vaccines Review
Biodegradable Polymeric Nanoparticles-Based Vaccine Adjuvants for Lymph Nodes Targeting Alice Gutjahr 1,2,3,† , Capucine Phelip 1,† , Anne-Line Coolen 1 , Claire Monge 1 , Anne-Sophie Boisgard 1 , Stéphane Paul 3 and Bernard Verrier 1, * 1
2 3
* †
Laboratoire de Biologie Tissulaire et d’Ingénierie Thérapeutique, UMR 5305, Université Lyon 1, CNRS, IBCP, Lyon 69007, France;
[email protected] (A.G.);
[email protected] (C.P.);
[email protected] (A.-L.C.);
[email protected] (C.M.);
[email protected] (A.-S.B.) InvivoGen, Toulouse 31400, France Groupe Immunité des Muqueuses et Agents Pathogènes, INSERM CIC1408 Vaccinologie, Faculté de Médecine de Saint-Etienne, Saint-Etienne 42270, France;
[email protected] Correspondence:
[email protected] These authors contributed equally to this work.
Academic Editor: Olga Borges Received: 23 August 2016; Accepted: 29 September 2016; Published: 12 October 2016
Abstract: Vaccines have successfully eradicated a large number of diseases. However, some infectious diseases (such as HIV, Chlamydia trachomatis or Bacillus anthracis) keep spreading since there is no vaccine to prevent them. One way to overcome this issue is the development of new adjuvant formulations which are able to induce the appropriate immune response without sacrificing safety. Lymph nodes are the site of lymphocyte priming by antigen-presenting cells and subsequent adaptive immune response, and are a promising target for vaccine formulations. In this review, we describe the properties of different polymer-based (e.g., poly lactic-co-glycolic acid, poly lactic acid . . . ) particulate adjuvants as innovative systems, capable of co-delivering immunopotentiators and antigens. We point out how these nanoparticles enhance the delivery of antigens, and how their physicochemical properties modify their uptake by antigen-presenting cells and their migration into lymph nodes. We describe why polymeric nanoparticles increase the persistence into lymph nodes and promote a mature immune response. We also emphasize how nanodelivery directs the response to a specific antigen and allows the induction of a cytotoxic immune response, essential for the fight against intracellular pathogens or cancer. Finally, we highlight the interest of the association between polymer-based vaccines and immunopotentiators, which can potentiate the effect of the molecule by directing it to the appropriate compartment and reducing its toxicity. Keywords: vaccine; adjuvant; immunogenicity; polymer; nanoparticles; nanodelivery; lymph node; antigen; molecular adjuvant
1. Why do We Need Adjuvants? Preventive vaccination is one of the major successes of medicine. It represents one of the most cost-effective health investments and, according to the World Health Organization, saves 2 to 3 million lives every year. However, infectious diseases (such as HIV, Chlamydia trachomatis, Bacillus anthracis or malaria) remain a leading cause of death worldwide. In the early days of vaccination, heterologous pathogens (nonpathogenic relative of the organism, such as cowpox virus for smallpox vaccination) [1], or attenuated pathogens (with decreased pathogenicity thanks to repeated culturing, such as the tuberculosis vaccine) were used to immunize populations. Those vaccines have a high intrinsic immunogenicity and usually induce asymptomatic infections that generate a life-long immunity similar
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to the one observed for individuals recovering from a natural infection. However, for many pathogens, this kind of vaccine has not been successfully developed, notably because of safety issues. Inactivated toxins (such as the inactivated tetanus toxin) or inactivated pathogens (such as the inactivated Polio virus), as well as synthetic peptides and recombinant protein subunits are now developed, but they have a poor immunogenicity. Therefore, they require a co-administration with adjuvants in order to elicit a robust immune response. Among various categories of adjuvants, delivery systems enhance antigen uptake by antigen-presenting cells and their migration into lymph nodes (LNs). Indeed, LNs house B and T lymphocytes, and are the site of lymphocyte priming by antigen-presenting cells for the induction of a subsequent adaptive immune response. Delivery systems can increase the persistence into the LNs and promote a mature immune response. There is a need for innovative adjuvants for prophylactic (preventive) vaccination, but also for therapeutic vaccines. Indeed, the use of vaccination to fight infections or cancer has been in the spotlight recently. The induction of a strong CD8+ T-cell immunity is central to elicit the destruction of infected or malignant cells [2], and the use of nanoparticulate vaccines seems to induce this kind of immune response through multiple pathways. For these reasons, adjuvantation is a promising strategy to address the challenges for the design of effective vaccines. 