Hadassah MedicalOrganization Phase 2 NCT00197301

VaxiSomewith CCSC[25]

InfluenzaCommercial split influenza virus andpolycationic liposome as adjuvant

(CCSC)

Intramuscularinjection NasVax Ltd Phase 2 NCT00915187

Fluzone withJVRS-100[26]

InfluenzaInactivated trivalent influenza virusvaccine administered with cationic

lipid-DNA complex adjuvant JVRS-100Intradermal

ColbyPharmaceuticalCompany and

JuvarisBioTherapeutics

Phase 2 NCT00936468

RTSS withAS01 [27] Malaria

Repeat sequences of the Plasmodiumfalciparum circumsporozoite protein

(RTSS) fused to the hepatitis Bsurface antigen with a liposome-basedadjuvant system that also containsmonophosphoryl lipid A (MPL) andQuillaja saponariaMolina fraction 21


KEMRI-WellcomeTrust CollaborativeResearch Program

andGlaxoSmithKline

Phase 3 NCT00872963


Table 1 Continued


IdentifierAmphomul[28]

Visceralleishmaniasis Amphotericin B lipid emulsion Intramuscular

injectionBharat Serums andVaccines Limited Phase 3 NCT00876824

of the vaccine In particular the charge and membranefluidity of the liposome can be fine-tuned by altering the lipidcomposition [42]

The properties of different phospholipids depend on boththeir polar headgroup and the nature of their fatty acid tailsThe characteristics of the lipid headgroup play a key role indetermining the surface properties and in particular the sur-face charge of the vesicles Naturally occurring phospholipidscan be categorized into 6 types according to their headgroupphosphatidylcholine (PC) phosphatidylethanolamine (PE)phosphatidylserine (PS) phosphatidylinositol (PI) phos-phatidylglycerol (PG) or phosphatidic acid (PA) WhereasPS PI PG and PA are negatively charged PC and PEare neutral but zwitterionic Further both the fluidity andpermeability of themembrane and in extension its resistanceto degradation here referred to as stability depend both onthe length and degree of saturation of the acyl chains of thetail as well as the charge of the headgroup All of these factorsinfluence the transition temperature of the lipid which is inturn decisive for whether the lipid membrane exists in gel-or fluid-phase at a certain temperature These factors alsodetermine the tendency of multicomponent membranes toundergo small-scale phase separations resulting in heteroge-neous distribution of different lipids A common modulatorof the membrane permeability and fluidity is cholesterolwhich also influences the liquid-to-gel phase temperature[43]

The possibility to chemically modify both the head-group and the tail region gives rise to the option of pro-ducing synthetic phospholipids tailored to specific require-ments For example positively charged liposomes have beenmade using synthetic cationic lipids such as 12-dioleoyl-3-trimethylammonium propane (DOTAP) 12-dimyristoyl-trimethylammonium propane (DMTAP) and dimethyldioc-tadecylammonium bromide (DDA) From this it followsthat the choice of lipid composition will greatly impact thebiophysical and chemical properties of the liposome Alsoby choosing lipids that naturally exist in cell membranesliposomes can be completely biodegradable nontoxic andnonimmunogenic in themselves [44 45] On the other handif components with an origin in archaeal bacterial or viralmembranes are chosen they could enhance the immuno-genicity of the formulation [35 37 46ndash50] Accordingly PSis naturally exposed on the surface of cells undergoingapoptosis and in this way liposomes containing PSmay effec-tively trigger phagocytosis by macrophages [51ndash53] Finallymany other modifications can be made to liposomes thatfurther extend the high versatility of these nanoparticlesSuchmodification includes the attachment of targeting agentssuch as galactose or APC-specific antibodies of polymersfor example poly(ethyleneglycol) or the addition of different

kinds of coatings such as chitosan [38 54ndash58] Furthermorewhen introduced into a physiological fluid nanoparticlessuch as liposomes acquire a dynamic layer of adsorbedproteins in a corona the nature of which is determined byboth the particlersquos size and surface properties as well as theshear stress the particle is exposed to [59ndash62] In practicethis means that carefully engineered surface properties maybe altered by the adsorption of a complex protein mixtureThis can have many diverse consequences for example theprotein corona may mask surface bound ligands and in thisway prevent receptor binding or it can cause complementactivation [63ndash65]

3 Physiochemical Properties ofLiposomal Vaccines

31 Surface Charge Although liposomes with different char-acteristics have been extensively used for vaccine deliveryit still remains to explain why the immune response ismodulated differently by different liposomal formulations Itis inherently difficult to dissect the contribution of differentproperties as changing one property usually influences oneor several others Surface charge can for instance be changedby altering the lipid composition however by changingthe lipid composition also other properties might changesuch as membrane fluidity rigidity and stability Hence itmay be difficult to directly assess the influence of changingdifferent physicochemical properties of liposomes on theimmune response Nevertheless many attempts have beenmade to describe the effects on immune responses afteraltering liposomal characteristics One of these parametersis the charge of the liposome which is assessed by thezeta potential a measure of the electrostatic potential at thelimit of what is called the diffuse electric double layer thatsurrounds the particle (Figure 3(a)) The double layer is adiffuse layer of differently charged ions spatially distributedat the surface of the particle which in this way becomesshielded The magnitude of the zeta potential thus dependson the concentration of ions within the double layer butalso other factors such as the ionic strength and pH ofthe dispersion medium This must be kept in mind whencomparing zeta potential values reported in different studiesand under different conditions

Because the cell surface as well as the mucus coating ofthe mucosal membrane is negatively charged it is frequentlyhypothesized that positively charged liposomes will exhibitstronger interactions with the cell membrane as well asan increased mucoadhesion The latter leads to reducedclearance rate that is slower removal from themucosalmem-branes Noteworthy both increased interactions with the cellmembrane and prolonged exposure time of the antigen atthe mucosal surface are thought to lead to increased cellular


The nature of theheadgroup influencesthe surface propertiesof the liposome most

notably the chargebut also the

membrane fluidity andpermeability

The length and degreeof saturation of the tails

affect how closely thelipids pack and in

extension the fluidityand permeability of

the membrane

Hydrophobictail

Glycerolbackbone

Hydrophilicheadgroup

POPCDOTAP DMPG

TAP

DA PI

PS

PA PE

PCPG

Positive Negative Neutral

OO

O O

HO

HO

O

O

OOO O O

O

OO

O

OPP O

O

H

HH

N+

N+

Ominus

Ominus

O

OPO

H

N+

Ominus

H

O

HO

HO

OO

OP

H

Ominus

H

O

HN+

OO

OO

HH

OO

OO

HHHO

OO

OOO

O

and combine them into custom liposomes

making up lipids with properties of your choice

and tails

Choose a headgroup

Figure 2 Generation of customized liposomes Lipids are polar molecules consisting of a hydrophilic headgroup and hydrophobic fatty acidtails Examples of positively charged headgroups are trimethylammonium propane (TAP) and dioctadecyl ammonium bromide (DA) whilenegatively charged headgroups are phosphatidylserine (PS) phosphatidylinositol (PI) phosphatidylglycerol (PG) or phosphatidic acid (PA)and finally neutral headgroups are phosphatidylcholine (PC) or phosphatidylethanolamine (PE) A headgroup can be combined with tailsof different nature to create lipids with the desired properties the examples shown are the lipid 12-dioleoyl-3-trimethylammonium propane(DOTAP) and the phospholipids dimyristoylphosphatidylglycerol (DMPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)Different lipids can then be combined into liposomes with different functional features which provide the basis for this highly diverse andversatile technology that is so excellently suited for vaccine development


