Elsevier

Advanced Drug Delivery Reviews

Volume 61, Issue 2, 27 February 2009, Pages 140-157
Advanced Drug Delivery Reviews

Nanoparticles for nasal vaccination

https://doi.org/10.1016/j.addr.2008.09.005Get rights and content

Abstract

The great interest in mucosal vaccine delivery arises from the fact that mucosal surfaces represent the major site of entry for many pathogens. Among other mucosal sites, nasal delivery is especially attractive for immunization, as the nasal epithelium is characterized by relatively high permeability, low enzymatic activity and by the presence of an important number of immunocompetent cells. In addition to these advantageous characteristics, the nasal route could offer simplified and more cost-effective protocols for vaccination with improved patient compliance.

The use of nanocarriers provides a suitable way for the nasal delivery of antigenic molecules. Besides improved protection and facilitated transport of the antigen, nanoparticulate delivery systems could also provide more effective antigen recognition by immune cells. These represent key factors in the optimal processing and presentation of the antigen, and therefore in the subsequent development of a suitable immune response. In this sense, the design of optimized vaccine nanocarriers offers a promising way for nasal mucosal vaccination.

Introduction

The development of vaccination has shaped the way we understand public health today. In fact, vaccination is often regarded as the most effective medical intervention introduced in human history [1]. In addition to the currently applied “classical” vaccines, the future of immunotherapeutics is directed towards the use of antigens with better immunogenicity/risk ratio for the development of effective vaccines against the most challenging pathogens and new diseases such as HIV [2]. Within this context, it is worth mentioning recent successful development of the first FDA-approved human papillomavirus vaccine (consisting of a major viral capsid protein) for cervix cancer prevention [3]. The field of vaccine development is also expanding towards the treatment of diseases without a pathogen but with a strong immunological component (e.g. Alzheimer or Parkinsons disease) [4].

In the current socioeconomic and sanitary framework, mucosal vaccination could bring many advantages to existing immunization strategies. First, there is an increased awareness by scientists, health professionals and regulatory agencies that immune responses are complex phenomena, and should not be just reduced to systemic antibody titers. Indeed, the development of secretory antibodies in mucosa is of great interest, as sites such as the respiratory or gastrointestinal mucosa represent the main gateway of entrance for many pathogens [5].

Mucosal immunity is best achieved by mucosal antigen administration, and recently the first product based on an intranasal vaccine has reached the market (Flu Mist®, MedImmune Vaccines, Inc., US, recently re-approved in refrigerated form [6]). Moreover, since different mucosal lymphoid tissues are functionally connected, it is feasible to build immune responses on distal mucosal sites [7]. This is particularly relevant when the target pathogen enters the body through mucosal surfaces where local vaccine administration has a low level of patient compliance (e.g. sexual transmitted diseases) and poor efficacy, as alternative mucosal sites can be used as immune response induction sites. Such an approach could have a major impact for future vaccination against pathogens such as HIV [8], [9].

From a socioeconomic perspective, mucosal vaccination offers many advantageous features. It is needle-free, and potentially, easy enough for self-administration. This eliminates the necessity of trained personnel for vaccine administration. Moreover, mucosal vaccines have higher patient compliance than parenteral ones. The combination of all this factors will most probably imply better patient adhesion to a fixed vaccination schedule — very important in an action where boosting doses are generally necessary [10]. Together with this possibility of self-administration, the reduced sterility concerns, and easier logistics offered by mucosal vaccination would result in reduced sanitary costs [11], [12]. These features are important enough for developed countries, but they claim their main significance for third world countries where vaccines with characteristics such as these mentioned above are currently a dire need [13], [14], [15].

Within the possibilities for mucosal vaccination, the nasal cavity is one of the most promising sites due to (i) its reduced enzymatic activity compared to other possible administration routes (e.g. oral route), (ii) its moderately permeable epithelium, and (iii) the high availability of immuno-reactive sites [16], [17], [18]. However, despite these encouraging characteristics, free antigens are usually unable to elicit protective responses following their intranasal (i.n.) administration due to multiple factors that limit their availability at the appropriate induction sites [19]. For this reason, vaccine adjuvants, including within this term also vaccine carriers, are necessary in order to improve the performance of existing and future antigens.

Among possible delivery systems, nanocarriers hold great promise because of their capacity to protect encapsulated biomacromolecules such as peptide-, protein- or nucleic acid-based antigens [20], to promote interaction of nanocarriers with mucosae [21], [22], and to direct antigens towards lymphoid tissues as potential inductive sites [23]. In the following sections, the main biological considerations and the principal barriers for nasal vaccine delivery are discussed. Afterwards, we analyze the interest of adjuvants for nasal vaccination, and discuss in detail the most promising vaccine delivery technologies based on nanocarriers.