2. Biodegradable Nanoparticles for Vaccine Delivery Biodegradable polymeric particles have been extensively studied during the past two decades. Biodegradable polymers are used in various medical applications, such as wound healing, tissue engineering, orthopedic devices, cardiovascular applications or vaccine administration. The use of nanoparticles (NPs) for vaccine administration was termed “nanovaccinology” in 2012 [3] and presents tremendous potential. Additionally, the use of biodegradable polymers for NP production is safe and reliable. They are indeed excellent adjuvants due to their physicochemical properties, which can be tuned to adapt to the desired antigen release profile and immunological response. Particle size can be tuned by changing the polymer concentration and the method of synthesis [4]. For these reasons, biodegradable NPs are ideal vectors for drug and protein delivery, and thus outstanding candidates for the future of vaccine administration [5–9]. 2.1. Various Polymers for Vaccine Application Biodegradable polymers are degraded in vivo by enzymatic processes, either hydrolysis or other mechanisms, and the degradation products are further eliminated by the normal metabolic pathways. This simple characteristic means that these materials have a great potential in medicine. The field of bioengineering offers a wide range of biodegradable polymers to produce NPs. Multiple polymers are already approved by the U. S. Food and Drug Administration (FDA) for some applications, even though no NP formulation has been approved for vaccination so far [10]. Synthetic or natural biodegradable polymers may be used and each family has attractive properties. However, the most popular biodegradable polymers used in vaccine applications are poly lactic-co-glycolic acid (PLGA), poly lactic acid (PLA) and polycaprolactone (PCL), three synthetic polymers. PLGA is a highly compatible co-polymer of PLA and poly glycolic acid (PGA), and is FDA-approved for diverse applications. It has emerged as an attractive polymer as it offers wide possibilities for sustained drug delivery. For vaccine applications, NPs of PLGA can carry the antigen by encapsulation or surface attachment by covalent or ionic bonding [11]. Together with PLGA, PLA is one of the most widely used polymers for particulate vaccine delivery, as a single polymer [12,13] or as a co-polymer when coupled to polyethylene glycol (PEG) [14] or PLGA [15]. This polymer is FDA-approved and its nanoformulation has already shown its efficacy in stimulating an efficient immune response after parenteral administration [16,17]. One of the major advantages of this anionic polymer is the possibility of encapsulation of hydrophobic molecules. It has been shown that PLA NPs can encapsulate or adsorb on their surface one or several antigens together with immunostimulant molecules like receptor ligands to improve their immunogenic potential [18].
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Like PLA and PLGA, PCL is an aliphatic polyester and is of interest for its safety, low cost and compatibility with other polymers [19]. PCL has safe degradation by-products after hydrolysis of its ester linkages. Indeed, unlike PLA and PLGA, its degradation does not lead to the formation of lactic acid, which could affect the bioactivity of the antigen [20]. PCL has often been used for long-term Vaccines 2016, 4,devices 34 Vaccines 3 of 15 implantable due its slow degradation rate [6]. 2016,to 4, 34 3 of 15 Vaccines 2016, 4, 34
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Like PLA and PLGA, PCL and is anPLGA, aliphatic polyester and is of interestand for its safety, lowfor cost LikeLike PLA PCL isAssociation an aliphatic polyester is of itsand safety, lowlow costcost andand 2.2. Polymers and Antigen/Immunopotentiator PLA [19]. and PLGA, PCL isdegradation an aliphatic polyester and isinterest ofhydrolysis interest for its compatibility with other polymers PCL has safe by-products after of its safety, compatibility with other polymers [19]. PCL has safe degradation by-products after hydrolysis of compatibility with other polymers [19]. PCL has safe degradation by-products after hydrolysisits of its ester linkages. Indeed, unlikeproperties PLA and PLGA, its degradation does not lead to the formation of lactic The physicochemical of biodegradable polymers allow several ways to associate NPs ester linkages. Indeed, unlike PLA and PLGA, its degradation does not lead to the formation of lactic ester linkages. Indeed, unlike PLA and PLGA, its degradation does not lead to the formation of lactic acid, which could affect the bioactivity ofthe thebioactivity antigen [20]. PCLantigen has often been used for long-term acid, which could affect of the [20].[20]. PCL has has often been used for for long-term and antigens, immunopotentiators or antigens/immunopotentiators together (Table 1).used The bioactive acid, which could affect the rate bioactivity of the antigen PCL often been long-term implantable devices due to itsdevices slow degradation [6]. implantable due to its slow degradation rate [6]. molecules can indeed be trapped in the encapsulation and implantable devices dueNPs to itsby slow degradation rate [6].be released during NP degradation.