O

OHO

HO

HO

HO

OOPO

O

O

OO

O

OO

OO

OOHNH

NH

OH

minusO

+

+

+

minus

minus

minus

(a) (b)

(c)

(d) (e)

(f)NH+

4

Figure 3 Various properties of liposome-based vaccines (a) The effect of altered surface charge on liposome function has been extensivelyexamined [32 33] (b)The lipid composition is critically influencing the immune response [34] (c) Also the localization of the antigen on orinside the liposome plays an important role in shaping the immune response to the vaccine There are several modes of antigen associationto liposomes Firstly antigens may be encapsulated in the aqueous core or they could be linked to the surface via covalent attachmentAlternatively a hydrophobic anchor can be used to attach the antigen to the surface via adsorption or through electrostatic interactionswith lipids of opposite charge For proteins with a hydrophobic region one may even successfully insert these in the liposomemembraneTheliposomemay also be used as an immunoenhancer simply by admixing the antigen and the liposomes (d)Only few studies have addressed theimpact of size or lamellarity [35 36] (e) Modifications of liposomes to increase their immunoenhancing effect can be done through attachingPAMPs such as lipid A (LPS) or through specific targeting strategies using cell-specific antibodies (anti-CD103 or -DEC205) [16 37] (f)Other modifications including addition of poly(ethylene glycol) (PEG) or different polymer coatings that increase the liposome penetrationof the mucosal barrier or to increase liposome resistance in biological fluids have also been developed [38]


uptake of antigen and stronger immune responses Howeverthis may not always be the case In general positively cationiccharged liposomes have been shown to be better retained andmore immunogenic at mucosal membranes than negativelycharged or neutral liposomes [66 67] Furthermore cationicliposomes were found to effectively deliver antigen to bothmucus and antigen presenting cells (APCs) as shown in an invitro model of the airway epithelium with liposomes madewith distearoylphosphatidylcholine (DSPC)trehalose 66-dibehenate (TDB) (neutral) and DSPCTDBDDA (positive)with varying amounts of DDA [68] Moreover cationicliposomes consisting of DOTAPChol DMTAPChol ormost prominently the polycationic sphingolipid ceramidecarbamoyl-spermine (CCS) and cholesterol were shown toeffectively stimulate systemic and mucosal humoral andcellular immune responses after in immunizations inmice [32] By contrast neutral liposomes with dimyris-toylphosphatidylcholine (DMPC) or anionic liposomes withDMPCdimyristoylphosphatidylglycerol (DMPG)were com-parably ineffective as immunogens [32] While a positivecharge appears to increase the immunogenicity of liposomesit still remains to be investigated in greater detail In factnegatively charged liposomes have been shown to be moreimmunogenic than both zwitterionic and positively chargedliposomes and it has even been postulated that anionic lipo-somes could exert an immunosuppressive effect on alveolarmacrophages and in this way promote an enhanced humoralimmune response [33 69ndash72] Hence it can be hypothesizedthat several mechanisms are modulated by the charge ofthe liposome It is also important to point out that alteredcharge of the liposomes by necessity involves modifying thelipid composition which most likely will change also otherproperties such as membrane heterogeneity fluidity andstability [73] Naturally also the charge of the liposome maydramatically influence the immunogenicity when given bydifferent routes as this may provide differentially chargedmicroenvironments

32 Lipid Composition The lipid composition (Figure 3(b))is known to influence the stability of the liposome amore stable formulation might lead to a larger amount ofbioavailable antigen and potentially also a depot effect Hanet al made liposomes from various combinations of Choldipalmitoylphosphatidylcholine (DPPC) dipalmitoylphos-phatidylserine (DPPS) and distearoylphosphatidylcholine(DSPC) and found that certain combinations impacted ontheir stability Liposomes with DSPC having a higher tran-sition temperature were more stable in vitro and likelyprotected antigen better from degradation in the gastroin-testinal tract [49] It was also found that stable lipo-somes containing DPPS induced stronger IgA responsescompared to formulations without DPPS [34] Combi-nations of both DPPCDMPG and DPPCPS have beenfound effective at targeting liposomes to macrophagesthough DPPCDMPG were found more immunogenic thanliposomes with DPPCpalmitoyl phosphatidylethanolamine(DPPE) orDPPCPS Noteworthy changing the lipid compo-sition also resulted in an altered charge with the DMPG andPS types being more negatively charged [73] Furthermore

cationic liposome-hyaluronic acid (HA) hybrid nanoparticlesystems have recently been developed and tested for DCmaturation It was found that primarily an upregulation ofcostimulatory molecules including CD40 CD86 and MHCclass II were responsible for an enhanced effect which greatlycontributed to an enhanced specific T cell and antibodyresponse following in vaccination [74]

As previouslymentioned using archaeal lipids liposomescan be made more immunogenic and liposomes compris-ing archaeal membrane lipids (archaeosomes) were foundconsiderably more potent than liposomes made with Eggphosphatidylcholine (EPC)Chol at inducing ovalbumin-(OVA-) specific IgGand IgA antibodies following oral admin-istration in a mouse model [46] This was likely due toincreased stability in the gastrointestinal tract and to the factthat the archaeosomes were better retained in the intestine[46] However the difference may also partly reflect thefact that the archaeosomes are negatively charged while theEPCChol-liposomes are neutral

33 Antigen Localization in Liposomal Formulations Thereare many ways of incorporating antigens into liposomesThis raises the question of whether some strategies aremore effective than others in the context of optimizing theimmunogenicity of the liposome Antigens can be hostedin the aqueous core of the liposome inserted into themembrane leaflet or bound to the surface by covalent bondsor intermolecular forces (Figure 3(c)) Hence a plethoraof combinations exist and those could be used to enhanceresistance against antigen degradation or facilitate antigenuptake Thus the liposome formulation may be tailoredfor specific needs and purposes If an oral vaccine is tobe designed one may hypothesize that encapsulating theantigen inside the liposomes should be an effective strat-egy to prevent enzymatic degradation However by hidingthe antigen in the liposome the immunogenicity may becompromised because the antigen will not be immediatelyaccessible for APCs Therefore choosing how to physicallyincorporate the antigen in the liposome may have criticalconsequences and could dramatically change the immuneresponse Unfortunately until now such aspects have notbeen addressed in a systematic manner When administeredorally encapsulated antigen may more effectively stimulatelocal IgA and serum IgG antibody responses comparedto when soluble antigen is admixed with the liposomes[73 75] On the other hand following in administrationadmixed antigen and liposome have been quite effectiveeven compared to liposome-encapsulated antigen [32 70]Interestingly liposomes have been found to exert an immu-noenhancing effect evenwhen administered 48 hours prior tothe antigen [70] Furthermore even liposome surface boundantigens rather than fully encapsulated antigens have beenfound to be more immunogenic following in immunization[71] Probably these observations underscore that the inroute is less sensitive to antigen degradation compared to theoral route Thus depending on the route of administrationit seems clear that antigens may or may not be immunogenicwhen exposed on the surface of the liposome and for manyformulations it may in fact be advantageous to have surface


bound as well as encapsulated antigens Indeed this may alsoapply to the adjuvant It was found that cholera toxin B-subunit (CTB) adjuvant bound to the surface of the liposomewas more effective compared to when encapsulated in theliposome [76] In fact a challenging question is what therelationship and localization should be between the antigenand the adjuvant in the liposome Theoretically it can beargued that since the adjuvant is included primarily topromote dendritic cells- (DC-) priming of the T cells it shouldbe encapsulated while the antigen should be both encap-sulated and surface bound to secure sufficient stimulationalso of naıve B cells Of note B cells normally recognize 3Dstructures with their receptors while T cell receptors react todegraded linear peptides However this interesting questionhas been poorly investigated and only few studies have beenpublished on this topic For example it has been observedthat by altering the lipid-to-antigen ratio the humoral andcellular immune responses can be differentially induced [3277] Thus it is likely that the immune response followingliposome administration is susceptible not only to the choiceof antigen and lipid components but also to their relativeproportions and localization in the liposome