The nasal passage is the first section of the respiratory system where air enters the body and the respiratory airflow begins. Because of this, nasal functions include warming, moisturizing and filtering the air before it reaches the lungs. The total surface of the nasal lumen is 160 cm2, divided between the two nasal cavities [24]. Nasal cavities are further subdivided into distinct anatomical and functional subunits: the nasal vestibule and atrium, respiratory region and olfactory region [16]. Posteriorly, the nasal passage is connected to the nasopharyngeal region.

The vestibule and the atrium form the anterior part of the nasal cavity, covered by stratified squamous and transitional non-ciliated epithelial cells, respectively [18]. This region presents low permeability due to its small relative surface area and low vasculature, and therefore, it is considered of low interest for drug delivery applications.

The respiratory region occupies the major part of the nose and is characterised by the presence of shelf-like structures called turbinates, which provides it with a high specific surface area [16]. This large surface area, together with rich vascularisation, renders the respiratory region with high permeability, which is of great relevance for nasal drug delivery [10]. This main part of the nasal passage is covered by pseudostratified airway epithelium, consisting mostly of columnar cells, but also including basal and goblet cells. Neighbouring epithelial cells are interconnected by tight junctions on the apical side and by interdigitations of the cell membrane on the lateral side [25].

The apical region of the columnar epithelial cells is densely covered by microvilli, which significantly expand the surface area of these cells towards the cavity. In addition, in the posterior part of the nasal cavity, the columnar cells also possess actively biting hair-like structures called cilia, which are larger and less densely distributed surface expansions than microvilli [25]. The active movement of these cilia is an important element in the removal of potentially harmful substances from the upper respiratory tract. Another key factor in this removal-mechanism (i.e. mucociliar clearance) is the nasal mucus, which covers the above described epithelial cells and provides them with a protective physical barrier. It is a viscoelastic mixture that contains glycoproteins (mucins), proteins (enzymes, immunoglobulins etc.), salts and lipidic components [26]. Nasal mucus is partly produced by goblet cells interspersed with the columnar cells, but mainly secreted by the serous and seromucous glands that are located in the connective tissue below the respiratory epithelium [27].

The active ciliar movement drives the overlying mucus layer continuously towards the nasopharynx. As a result, inhaled particulates trapped in the mucus are efficiently cleared from the nasal passage. Environmental factors (e.g. low temperature, air pollution) and pathological conditions (e.g. common cold, inflammation) can hamper mucociliary clearance by altering the physico-chemical properties of the mucus layer or the number/activity of ciliated cells [16], [17].

The olfactory epithelium is a small region of specialized pseudostratified ciliated cells located on the upper part of the nasal cavity. This epithelium with interspersed neuronal terminations is directly involved in smell perception. The primary olfactory neurons are in contact with the environment in the nasal cavity and they are also in communication through their axons with the olfactory bulb in the brain [28]. Despite the small surface area (2–400 mm2) of the olfactory region, there are two important considerations of this region in relation to drug delivery. First, the basic smell detecting capacity should not be compromised when administering any substance to the nasal mucosa. Second, the olfactory epithelium represents a unique pathway for direct nose-to-brain delivery via the olfactory bulb [29], [30]. Although there is evidence for direct brain delivery by olfactory epithelial deposition, the mechanism of this pathway has still to be further investigated for better understanding.

In general, organised mucosal inductive sites for protective immune reactions are present where pathogens frequently enter the body. The mucosal surface of the nasal passage is separated from the external environment by the previously described epithelial barrier, which protects it by non-specific defence mechanisms (mucosal secretion, mechanical cleaning etc.). A more specific protective mechanism is provided by immunological functions of the mucosa-associated lymphoid tissue (MALT) [5] (Fig. 1).

In general, the degree of development of MALT components is species- and age dependent. Although these lymphoid tissues share many structural and functional characteristics throughout the body, each of them is adapted to its specific anatomical location. In a strict sense, nasal-associated lymphoid tissue (NALT) is defined by the presence of organized lymphoid aggregates and the infiltration of the overlying epithelium in the nasal cavity [31]. Such structure has been first described in rodents as a pair of lymphoid aggregates located at the entrance of the pharyngeal duct. In humans, this tissue exists in a less distinct form, as a so-called “diffuse NALT” consisting of a collection of isolated subepithelial lymphoid follicles [31], [32]. In addition to that, highly organised lymphoid tissues are present in the human nasopharynx and oropharynx, incorporating the lingual, palatine and nasopharyngeal tonsils (adenoids). This assembly of lymphoid tissues is denominated as Waldeyer's ring and it plays an important role in respiratory immune defence [33]. Indeed, most particles entrapped in the mucus layer are carried to this region by the mucociliar clearance mechanism [16].