The of2.2. interest can also simply be adsorbed on the surface of the NPs by electrostatic or 2.2.molecule Polymers and Antigen/Immunopotentiator Association Polymers and and Antigen/Immunopotentiator Association 2.2. Polymers Antigen/Immunopotentiator hydrophobic interactionsproperties [21]. This association ispolymers easy to Association perform butways onlytoprovides a weak interaction The physicochemical of biodegradable allow several associate NPs The physicochemical properties of biodegradable polymers allow several ways to associate NPsNPs properties of biodegradable allow several ways to associate with NPs. immunopotentiators This typeThe of physicochemical association could be interesting if the polymers application requires a rapid release andthe antigens, or antigens/immunopotentiators together (Table 1). The bioactive andand antigens, immunopotentiators or antigens/immunopotentiators together (Table 1). The bioactive antigens, immunopotentiators or antigens/immunopotentiators together (Table 1). The bioactive can indeed be can trapped in the NPs by in encapsulation be released during NP of molecules the immunomodulators. Aindeed chemical conjugation provides a slight to strong with NP the molecules be be trapped the NPsNPs byand encapsulation and beassociation released during molecules can indeed trapped in adsorbed the by encapsulation and be released during NP degradation. The molecule of interest can also simply be on the surface of the NPs by NPs by either adding a chemical cross-linker such unmodified or modified polyethylene glycol degradation. TheThe molecule of interest canas also simply be adsorbed on the surface of the the NPs by by degradation. molecule of association interest can also simply be adsorbed on the surface NPs electrostatic or hydrophobic interactions [21]. interactions This is easy to perform but only provides a onlyof electrostatic or hydrophobic [21]. This association is easy to perform but provides a a (PEG), (interesting because of its thiol reactive maleimide), or by direct group association of the antigen electrostatic or hydrophobic interactions [21]. This association is easy to requires perform but only provides weak interaction weak with the NPs. This type ofNPs. association could be interesting if the interaction with the ThisThis type of association could be application interesting if the application requires weak interaction with the NPs. type of association could be interesting if the application requires with the carboxylic group of the NPs [22].A However, due to theprovides aliphatic naturetoofstrong these biopolymers, a rapid release aof rapid the immunomodulators. chemical conjugation a slight release of the immunomodulators. A chemical conjugation provides a slight to strong aNPs rapid release of the immunomodulators. Asuch chemical conjugation provides a slight toofstrong theassociation strategieswith forassociation conjugation are limited [23], which is why there is no reference to conjugation an the by with either adding a chemical cross-linker as unmodified or modified the NPs by either adding a chemical cross-linker such as unmodified or modified association with the NPs by either adding a chemical cross-linker such as unmodified or modified polyethylene glycol (PEG), (interesting because of its thiol reactive maleimide), or by direct group antigen with PCL nanoparticles in the literature. Yet, although no examples of the use of conjugated polyethylene glycol (PEG), (interesting because of its thiol reactive maleimide), or by group polyethylene (PEG), (interesting because its thiol reactive maleimide), or direct by direct group association of theassociation antigen with theglycol carboxylic group of the NPs [22].of However, due toHowever, the aliphatic of the antigen with the carboxylic group of the NPs [22]. due to the aliphatic PCL nanoparticles for immunotherapy were found, conjugation by amide group could be possible. association of the antigen with the carboxylic group of the NPs [22]. However, due to the aliphatic nature of these biopolymers, thebiopolymers, strategies forthe conjugation are limited [23], which is why there is nois why there is no nature ofsingle these strategies for conjugation limited [23], which The ease of