34 Size and Lamellarity A broad range of unilamellar andmultilamellar liposomes with varying sizes have been foundto have variable effects following mucosal immunization(Figure 3(d)) While unfortunately the degree of multil-amellarity is not routinely reported the influence of sizeandor lamellarity on the immunogenicity of the liposomeis yet to be determined For example a comparative studybetween unilamellar archaeosomes 100 nm in diameter orlarge multilamellar aggregates of these clearly identifiedbetter immunogenicity of the multilamellar aggregates [35]Noteworthy not only the size but also the lipid assemblywas different between the unilamellar and multilamellarconstructs in this example On the other hand anotherstudy reported that a ldquodouble liposomerdquo consisting of small(sim250 nm) liposomes made from SoyPC DPPC Chol andSA encapsulated into a bigger (1 to 10120583m) outer liposomemade from DMPC and DMPG was found only marginallymore immunogenic than the small liposomes given alone byoral administration [36] Taken together constructing homo-geneous monodisperse and unilamellar liposomes is highlychallenging and various degrees of multilamellar constructsmay coexist making interpretations of experimental resultsdifficult but recent advancements in this technology mayallow for more accurate comparisons of the influence of sizelamellarity and overall structure in the future [78]

35 Modifications Increasing the Bioavailability of LiposomalAntigens The microenvironment at mucosal surfaces oftenpromotes a high clearance rate of liposomes Thereforevarious strategies have been tested to enhance mucus pen-etration or to increase membrane adhesion to facilitatebioavailability of the vaccine antigens (Figure 3(f)) Layer-by-layer deposition of polyelectrolytes onto the liposome forexample has been used as a liposome-stabilizing approachwhich resulted in higher specific IgA and IgG antibody levelsas well as an increased T cell response [79] Polyvinyl alcohol

or chitosan has been tested to enhance bioadhesive propertiesof the liposome and it has been observed that chitosan-loaded liposomes indeed stimulated enhanced IgG antibodyresponses [58] Chitosan is a positively charged polysaccha-ride that can form strong electrostatic interactions with cellsurfaces and mucus and therefore increase retention timeand facilitate interactions between the liposome and APCsin the mucosal membrane Alternatively such modificationscan also transiently open tight junctions between epithelialcells to allow for transmucosal transport of the liposomes[80ndash82] In fact chitosan-coated liposomes have been shownto give better serum IgG antibody levels compared to otherbioadhesive polymers such as hyaluronic acid or carbopolcoated liposomes and host much better immunogenicitythan uncoated negative neutral or positively charged lipo-somes [38 56ndash58]

Considerable attention has been given to studying howliposomes are retained by andor taken up across themucosal membranes Liposome interactions with the intesti-nal mucosa have been studied in vivo and ex vivo usingvarious in vitro models [46 79 83 84] The latter mod-els have addressed whether passage of liposomes betweenthe tight junctions of epithelial cells can be achievedIndeed tight junctions were reported to be open whenusing PCChol-liposomes or Tremella-coated liposomes[84] Enhanced immune responses were also observedwith mucus-penetrating liposomes made with poly(ethyleneglycol) (PEG) or the PEG-copolymer pluronic [38] Sig-nificantly higher specific IgA and IgG antibody levelswere found with PEGylated than un-PEGylated liposomesCharge-shielding modifications with PEG or Pluronic F127also proved useful in preventing liposome aggregation toobtain small (lt200 nm) chitosan-coated liposomes In factthese shielded chitosan-coated and PEGylated liposomesyielded the highest functional serum antibody titers of allthe formulations tested and the strongest IgA responses[38]

36 Cell-Targeting Modifications of Liposomes Modifica-tions aimed at increasing liposome stability andor uptakehave indeed proven effective One of the most exploredmodifications is aimed at targeting the delivery of lipo-somes to subsets of cells Liposomes can be equippedwith various targeting elements aiming at enhancing theirimmunogenicity (Figure 3(e)) For example additional tar-geting components may enhance the uptake by APCs orthe penetration of the liposome through the mucus layerThe strongly GM1-ganglioside-binding molecule CTB hasbeen reported to enhance liposome immunogenicity More-over when monophosphoryl lipid A acting through theTLR4 receptors was added to liposomes their ability tostimulate the innate immune response was dramaticallyimproved [34 37 49 85 86] Other Toll-like receptor ago-nists or Escherichia coli heat-labile toxin (LT) have also beenused in combination with liposomes as adjuvants [47 87]Furthermore linking CpG which acts through TLR9 sig-naling or Bordetella pertussis filamentous hemagglutinin tothe liposome has all been found to enhance immunogenicity[88 89]


In factmany different liposome cell-targeting approacheshave been investigated To this end specific antibodies havebeen found to enhance binding to M cells thereby targetingthe liposome to the follicle associated epithelium (FAE)This is the thin epithelial cell layer that is responsible forantigen uptake from the luminal side such as the epitheliumthat overlays Peyerrsquos patches (PP) in the small intestine [16]Similarly lectin Agglutinin I from Ulex europaeus coatedliposomes were shown to improve M cell-targeting andantigen uptake [83 90 91] Also galactosylation of liposomesresulted in higher specific IgA and IgG antibody levelscompared to unmodified liposomes [54] Moreover lipo-somes coated with the influenza virus protein hemagglutininwere more immunogenic than uncoated liposomes [50] Inaddition mannosylated lipids or anti-CD40 antibody-coatedliposomes were found to host an enhanced ability to targetDCs and thereby greatly promoted a stronger immuneresponse [55 87] Furthermore the identification of Minclea receptor for the mycobacterial cord factor trehalose 661015840-dimycolate (TDM) on innate immune cells led to that TDManalogs were found to be effective stimulants of the produc-tion of G-CSF in macrophages Indeed immunizations inmice with cationic liposomes containing the analogues TDMdemonstrated a superior adjuvant activity [92]

Many strategies have indeed been applied to achievecell-targeting of liposomes with varying degrees of improvedfunction Needless to say there exist a plethora of possibilitiesto explore when it comes to targeting liposomes to the cells ofthe mucosal immune system If analytical tools are combinedwith suitable in vitro and in vivo assay systems it willgreatly help identifying the relative importance of liposometargeting and how composition such as size lamellaritysurface charge and fluidity of the membrane can influencethe immune response

4 Mucosal Immune Responses to Liposomes

Prior to an adaptive immune response innate immuneactivation must have occurred leading to the productionof proinflammatory molecules and the expression by APCof costimulatory and immunomodulating molecules thatis chemokines cytokines and the costimulatory moleculesCD80 CD86 CD40 and others Innate immune activatorscan be classified into several categories including the domi-nant ones Toll-like receptors (TLRs) C-type lectin receptors(C-LRs) and non-Toll-like receptors (NLRs) [93 94] Thesereceptors recognize pathogen-associated molecular patterns(PAMPs) such as bacterial cell-wall components (eg pepti-doglycan lipoteichoic acid and flagellin) and different formsof microbial nucleic acids (eg double-stranded RNA high-CpG-content DNA) The role of an adjuvant in a vaccineformulation is thus to activate innate immunity and there-fore most vaccine adjuvants are derived from PAMPs Alsofor mucosal vaccines it is critical to induce a sufficient andappropriate innate immune response preferentially withoutcausing unwanted side effects such as tissue damage Thus asuccessful mucosal vaccine must be capable of inducing notonly an adaptive immune response but also a strong innateimmune response [13] Fortunately liposomes can do both