The NALT has full range of immunocompetent cells, including subepithelial B-lymphocytes, CD4+ and CD8+ T-lymphocytes, phagocytic antigen presenting cells (APC) such as macrophages and diverse subsets of dendritic cells (DCs) [34]. In addition, the overlying epithelium of mucosal follicles forms a specialised cell layer (i.e. follicle associated epithelium), which has a more loose structure that enables the contact between antigens and immune cells. More importantly, the follicle associated epithelium also incorporates microfold cells (M cells) characterised by a basolateral cytoplasmic invagination that forms an intraepithelial pocket, also containing lymphocytes and some phagocytic cells [5]. M cells possess a high capacity to transport a wide range of materials by transcellular vesicular transport to these underlying intraepithelial cells. Alternatively, in the regions where organised follicles and M cells are absent, dendritic cells can traffic close to the epithelial layer and establish contact with antigens through interaction with epithelial cells [7]. It has been indicated by several reports that, in contrast to soluble antigens, particulates could be preferentially taken up by M cells following nasal administration [35].

Following their contact with pathogens, different subsets of intraepithelial or subepithelial antigen presenting cells can stimulate local adaptive immune responses by presenting the antigen to neighbouring lymphocytes in the context of the major histocompatibility complex (MHC). Alternatively, dendritic cells can also migrate and carry the antigen to proximal draining lymph nodes and disseminate immune responses to distant sites of the body [36].

Immune responses in the mucosal tissue are dependent on the nature of the antigen and also on the type of the antigen presenting cells involved. Ideally, adaptive immune responses should comprise both cellular and humoral immune responses against the pathogens. Cellular immune defence is mostly effected by cytotoxic T lymphocytes (CTL) and antibody dependent cell-mediated cytotoxicity through natural killer cells. Since these mechanisms can directly destroy specific cells, this type of immune response is crucial for the clearance of viruses and intracellular parasites [7], [37].

Humoral immune defence at the mucosal surface is principally mediated by the production of immunoglobulin A (IgA) following activation of B cells. IgA is found in mucosal secretions in specific forms, as dimeric or multimeric IgA. In contrast to other antibody isotypes, this so-called secretory IgA is relatively resistant to enzymatic degradation, which makes it especially and uniquely suitable for mucosal defence [38]. The principal role of the secretory antibody system is to inhibit invasion and colonization of pathogens (immune exclusion), in cooperation with the innate immune system [7]. In addition, mucosal immunisation can also result in the production of serum IgA and serum IgG antibodies. This is related to the above mentioned migration capacity of different subsets of immune cells that allows their contact with systemic inductive sites [39], [40].

In summary, cells of the nasal associated lymphoid tissue can be involved in the close regulation of both cellular and humoral immune responses locally and also at distant sites. Since this is not always the case for other mucosal sites (e.g. oral or vaginal mucosal tissues), this feature makes the nasal route particularly attractive for the administration of vaccines.

The ultimate goal of vaccination is to facilitate the formation of long-lived protective immune responses. This is achieved by inducing specific T- and B-memory cells, as well as some readily available circulating antibodies. This objective was typically fulfilled by parenteral injection of attenuated viruses, an approach that carries the risk of reversion of the microorganism to a pathogenic state. Current purified antigens and custom-made epitopes are safer, but usually unable to build sufficient immune responses. This lack of potency is further aggravated when considering a mucosal route for vaccination. Vaccine adjuvants are compounds that are co-administered with antigens as immunostimulants and/or immunomodulators, and therefore, they are of great interest for challenging applications in immunology [41], [42]. Herein, we cover some of the mechanisms through which adjuvants can enhance or modulate immune responses from the perspective of nasal vaccination:

  • 1)

    Enhancement of antigen immunoavailability.

  • 2)

    Recognition of “foreign/danger” signals.

  • 3)

    Immunomodulation.