adsorbing orbiopolymers, multiple antigens or for ligands inare PLA/PLGA particles (compared nature ofanthese the strategies are limited [23], which why there is no reference to conjugation ofto antigen with PCL nanoparticles inconjugation the literature. Yet, although noYet,is although reference conjugation of an antigen with PCL nanoparticles in the literature. no no reference to conjugation of an antigen with PCL nanoparticles in the literature. Yet, although with others adjuvants) explains the renewed interest in vaccine approaches using these polymeric examples of the examples use of conjugated PCL nanoparticles fornanoparticles immunotherapy were found, conjugation of the use of conjugated PCL for immunotherapy were found, conjugation examples of theThe use ease of conjugated PCLsingle nanoparticles for antigens immunotherapy were conjugation nanoparticles [24]. by amide group could be possible. of adsorbing or multiple or ligands in found, by amide group could be possible. The ease of adsorbing single or multiple antigens or ligands in by(compared amide group be adjuvants) possible. The ease ofthe adsorbing single or multiple antigens or ligands in PLA/PLGA particles withcould others explains renewed interest in vaccine PLA/PLGA particles (compared withwith others adjuvants) explains the the renewed interest in vaccine PLA/PLGA particles (compared others adjuvants) explains renewed interest in vaccine approaches these polymeric nanoparticles [24]. Table 1. using Possibilities to associate antigens or nanoparticles immunopotentiators approaches using these polymeric [24].[24]. to nanoparticles. Encapsulation is approaches using these polymeric nanoparticles
performed by mixing the bioactive molecule with the polymer during synthesis and leads to very few Table 1. Possibilities to associate antigens or immunopotentiators to nanoparticles. Encapsulation is Encapsulation is Table 1. Possibilities to associate antigens or immunopotentiators to nanoparticles. physical interactions with nanoparticles (NP). Adsorption of the immunomodulators occurs via is Table 1.the Possibilities to associate antigens or immunopotentiators to Encapsulation performed by mixing the bioactive molecule with the polymer during synthesis and leadssynthesis to nanoparticles. very few performed by mixing the bioactive molecule with the polymer during and leads to very few electrostatic or hydrophobic interactions and provides a weak association. A stronger association isfew performed by mixing the bioactive molecule with the polymer during synthesis and leads to very physical interactions with the nanoparticles Adsorption of the Adsorption immunomodulators occurs via physical interactions with (NP). the nanoparticles (NP). of the immunomodulators occurs via physical interactions with the nanoparticles (NP). Adsorption of the immunomodulators occurs via provided by the chemical conjugation that links the immunomodulator and the NPs via a cross-linker. electrostatic or hydrophobic interactions and provides a weak association. A stronger association is electrostatic or hydrophobic interactions and provides a weak association. A stronger association is electrostatic or that hydrophobic interactions and provides weak A stronger association is provided by the chemical conjugation links the immunomodulator and theaNPs viaassociation. a cross-linker. provided by the chemical conjugation that links the immunomodulator and the NPs via a cross-linker. provided by the chemical conjugation that links the immunomodulator and the NPs via a cross-linker. Association Type of Interaction Polymers Involved Association Type of interaction involvedPolymers involved Association Type of Polymers interaction Association Type of interaction Polymers involved
/
Encapsulation Encapsulation
/
PLA, PLGA, PCL PLA, PLGA, PCL / PLA, PLGA, PCL / PLA, PLGA, PCL
Encapsulation Encapsulation
Electrostatic or hydrophobic PLA, PLGA, PCL PLA, PLGA, PCL Electrostatic or hydrophobic Electrostatic orElectrostatic hydrophobic PLA, PLGA, PCL PCL or hydrophobic PLA, PLGA, Adsorption
Adsorption
Adsorption Adsorption
Chemical cross-linking PLA, PLGA Chemical cross-linking PLA, PLGA Chemical cross-linking PLA, PLGA PLA, PLGA Chemical cross-linking Conjugation
3.