They can both serve as delivery vehicles for vaccine antigensand act as immunomodulators triggering both innate andadaptive immune responses Indeed many modifications ofthe liposome itself can dramatically influence the innateimmune response and thereby augment or qualitativelymodify also the adaptive immune response To this endaltered lipid composition charge particle size or addedtargeting elements can all be utilized to tailor the immuneresponse to the liposome and stimulate the required anti-infectious immune response (Figure 3) In addition to get aneven stronger activation of innate immunity liposomes canbe equipped with specific adjuvantsPAMPs such as flagellinor CpG as we have already discussed

Mucosal immunizations as opposed to systemic immu-nizations effectively support IgA class switch recombination(CSR) and production of sIgA at mucosal sites [95] Inthe intestine this occurs in the organized gut associatedlymphoid tissues (GALT) and in particular in Peyerrsquos patches(PP) which are the dominant sites for IgA CSR in the gutIn the upper respiratory tract the most active inductive siteis the nasal associated lymphoid tissues (NALT) but alsocervical lymphnodes and themediastinal lymphnode (mLN)are central to the mucosal immune response Ultimatelytargeting of the liposome to these sites could be a muchpreferred strategy in future vaccine formulations as alreadydiscussed Following antigen recognition and activation ofspecific B cells inGALT andNALT these cells undergo strongexpansion in the germinal centers (GC) in the B cell folliclesMost immune responses are T cell dependent and thusthe expanding B cells require the participation of follicularhelper T cells (TFH) in the GC to differentiate into long-livedplasma cells and memory B cells These CD4+ T cells aregenerated through the peptide-priming event that the DCsorchestrate in the T cell zone in the lymph node In thisway the DC is a key player in the immune response andit impacts also the ability of the activated lymphocytes tomigrate to the effector tissue from where the DC originatedThe plasma cells thus eventually migrate from the inductivesite via the lymph and blood back to the lamina propria in themucosal membrane where they produce sIgA Thus there isa complex series of events that need to be completed before aproductive sIgA response can be found in the lamina propriaof the mucosal membrane (Figure 1) Hence liposomes canbe made to impact or modulate many different steps in thisseries of events A critical question is how we can assess andcompare different liposome formulations for their efficacyand characteristic impact when multiple steps are involvedWe would advocate a reductionist approach where differentliposomes are evaluated for their effect in different stages ofthe buildup of an immune response Therefore in the nextsection we focus on the interaction of liposomes with DCsand APCs in general

41 Innate and Adaptive Immunity against Liposome Vac-cination Although significant progress has been made inunderstanding antigen uptake and processing of liposomedelivered antigens the fine details of these processes arestill poorly known Liposomes that enhance cell membranefusion that is fusogenic liposomes deliver their antigenic


content to the cytoplasm of the APC which enables MHCclass II presentation to CD4+ T cells and in some DCsubsets also allows for cross-presentation to MHC class Irestricted CD8+ T cells Of note liposomes that are takenup via scavenger receptors (CD68 CD36 and Clec LOX-1) or other innate immune receptors are usually restrictedto prime CD4+ T cells through MHC class II presentationThus targeting of liposomes to different DC subsets oruptake mechanisms can provide a means to specifically tailorthe immune response to a certain antigen or facilitate thedevelopment of a distinct type of immune response [9697] For instance whereas zwitterionic or anionic liposomeshave not been reported to drive inflammation cationicliposomes have been shown to stimulate proinflammatoryresponses in DCs leading to an upregulation of costimu-latory molecules CD80 and CD86 and proinflammatorycytokines [98] Furthermore Yan et al reported that DCstimulation by cationic liposomes composed of DOTAP (12-dioleoyl-3-trimethylammonium propane) also stimulatedreactive oxygen species (ROS) which activated extracellularsignal-regulated kinase (ERK) and p38 and downstreamproinflammatory cytokineschemokines interleukin-12 (IL-12) and chemokine (C-C motif) ligand 2 (CCL2) [99] Inaddition DOTAP liposomes were shown to induce tran-scription of monocyte chemoattractant protein-1 (MCP-1CCL2) macrophage inflammatory protein-1 alpha (MIP-1120572CCL3) and macrophage inflammatory protein-1 beta(MIP-1120573CCL4) [100] Also DiC14-amidine cationic lipo-somes can induce the secretion of IL-1120573 IL-6 IL-12p40interferon-120573 (IFN-120573) interferon-120574-inducible protein 10 (IP-10) and TNF-120572 by human and mouse myeloid DCs [101]While anionic liposomes in general are poorly proinflam-matory modifications such as using mannosylated lipidscould make these liposomes much more proinflammatoryand effective at stimulating DCs [102 103] With regard tomacrophages it has been reported that galactose-modifiedliposomes can stimulate TNF-120572 and IL-6 production whichwas associated with significantly higher specific sIgA anti-body levels in the nasal and lung tissues and increased serumIgG antibodies [54]

Carefully analyzing the literature it appears unclearhow different liposomes stimulate strong innate immuneresponses A high density of positive charges on liposomesis considered beneficial while negatively charged or neutrallipids are likely to lower this capacity [104 105] Asmentionedpreviously liposomes effectively stimulate both T and Bcell responses but it is their direct impact on the DCsthat matters for the adaptive immune response In fact theuptake of liposomes by DCs has an important effect on thedevelopment of different CD4+ T cell subsets These CD4+ Tcell subsets have both distinct and overlapping functions buttheir individual impact on the immune response is criticalFor example if protection against a pathogen requires IFN-120574 production (Th1 cells) then the development of exclusiveTh2-dominated responses can be detrimental Therefore inthis context monophosphoryl lipid A (MPL) (a TLR4 ligand)or monomycoloyl glycerol (MMG) combined with DDA ina liposome will consistently promote IFN-120574 production thatis a Th1-biased immune response [106] Moreover trehalose

dibehenate (TDB) liposomes together with the combinedAg85B-ESAT-6 vaccine antigen enhanced antituberculosisspecific IFN-120574 and IL-17 production as well as increasedspecific serum IgG2 antibody levels [107] While the com-position of lipids matters for the subset of CD4+ T cellthat the DC can prime also a larger size of the liposomemay influence the generation of Th1 CD4+ T cells [108]However the mechanism for this effect is unclear but couldrelate to the fact that different subsets of DCs or other APCslike macrophages are involved in processing differently sizedliposomes such that small sized liposomes preferentiallystimulate Th2 CD4+ T cell responses On the other handit has been claimed by many investigators that protection isbest achieved with a balanced Th1Th2 response Liposome-based microneedle array (LiposoMAs) and a mannose-PEG-cholesterol (MPC)lipid A-liposome (MLLs) system areboth examples of liposome formulations with a balancedTh1Th2 response [109ndash111] An even more complex vector isthe combination of nanoparticle technology and liposomeswith biodegradable poly(DL-lactic-co-glycolic acid) (PLGA)cationic surfactant dimethyldioctadecylammonium (DDA)bromide and the immunopotentiator TDB which promotesTh1 and Th17 CD4+ T cell responses and enhanced specificserum antibodies [112] Moreover the mycobacterial cell-wall lipid monomycoloyl glycerol analog has been usedin combination with DDA This combination resulted ina promising vaccine delivery system that induced strongantigen-specific Th1 andTh17 responses [113]