It is now well known that adaptive immune responses require the activation of T-helper lymphocytes (Th), which amplify the effector immune response and regulate microbiocidal effector cells, microbiocidal macrophages, cytotoxic T-cells (CTLs) and B-cells through membrane-bound cell receptors and secretory cytokines [43]. In some cases, Th-cells can be primed directly by antigens; however, T-cells are “geographically” restricted to the lymphoid areas of the body to where antigens need to be delivered [44]. In the case of the nasal cavity, these lymphoid tissues comprise not only draining lymphatics, but also the NALT (see Section 1.1.2). Alternatively, the antigen can also be sampled and processed by professional antigen presenting cells, of which DCs are the most relevant cellular type. Naïve DCs circulate in mucosal tissues, where they can take up antigens that induce their maturation. As DCs mature, they migrate to secondary lymphoid tissues were they can start a Th or CTL response [45]. Antigens should reach the secondary lymphoid tissue in sufficient quantities (either in soluble form or processed by APCs) to induce immune responses, which has led to the term “immunoavailability” [19], [46].

In order for the antigen to reach the secondary lymphoid tissues after i.n. administration, it needs to overcome several barriers; consequently, vaccine carriers are required in order to achieve this objective. The first barrier to nasal antigen delivery is the degradation of the antigen by enzymes. This is a main issue during the time of residence of the antigen in the nasal cavity, but also once it has been absorbed in the body. Many works have suggested that antigen stability in biological environment can be enhanced by its inclusion within the matrix of a suitable nanocarrier [47].

Another important barrier in vaccine delivery is the mucus layer covering the epithelium. For antigens of small molecular weight, the mucus layer is a delivery barrier mainly because of its high content in degradative enzymes [48] rather than for its capacity to reduce protein diffusion [49]. For antigens associated to nanocarriers, mucus represents a physical barrier for penetration [50], [51]. This is a main consideration for vaccine carriers to reach subepithelial lymphoid follicles (“diffuse NALT”), because they need to diffuse through the thick mucus layer just before arriving to a second physical barrier: the epithelial membrane [52]. There is now evidence that the transit of carriers through the mucus layer can be improved by careful selection of the surface characteristics of the carrier [21], [51], [53]. Mucus and their associated mechanisms can also bring some advantages to nasal vaccine delivery. For instance, as it has been previously mentioned, mucociliar clearing in humans drives most particles entrapped in the mucus to the Waldeyer's ring, the main site for antigen sampling in the nasal cavity [16].

Further on, antigens alone or loaded in vaccine delivery systems should cross the nasal epithelium and circulate into the lymphoid system or interact with APCs and induce their migration to this tissue. Again, physicochemical characteristics of vaccine carriers result critical at this step. The interaction of particle carriers with epithelium is generally better for nano-sized systems [21], [54], [55]. On the other hand, several authors have indicated that small microparticles might be the more favourable for APC uptake [56]. This size discrimination comes from the fact that APCs can phagocyte particles while epithelial cells are restricted to other processes only applicable to smaller carriers (pinocytosis, endocytosis, etc.).

Surface characteristics of the nanocarriers also play a crucial role in their internalization and processing. For instance, enhanced immunoavailabity after nasal antigen delivery was achieved with carriers comprising hydrophilic polymers such as chitosan and PEG [21], [57]. Targeting to APCs and improved cell penetration can also be achieved by including biological moieties in the surface that can interact with the cells of the immune system [58]. Examples of these targeting moieties are mannose-derived polymers [59], [60], complex biomolecules such as influenza virus hemagglutinin and neuraminidase present on the surface of virosomes [61] and inactivated viral proteins [8], [9]. Cytokines such as granulocyte macrophage–colony-stimulating-factor and fetal liver tyrosine kinase 3 ligand can enhance the migration of DCs to the carrier site [62], [63], and therefore have a positive effect on antigen or carrier uptake by these cells.

The necessity of certain antigen availability in lymphoid tissues is not only dependent on spatial considerations, but also on temporal ones. The optimal kinetic profile for antigen presentation at the induction sites was under discussion during the last decades. The traditional consideration was that continuous delivery of antigen leads to tolerance [64]. However, in the last decade it has become increasingly accepted that continuous delivered antigen can elicit adequate immune responses [65].

A final consideration is antigen structure. Similitude with pathogen antigen-disposition leads to higher immugenicity of the antigen. For that reason, it has been suggested that surfaces with highly repetitive antigen sequences can crosslink B-cell receptors and thereby they initiate efficient humoral immune responses [66], [67].

Examples of adjuvants that enhance antigen immugenicity by improving its immunoavailability are most vaccine carriers such as micro- and nanoparticles, liposomes, etc. Having an antigen delivery system is most critical when considering a route of administration such as the nasal, where an important fraction of the antigen is eliminated by natural protective mechanisms and thus, will not reach the target tissues.