Conjugation Conjugation
Conjugation 3. Influence of Particle Characteristics on APCs Uptakeon and Targeting toand Lymph Nodesto Lymph Nodes 3. Influence of Particle Characteristics Uptake Targeting 3. Influence of Particle Characteristics APCs on APCs Uptake and Targeting to Lymph Nodes LNs are target organs for target vaccine delivery, where Band T-lymphocytes reside and activated Influence of Particle Characteristics on APCs Uptake andB-Targeting to are Lymph Nodes LNsLNs are organs for vaccine delivery, where and T-lymphocytes reside andand are are activated areTo target organs vaccine delivery, whereenter B- anddirectly T-lymphocytes reside activated in the presence in of the an antigen. access thefor LN, antigens can either orenter through presence of an antigen. To access the LN, antigens can either directly or through in the presence of andelivery, antigen. where To access the LN, antigens can either directly or through LNs are target organs for vaccine B- and T-lymphocytes resideenter and are activated in
the presence of an antigen. To access the LN, antigens can either enter directly or through intermediate
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intermediate antigen-presenting Two major types of APCs been described for their antigen-presenting cells (APCs).cells Two(APCs). major types of APCs have been have described for their ability to ability to naturally uptake and process antigens: dendritic cells (DCs) and macrophages. They have naturally uptake and process antigens: dendritic cells (DCs) and macrophages. They have similar similar functions, DCs to aremigrate able tofrom migrate from tissues LNsnaïve and T prime naïve T functions, but onlybut DCsonly are able tissues to LNs and to prime lymphocytes. lymphocytes. In addition to their ability to migrate to the LNs, DCs have capacity to coordinate innate In addition to their ability to migrate to the LNs, DCs have capacity to coordinate innate and adaptive and adaptive immune responses in vivo and are involved in vaccination strategies [25]. Polymeric immune responses in vivo and are involved in vaccination strategies [25]. Polymeric NPs enhance NPs enhance vaccine accumulation into LNs superior and promote superior cellularimmunity and humoral vaccine accumulation into LNs and promote cellular and humoral to a immunity variety of to a variety of antigens in mice relative to soluble forms of protein and peptide vaccines [26]. Many antigens in mice relative to soluble forms of protein and peptide vaccines [26]. Many studies have studies have reported that nanoparticle characteristics such as size, shape or surface properties can reported that nanoparticle characteristics such as size, shape or surface properties can significantly significantly biological [27,28]. Forparameters example, can these parameters affect influence theirinfluence biologicaltheir activity [27,28]. activity For example, these affect targeting can to specific targeting to specific cells, antigen uptake and the type of immune response induced. In this part, we cells, antigen uptake and the type of immune response induced. In this part, we discuss the influence discuss those properties ontargeting. APC uptake and LN targeting. of thosethe NPinfluence propertiesofon APCNP uptake and LN 3.1. Targeting 3.1. Effect Effect of of Nanoparticle Nanoparticle Size Size for for APC APC Uptake Uptake and and LN LN Targeting Nanoparticles Nanoparticles or or exogenous exogenous pathogens pathogens can can be be taken taken up up by by cells cells through through various various pathways. pathways. Phagocytosis and pinocytosis (including clathrin-mediated endocytosis, caveolae-mediated Phagocytosis and pinocytosis (including clathrin-mediated endocytosis, caveolae-mediated endocytosis endocytosis and and macropinocytosis) macropinocytosis) are are the the two two main main endocytic endocytic pathways pathwaysused used for for NP NP uptake uptake (Figure (Figure 1). 1). These different pathways differ in the composition of the coat, the size of the vesicles and the fate of These different pathways differ in the composition of the coat, the size of the vesicles and the fate of the the internalized molecule Macropinocytosis is a process the endocytosis of extracellular internalized molecule [29]. [29]. Macropinocytosis is a process for the for endocytosis of extracellular material material (0.5–5 µ m) membrane through membrane protrusions. Phagocytosis is involved in large size endocytic (0.5–5 µm) through protrusions. Phagocytosis is involved in large size endocytic material material size rangesthan greater thanClathrin-mediated 500 nm. Clathrin-mediated endocytosis induces of with sizewith ranges greater 500 nm. endocytosis induces the uptakethe of uptake NPs with NPs with a size under 150 nm. Caveolae-mediated endocytosis allows different cellular processes a size under 150 nm. Caveolae-mediated endocytosis allows different cellular processes including including protein endocytosis. Generally, caveolae-vesicles induce themigration intracellular migration of protein endocytosis. Generally, caveolae-vesicles induce the intracellular of materials with materials with a size of 50–80 nm. NPs with a similar size as pathogens are efficiently recognized and a size of 50–80 nm. NPs with a similar size as pathogens are efficiently recognized and taken up by taken up for theofinduction of theresponse immune[30]. response with a size between 20 and APCs forby theAPCs induction the immune NPs [30]. withNPs a size between 20 and 200 nm 200 are nm are preferentially taken up by DCs, through the pinocytosis mechanism, while macrophages preferentially taken up by DCs, through the pinocytosis mechanism, while macrophages uptake larger uptake larger from 0.5 to 5macropinocytosis µ m, through macropinocytosis and[30]. phagocytosis [30]. NPs, from 0.5 NPs, to 5 µm, through and phagocytosis
Figure 1. Pathways of endocytosis of exogenous particles, molecules or pathogens. According to the size of an extracellular molecule or particle, different endocytosis pathways take place to engulf it into Particles between between 50 50 and and 80 80nm nmare aretaken takeninto intothe thecell cellthrough throughcaveolin-mediated caveolin-mediatedendocytosis, endocytosis, the cell. Particles