Protection against most infections requires both anti-bodies and adequate T cell immunity However for mostinfectious diseases we have given strongest attention to anti-bodies as correlates of protectionHowever this scenariomaychange when we will identify more and more T cell mediatedparameters that correlate with protection Recently it wasidentified that in influenza the best correlate of protection ina study with healthy human volunteers was the preexistinginfluenza-specific CD4+T cells [114] It has been found thathigh density antigen coating onto liposomes often stimulatesbetter antibody responses than encapsulated antigens but acombination such as the DOTAP-PEG-mannose liposomes(LP-Man) will enhance not only antibody responses butalso APC antigen uptake and strong memory CD4+ T celldevelopment [115ndash118] This and other progress in the fieldhave to be considered when developing programs for theevaluation of vaccine efficacy For example with liposomevaccines as novel universal broadly protective influenzavaccines against also heterosubtypic virus strains it will beimportant to also assess and evaluate CD4+ T cell immunityas a correlate of protection Therefore we need to betterdefine how specific CD4+ T cells and for that matter CD8+T cells correlate to impaired viral replication or bacterialgrowth and reduced transmission of infection

5 Concluding Remarks andFuture Perspectives

Here we have described and discussed recent progressin nanoparticle formulations using liposomes for mucosal


vaccine delivery against infectious diseases We have under-scored the complexity of the liposome formulation andpointed to many combinations and plasticity of the liposomenanoparticle as a carrier of vaccine antigens and adjuvantsNeedless to say when considering liposome-based vaccinesit is important to consider all the properties of the for-mulation the route of administration and the biologicalresponseThus liposome size lamellarity and surface chargeas well as lipid composition and fluidity of the membranecan all influence the immune response following vaccinationImportantly the choice of antigen with its own inherentphysicochemical properties and the position of the antigenand the adjuvant in the liposome critically affect the functionof the liposome The antigenlipid ratio and properties of theadded adjuvant are also important parameters that change theimmunogenicity and stability of the liposome

Most studies using liposome-based vaccines have focusedon the end result of immunization namely the magnitudeand the quality of the immune response Few studies haveattempted to systematically identify mechanism of action atthe different stages of an immune response For mucosalimmunizations liposome vaccines have been thoroughlyinvestigated for their stability in intestinal fluids theirmucoadhesive properties or the efficiency in targeting anduptake by DCs and other APCs [46 49 58 79 119 120]The site of liposomal penetration of the mucosal barrier isimportant to determine If occurring at the duodenal sitethe outcome may be significantly different from penetrationthat takes place in the ileum or colon of the gut Similarlyantigen uptake in the NALT may be more effective than ifthe liposome reaches deeper into the respiratory tract Thisis not just because of the stability of the liposomal antigen ata distinct location but also theDC subsets that are exposed tothe antigen may differ dramatically and hence the outcomeof the vaccination may vary At the cellular level we largelylack studies that investigate how liposome delivered antigensare processed and presented byDCs the kinetics of these pro-cesses and whether the formulation will affect the migrationand function of the DC in the draining lymph node Morework is needed to generalize the principles for an optimaldesign of the liposome in this regard For example it stillremains unclear whether liposomes that rapidly penetrate themucosal barrier are also good inducers of a mucosal immuneresponse or alternatively shouldmucoadhesive liposomes beused to provide a depot of antigen for an extended loadingof DCs with antigen Simple screening systems should bedeveloped to address these questions To assess whether aliposome efficiently delivers peptide for CD8+ or CD4+ Tcell priming one could focus on surface expression on theDC of complexes with MHC class I and II molecules pluspeptide using specific antibodies that detect these complexesand flow cytometry analysis [121] In this way screening ofliposome-antigen formulations could be evaluated on thebasis of effective expression of such complexes on the surfaceof a target DC population in vitro and in vivo The densityof such complexes most likely will relate to the ability of theDC to effectively prime the T cells in the lymph node asthe T cell receptor does not recognize the peptide but thecomplex

Identifying and standardizing liposome-stimulated im-mune responseswould greatly aid in the prospects of compar-ing different liposome vaccine formulations for their efficacyThis would also contribute to a more rational design ofeffective liposome-based mucosal vaccines Currently thereis no agreed protocol or procedure on how to evaluate andcharacterize the immune response to liposomes Hence moststudies are performed without relevant comparisons and theevaluation of whether the liposome formulation is more orless effective compared to other types of formulations isimpossible to assess Therefore it would be an advantageif investigators could agree on using some standardizationprotocol perhaps using cholera toxin or some other strongsoluble adjuvant to admix with antigen and compare theimmune response to those of the liposomes Thus it is fairto say that we still lack comprehensive comparisons to othermodes of formulating antigens for mucosal vaccination

The liposome technology applied to the development ofthe next generation ofmucosal vaccines holdsmuch promiseIt is conceivable that better targeted liposomes with addedadjuvant capacity will prove to be effective in stimulatingmucosal immune responses that are protective against mul-tiple infectious diseases It is likely that these liposomeshave both surface anchored antigen and encapsulated antigento optimize B cell as well as T cell priming The lipidcompositions that yield higher gel-liquid crystal transitiontemperatures will be preferred as they have been foundto stimulate stronger immune responses Cationic ratherthan neutral liposomes being more proinflammatory will beselected for mucosal immunization It will be important inthe future to apply the better understanding of the liposomemanufacturing technology and the principles for induction ofmucosal immune responses to the design and development ofthe next generation of mucosal vaccines

Competing Interests

The authors have no conflict of interests

Authorsrsquo Contributions

Valentina Bernasconi and Karin Norling contributed equallyto this work

References

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[2] E J Ryan L M Daly and K H G Mills ldquoImmunomodulatorsand delivery systems for vaccination by mucosal routesrdquo Trendsin Biotechnology vol 19 no 8 pp 293ndash304 2001

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[4] N Lycke ldquoRecent progress in mucosal vaccine developmentpotential and limitationsrdquo Nature Reviews Immunology vol 12no 8 pp 592ndash605 2012


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[7] N Grimaldi F Andrade N Segovia et al ldquoLipid-basednanovesicles for nanomedicinerdquo Chemical Society Reviews vol45 no 23 pp 6520ndash6545 2016

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[9] R Mischler and I C Metcalfe ldquoInflexal V a trivalent virosomesubunit influenza vaccine productionrdquoVaccine vol 20 supple-ment 5 pp B17ndashB23 2002

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[48] M I de Jonge H J Hamstra W Jiskoot et al ldquoIntranasalimmunisation of mice with liposomes containing recombinantmeningococcal OpaB and OpaJ proteinsrdquo Vaccine vol 22 no29-30 pp 4021ndash4028 2004

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[52] I J Fidler S Sone W E Fogler et al ldquoEfficacy of liposomescontaining a lipophilic muramyl dipeptide derivative for acti-vating the tumoricidal properties of alveolar macrophages invivordquo Journal of Biological Response Modifiers vol 1 no 1 pp43ndash55 1982

[53] N C Phillips J Rioux and M S Tsao ldquoActivation of murineKupffer cell tumoricidal activity by liposomes containing lipo-philic muramyl dipeptiderdquo Hepatology vol 8 no 5 pp 1046ndash1050 1988