The immune system has evolved to recognize some of the molecular patterns characteristic from pathogens. DCs recognise these molecular fingerprints, termed pathogen associated molecular patters (PAMP, markers of “foreign”), through pathogen recognition receptors such as Toll-like receptors (TLR), mannose receptors and complement receptors [68], [69], [70]. Activation of these receptors induces the upregulation of several co-stimulatory molecules, some as soluble factors, others bound to the cell membrane. Inflammatory cytokines, in turn, activate the expression of different TLR in APCs ensuring that amplification of the inflammatory response is achieved in presence of a pathogen [71]. Some authors also support that APCs activate co-stimulatory signaling in response to molecules present in damaged or necrotic tissue (“danger” signals) [72], [73].

It is interesting to note that co-stimulatory signaling was considered essential for powerful immune responses; however, some recent data is questioning again this hypothesis [74], [75]. In any case, it is clear that co-stimulation is an important pathway for building immune responses and therefore, should be taken into account when aiming at challenging vaccine delivery applications. Examples of adjuvants that enhance co-stimulatory signaling by presenting foreign/danger signals are: Freund's adjuvant, certain oligonucleotide sequences such as CpG, double stranded RNA such as poly I:C, bacterial lipopolysaccarides, monophosphoryl lipid A, and mannose [76], [77], [78], [79]. Other polysaccharides used in drug delivery such as chitosan have also been shown to interact with these receptors [79].

When T-cells are exposed to antigen epitopes, and possibly to some other co-stimulatory molecules, they amplify adaptive immune responses. T-helper lymphocytes are divided into categories, 1 and 2 depending on the kind of immune response they favour. Helper T-lymphocytes type 1 (Th1) are potent secretors of interferon-gamma (IFN-γ), and they promote processes of cellular immunity: proliferation of macrophages, enhancement of the microbiocidal activity of this cell population, etc. Helper T-lymphocytes type 2 (Th2) produce interleukin-4 as their most distinguishable cytokine, and they stimulate humoral responses. The quality of the immune responses (Th1/Th2 ratio) can be assessed based on the ratio between the IgG subtypes IgG2a and IgG1 (for Th1 and Th2 respectively) [80]. Th1/Th2 ratio is dependent on the antigen, its structure, and the presence of some other co-stimulatory molecules [81]. Therefore it is not surprising that adjuvants can sometimes shift the Th1/Th2 ratio of immune responses. Many adjuvants that act as vaccine delivery systems polarize the response into Th2 type. On the other hand, other adjuvants that induce co-stimulatory molecules polarize it into a Th1 type [82]. Nevertheless, this is not a clear-cut division; ISCOMs, for instance shift the response towards a Th1 [83], while cholera toxin or some polysaccharides shift it towards a Th2 [84].

Recent works have identified DC subpopulations of high relevance for vaccine delivery. A study from Dudziak et al. [85] suggested that different subpopulations of dendritic cells handle the activation of helper T- and cytotoxic T-cell responses respectively. In another study, a DC cell lacking its classic morphology has been identified as the major APC dedicated to the detection of viruses [86]. Clearly, there will be important advances in vaccinology once carriers are designed to target specific APC populations. With this technology, the possibility to control the main pathway favoured for immune response will be a closer reality.

Section snippets

Nanocarriers for nasal antigen delivery

In this section of the manuscript we will cover in higher detail a specific group of adjuvants, i.e. nanocarriers. Readers interested in other adjuvants (salts, bacteria, polysaccharides, etc) can find an excellent review on this topic from Aguilar et al. [87].

Nanocarriers present several characteristics that makes them unique as vaccine adjuvants [88]. First, nanocarriers are one of the only adjuvants that can effectively increase the amount of antigen that reaches the immune system. Secondly,

Conclusions

The use of nanometric drug delivery systems for nasal vaccination has been investigated since the end of the 80s, but it has only been recently that the first nanocarrier-based vaccine candidates have reached clinical trials. Although many challenges remain ahead, the increasing knowledge about the immune system, the growing field of nanomedicine and the application of new nanotechnologies together with a more favourable view towards the benefits of mucosal vaccination by regulatory

Acknowledgements

The authors would like to thank the “Bill and Melinda Gates Foundation” for the financial support of the project entitled “Surface-modified nanostructures as delivery vehicles for transmucosal vaccination” (reference no 37866) within the frame of the “Grand Challenges in Global Health” programme.

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    This review is part of the Advanced Drug Delivery Reviews theme issue on “Drug and Gene Delivery to Mucosal Tissues: The Mucus Barrier”.

    1

    These authors contributed equally to this manuscript.

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