[54] H-W Wang P-L Jiang S-F Lin et al ldquoApplication ofgalactose-modified liposomes as a potent antigen presentingcell targeted carrier for intranasal immunizationrdquo Acta Bioma-terialia vol 9 no 3 pp 5681ndash5688 2013

[55] A Ninomiya K Ogasawara K Kajino A Takada and HKida ldquoIntranasal administration of a synthetic peptide vaccineencapsulated in liposome together with an anti-CD40 antibodyinduces protective immunity against influenza a virus in micerdquoVaccine vol 20 no 25-26 pp 3123ndash3129 2002

[56] M Amin M R Jaafari and M Tafaghodi ldquoImpact of chitosancoating of anionic liposomes on clearance rate mucosal andsystemic immune responses following nasal administration inrabbitsrdquo Colloids and Surfaces B Biointerfaces vol 74 no 1 pp225ndash229 2009

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[61] G Caracciolo ldquoLiposome-protein corona in a physiologicalenvironment challenges and opportunities for targeted deliveryof nanomedicinesrdquo Nanomedicine Nanotechnology Biologyand Medicine vol 11 no 3 pp 543ndash557 2015

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[63] V Mirshafiee M Mahmoudi K Y Lou J J Cheng and ML Kraft ldquoProtein corona significantly reduces active targetingyieldrdquoChemical Communications vol 49 no 25 pp 2557ndash25592013

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[65] S N Thomas A J van der Vlies C P OrsquoNeil et al ldquoEngineer-ing complement activation on polypropylene sulfide vaccinenanoparticlesrdquo Biomaterials vol 32 no 8 pp 2194ndash2203 2011

[66] M E Baca-Estrada M Foldvari and M Snider ldquoInductionof mucosal immune responses by administration of liposome-antigen formulations and interleukin-12rdquo Journal of Interferonand Cytokine Research vol 19 no 5 pp 455ndash462 1999

[67] B Guy N Pascal A Francon et al ldquoDesign characterizationand preclinical efficacy of a cationic lipid adjuvant for influenzasplit vaccinerdquo Vaccine vol 19 no 13-14 pp 1794ndash1805 2001

[68] P T Ingvarsson I S Rasmussen M Viaene P J Irlik HM Nielsen and C Foged ldquoThe surface charge of liposomal


adjuvants is decisive for their interactions with the Calu-3 andA549 airway epithelial cell culture modelsrdquo European Journalof Pharmaceutics and Biopharmaceutics vol 87 no 3 pp 480ndash488 2014

[69] A de Haan H J Geerligs J P Huchshorn G J M van Schar-renburg A M Palache and J Wilschut ldquoMucosal immunoad-juvant activity of liposomes induction of systemic IgG andsecretory IgA responses in mice by intranasal immunizationwith an influenza subunit vaccine and coadministered lipo-somesrdquo Vaccine vol 13 no 2 pp 155ndash162 1995

[70] J Wilschut A D Haan H J Geerligs et al ldquoLiposomes asa mucosal adjuvant system an intranasal liposomal influenzasubunit vaccine and the role of iga in nasal anti-influenzaimmunityrdquo Journal of Liposome Research vol 4 no 1 pp 301ndash314 1994

[71] Y Aramaki Y Fujii K Yachi H Kikuchi and S Tsuchiya ldquoActi-vation of systemic and mucosal immune response followingnasal administration of liposomesrdquo Vaccine vol 12 no 13 pp1241ndash1245 1994

[72] A De Haan G Groen J Prop N Van Rooijen and JWilschut ldquoMucosal immunoadjuvant activity of liposomes roleof alveolar macrophagesrdquo Immunology vol 89 no 4 pp 488ndash493 1996

[73] N C Phillips L Gagne N Ivanoff and G Riveau ldquoInfluence ofphospholipid composition on antibody responses to liposomeencapsulated protein and peptide antigensrdquoVaccine vol 14 no9 pp 898ndash904 1996

[74] Y Fan P Sahdev L J Ochyl J Akerberg and J J MoonldquoCationic liposome-hyaluronic acid hybrid nanoparticles forintranasal vaccination with subunit antigensrdquo Journal of Con-trolled Release vol 208 pp 121ndash129 2015

[75] Y Fujii Y Aramaki T Hara K Yachi H Kikuchi and STsuchiya ldquoEnhancement of systemic and mucosal immuneresponses following oral administration of liposomesrdquoImmunology Letters vol 36 no 1 pp 65ndash69 1993

[76] E Harokopakis G Hajishengallis and S M Michalek ldquoEffec-tiveness of liposomes possessing surface-linked recombinant Bsubunit of cholera toxin as an oral antigen delivery systemrdquoInfection and Immunity vol 66 no 9 pp 4299ndash4304 1998

[77] S Minato K Iwanaga M Kakemi S Yamashita and N OkuldquoApplication of polyethyleneglycol (PEG)-modified liposomesfor oral vaccine effect of lipid dose on systemic and mucosalimmunityrdquo Journal of Controlled Release vol 89 no 2 pp 189ndash197 2003

[78] Y Yang J Wang H Shigematsu et al ldquoSelf-assembly of size-controlled liposomes on DNA nanotemplatesrdquo Nature Chem-istry vol 8 no 5 pp 476ndash483 2016

[79] H Harde A K Agrawal and S Jain ldquoTetanus toxoid-loaded layer-by-layer nanoassemblies for efficient systemicmucosal and cellular immunostimulatory response followingoral administrationrdquo Drug Delivery and Translational Researchvol 5 no 5 pp 498ndash510 2015

[80] L Illum I Jabbal-Gill M Hinchcliffe A N Fisher and S SDavis ldquoChitosan as a novel nasal delivery system for vaccinesrdquoAdvancedDrugDelivery Reviews vol 51 no 1-3 pp 81ndash96 2001

[81] G Borchard H L Lueszligen A G de Boer J C Verhoef C-M Lehr and H E Junginger ldquoThe potential of mucoadhesivepolymers in enhancing intestinal peptide drug absorption IIIeffects of chitosan-glutamate and carbomer on epithelial tightjunctions in vitrordquo Journal of Controlled Release vol 39 no 2-3 pp 131ndash138 1996

[82] VDodaneMAminKhan and J RMerwin ldquoEffect of chitosanon epithelial permeability and structurerdquo International Journalof Pharmaceutics vol 182 no 1 pp 21ndash32 1999

[83] K Li X Zhao S Xu D Pang C Yang and D Chen ldquoApplica-tion of Ulex europaeus agglutinin I-modified liposomes for oralvaccine Ex vivo bioadhesion and in Vivo immunityrdquo Chemicaland Pharmaceutical Bulletin vol 59 no 5 pp 618ndash623 2011

[84] H-C Cheng C-Y Chang F-I Hsieh et al ldquoEffects of tremella-alginate-liposome encapsulation on oral delivery of inactivatedH5N3 vaccinerdquo Journal of Microencapsulation vol 28 no 1 pp55ndash61 2011

[85] J Vadolas J K Davies P J Wright and R A StrugnellldquoIntranasal immunization with liposomes induces strong mu-cosal immune responses in micerdquo European Journal ofImmunology vol 25 no 4 pp 969ndash975 1995

[86] T Lian T Bui and R J Y Ho ldquoFormulation of HIV-envelopeprotein with lipid vesicles expressing ganglioside GM1 associ-ated to cholera toxin B enhances mucosal immune responsesrdquoVaccine vol 18 no 7-8 pp 604ndash611 1999

[87] B Heurtault P Gentine J-SThomann C Baehr B Frisch andF Pons ldquoDesign of a liposomal candidate vaccine against Pseu-domonas aeruginosa and its evaluation in triggering systemicand lung mucosal immunityrdquo Pharmaceutical Research vol 26no 2 pp 276ndash285 2009

[88] A Joseph I Louria-Hayon A Plis-Finarov et al ldquoLiposomalimmunostimulatory DNA sequence (ISS-ODN) an efficientparenteral and mucosal adjuvant for influenza and hepatitis Bvaccinesrdquo Vaccine vol 20 no 27-28 pp 3342ndash3354 2002

[89] O Poulain-Godefroy N Mielcarek N Ivanoff et al ldquoBor-detella pertussis filamentous hemagglutinin enhances theimmunogenicity of liposome-delivered antigen administeredintranasallyrdquo Infection and Immunity vol 66 no 4 pp 1764ndash1767 1998

[90] H M Chen V Torchilin and R Langer ldquoLectin-bearingpolymerized liposomes as potential oral vaccine carriersrdquo Phar-maceutical Research vol 13 no 9 pp 1378ndash1383 1996

[91] K Li D Chen X Zhao H Hu C Yang and D PangldquoPreparation and investigation of Ulex europaeus agglutininI-conjugated liposomes as potential oral vaccine carriersrdquoArchives of Pharmacal Research vol 34 no 11 pp 1899ndash19072011

[92] AHuber R S Kallerup K S Korsholm et al ldquoTrehalose diesterglycolipids are superior to the monoesters in binding toMincleactivation ofmacrophages in vitro and adjuvant activity in vivordquoInnate Immunity vol 22 no 6 pp 405ndash418 2016

[93] RMedzhitov ldquoToll-like receptors and innate immunityrdquoNatureReviews Immunology vol 1 no 2 pp 135ndash145 2001

[94] T Maekawa T A Kufer and P Schulze-Lefert ldquoNLR functionsin plant and animal immune systems so far and yet so closerdquoNature Immunology vol 12 no 9 pp 818ndash826 2011

[95] M E Lamm ldquoInteraction of antigens and antibodies atmucosalsurfacesrdquo Annual Review of Microbiology vol 51 pp 311ndash3401997

[96] C Qiao J Liu J Yang et al ldquoEnhanced non-inflammasomemediated immune responses by mannosylated zwitterionic-based cationic liposomes for HIV DNA vaccinesrdquo Biomaterialsvol 85 pp 1ndash17 2016

[97] E Yuba C Kojima A Harada Tana S Watarai and KKono ldquopH-sensitive fusogenic polymer-modified liposomesas a carrier of antigenic proteins for activation of cellularimmunityrdquo Biomaterials vol 31 no 5 pp 943ndash951 2010


[98] C Lonez M Vandenbranden and J-M Ruysschaert ldquoCationiclipids activate intracellular signaling pathwaysrdquo Advanced DrugDelivery Reviews vol 64 no 15 pp 1749ndash1758 2012

[99] W Yan W Chen and L Huang ldquoReactive oxygen species playa central role in the activity of cationic liposome based cancervaccinerdquo Journal of Controlled Release vol 130 no 1 pp 22ndash282008

[100] W Yan W Chen and L Huang ldquoMechanism of adjuvantactivity of cationic liposome phosphorylation of aMAP kinaseERK and induction of chemokinesrdquoMolecular Immunology vol44 no 15 pp 3672ndash3681 2007

[101] T Tanaka A Legat E Adam et al ldquoDiC14-amidine cationicliposomes stimulate myeloid dendritic cells through toll-likereceptor 4rdquo European Journal of Immunology vol 38 no 5 pp1351ndash1357 2008

[102] C Foged C Arigita A Sundblad W Jiskoot G Storm and SFrokjaer ldquoInteraction of dendritic cells with antigen-containingliposomes effect of bilayer compositionrdquo Vaccine vol 22 no15-16 pp 1903ndash1913 2004

[103] Y Ma Y Zhuang X Xie et al ldquoThe role of surface charge den-sity in cationic liposome-promoted dendritic cell maturationand vaccine-induced immune responsesrdquo Nanoscale vol 3 no5 pp 2307ndash2314 2011

[104] R F Coburn ldquoPolyamine effects on cell function possiblecentral role of plasma membrane PI(45)P2rdquo Journal of CellularPhysiology vol 221 no 3 pp 544ndash551 2009

[105] M K Dymond and G S Attard ldquoCationic type i amphiphilesas modulators of membrane curvature elastic stress in vivordquoLangmuir vol 24 no 20 pp 11743ndash11751 2008

[106] E M Agger J P Cassidy J Brady K S Korsholm CVingsbo-Lundberg and P Andersen ldquoAdjuvant modulation ofthe cytokine balance in Mycobacterium tuberculosis subunitvaccines immunity pathology and protectionrdquo Immunologyvol 124 no 2 pp 175ndash185 2008

[107] J Davidsen I Rosenkrands D Christensen et al ldquoCharac-terization of cationic liposomes based on dimethyldioctadecy-lammonium and synthetic cord factor from M tuberculosis(trehalose 661015840- dibehenate)mdasha novel adjuvant inducing bothstrong CMI and antibody responsesrdquo Biochimica et BiophysicaActa vol 1718 no 1-2 pp 22ndash31 2005

[108] J F S Mann E Shakir K C Carter A B Mullen J Alexanderand V A Ferro ldquoLipid vesicle size of an oral influenza vaccinedelivery vehicle influences the Th1Th2 bias in the immuneresponse and protection against infectionrdquo Vaccine vol 27 no27 pp 3643ndash3649 2009

[109] T Wang and N Wang ldquoPreparation of the multifunctionalliposome-containing microneedle arrays as an oral cavitymucosal vaccine adjuvant-delivery systemrdquo in Vaccine Designvol 1404 ofMethods inMolecular Biology pp 651ndash667 Springer2016

[110] S S Joshi and V A Arankalle ldquoDifferential immune responsesin mice immunized with recombinant neutralizing epitopeprotein of hepatitis E virus formulated with liposome and alumadjuvantsrdquo Viral Immunology vol 29 no 6 pp 350ndash360 2016

[111] Y Zhen N Wang Z Gao et al ldquoMultifunctional lipo-somes constituting microneedles induced robust systemic andmucosal immunoresponses against the loaded antigens via oralmucosal vaccinationrdquo Vaccine vol 33 no 35 pp 4330ndash43402015

[112] F Rose J E Wern P T Ingvarsson et al ldquoEngineering of anovel adjuvant based on lipid-polymer hybrid nanoparticles a

quality-by-design approachrdquo Journal of Controlled Release vol210 pp 48ndash57 2015

[113] B Martin-Bertelsen K S Korsholm C B Roces et al ldquoNano-self-assemblies based on synthetic analogues of mycobacterialmonomycoloyl glycerol and DDA supramolecular structureand adjuvant efficacyrdquo Molecular Pharmaceutics vol 13 no 8pp 2771ndash2781 2016

[114] T M Wilkinson C K F Li C S C Chui et al ldquoPreexistinginfluenza-specific CD4+ T cells correlate with disease protec-tion against influenza challenge in humansrdquo Nature Medicinevol 18 no 2 pp 274ndash280 2012

[115] J Ingale A Stano J Guenaga et al ldquoHigh-density array ofwell-orderedHIV-1 spikes on synthetic liposomal nanoparticlesefficiently activate B cellsrdquo Cell Reports vol 15 no 9 pp 1986ndash1999 2016

[116] C Wang P Liu Y Zhuang et al ldquoLymphatic-targeted cationicliposomes a robust vaccine adjuvant for promoting long-termimmunological memoryrdquo Vaccine vol 32 no 42 pp 5475ndash5483 2014

[117] S A Plotkin ldquoImmunologic correlates of protection induced byvaccinationrdquo Pediatric Infectious Disease Journal vol 20 no 1pp 63ndash75 2001

[118] M Pihlgren A B Silva R Madani et al ldquoTLR4- and TRIF-dependent stimulation of B lymphocytes by peptide liposomesenables T cell-independent isotype switch in micerdquo Blood vol121 no 1 pp 85ndash94 2013

[119] H Harde A K Agrawal and S Jain ldquoTrilateral rsquo3Prsquo mechanicsof stabilized layersomes technology for efficient oral immuniza-tionrdquo Journal of Biomedical Nanotechnology vol 11 no 3 pp363ndash381 2015

[120] P TWong P R Leroueil DM Smith et al ldquoFormulation highthroughput in vitro screening and in vivo functional charac-terization of nanoemulsion-based intranasal vaccine adjuvantsrdquoPLoS ONE vol 10 no 5 Article ID e0126120 2015

[121] D BMurphy D Lo S Rath et al ldquoA novelMHC class II epitopeexpressed in thymic medulla but not cortexrdquo Nature vol 338no 6218 pp 765ndash768 1989

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Epithelial cells

Dendritic cell

MHC IIpeptide

TCR

B cell

Mucosal surface

Inductive site Effector site

Secretory IgA

NALT

T cell zone

Follicular DC

B cell follicle

TFH cell

p p

TFH cell

B cell

Germinal centre

Lymph node

Plasma cell

IgA B cell

Blood stream

Vaccine

FAE cells

CD8+ T cell

CD4+ T cell

CD4+ and CD8

+ effectorand memory T cells

Figure 1 Principles for induction of mucosal immune responses after intranasal vaccination The respiratory mucosal immune systemconsists of clusters of lymphoid cells beneath the mucosal epithelium hosting both innate and adaptive immune cells [29] There is a cleardistinction between inductive and effector sites and these are also physically separated Inductive sites are organized lymphoid tissues whereantigen is taken up by DCs and other APCs The effector sites on the other hand are tissues that provide protection against infection wherespecific antibodies and CD4+ and CD8+ effector and memory T cells reside [30] The main inductive sites for mucosal immune responsesafter intranasal vaccination are known as nasopharynx-associated lymphoid tissue (NALT) which harbors B cell follicles and T cell zones inwell demarked microanatomical areas [31] Antigens are taken up by DCs that get access to the luminal content either through direct uptakethrough the epitheliumor via the follicle associated epithelium (FAE) that overlay theNALT After antigen uptake the immatureDCs undergomaturation and subsequently leave themucosal tissue for the draining lymph nodes alternatively if already in the NALT the DCs will directlyprime naive CD4+ or CD8+ T cells Activated CD4+ T cells differentiate into various subsets T helper 1 (Th1)Th2 orTh17 cells regulatory Tcells (Tregs) or follicular helper T cells (TFH) The latter are critically needed for the expansion and differentiation of the activated B cells inthe germinal center (GC) which is formed in the B cell follicle in the lymph node after vaccination TFH cells are involved in the developmentof long-lived plasma cells and memory B cells in the GC

the next generation of effective mucosal vaccines is muchneeded

Liposomes have been extensively used as delivery vehi-cles for vaccine antigens some of the advantages of these

formulations are (a) protection against antigen degradation(b) tissue depot effects or slow release of antigen and (c)facilitated uptake of antigen by antigen presenting cells (APC)[5 6] Phosphatidylcholines are the most common lipids













Identifier









adjuvant



Phase 1 NCT01502358


Tuberculosis








AIntradermal




Malaria
















(CCSC)







Phase 2 NCT00936468






andGlaxoSmithKline

Phase 3 NCT00872963


Table 1 Continued



















the membrane

Hydrophobictail

Glycerolbackbone


POPCDOTAP DMPG

TAP

DA PI

PS

PA PE

PCPG


OO

O O

HO

HO

O

O

OOO O O

O

OO

O

OPP O

O

H

HH

N+

N+

Ominus

Ominus

O

OPO

H

N+

Ominus

H

O

HO

HO

OO

OP

H

Ominus

H

O

HN+

OO

OO

HH

OO

OO

HHHO

OO

OOO

O



and tails

Choose a headgroup



O

OHO

HO

HO

HO

OOPO

O

O

OO

O

OO

OO

OOHNH

NH

OH

minusO

+

+

+

minus

minus

minus

(a) (b)

(c)

(d) (e)

(f)NH+

4



































Competing Interests




References






































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























Identifier









adjuvant



Phase 1 NCT01502358


Tuberculosis








AIntradermal




Malaria
















(CCSC)







Phase 2 NCT00936468






andGlaxoSmithKline

Phase 3 NCT00872963


Table 1 Continued



















the membrane

Hydrophobictail

Glycerolbackbone


POPCDOTAP DMPG

TAP

DA PI

PS

PA PE

PCPG


OO

O O

HO

HO

O

O

OOO O O

O

OO

O

OPP O

O

H

HH

N+

N+

Ominus

Ominus

O

OPO

H

N+

Ominus

H

O

HO

HO

OO

OP

H

Ominus

H

O

HN+

OO

OO

HH

OO

OO

HHHO

OO

OOO

O



and tails

Choose a headgroup



O

OHO

HO

HO

HO

OOPO

O

O

OO

O

OO

OO

OOHNH

NH

OH

minusO

+

+

+

minus

minus

minus

(a) (b)

(c)

(d) (e)

(f)NH+

4



































Competing Interests




References






































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1 Continued



















the membrane

Hydrophobictail

Glycerolbackbone


POPCDOTAP DMPG

TAP

DA PI

PS

PA PE

PCPG


OO

O O

HO

HO

O

O

OOO O O

O

OO

O

OPP O

O

H

HH

N+

N+

Ominus

Ominus

O

OPO

H

N+

Ominus

H

O

HO

HO

OO

OP

H

Ominus

H

O

HN+

OO

OO

HH

OO

OO

HHHO

OO

OOO

O



and tails

Choose a headgroup



O

OHO

HO

HO

HO

OOPO

O

O

OO

O

OO

OO

OOHNH

NH

OH

minusO

+

+

+

minus

minus

minus

(a) (b)

(c)

(d) (e)

(f)NH+

4



































Competing Interests




References






































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























the membrane

Hydrophobictail

Glycerolbackbone


POPCDOTAP DMPG

TAP

DA PI

PS

PA PE

PCPG


OO

O O

HO

HO

O

O

OOO O O

O

OO

O

OPP O

O

H

HH

N+

N+

Ominus

Ominus

O

OPO

H

N+

Ominus

H

O

HO

HO

OO

OP

H

Ominus

H

O

HN+

OO

OO

HH

OO

OO

HHHO

OO

OOO

O



and tails

Choose a headgroup



O

OHO

HO

HO

HO

OOPO

O

O

OO

O

OO

OO

OOHNH

NH

OH

minusO

+

+

+

minus

minus

minus

(a) (b)

(c)

(d) (e)

(f)NH+

4



































Competing Interests




References






































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

OHO

HO

HO

HO

OOPO

O

O

OO

O

OO

OO

OOHNH

NH

OH

minusO

+

+

+

minus

minus

minus

(a) (b)

(c)

(d) (e)

(f)NH+

4



































Competing Interests




References






































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































Competing Interests




References






































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Review Article Mucosal Vaccine Development Based on...

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Transcript of Review Article Mucosal Vaccine Development Based on...