Recent Advances in Mucosal Immunization Using Virus-like Particles

Review pubs.acs.org/molecularpharmaceutics

Recent Advances in Mucosal Immunization Using Virus-like Particles Gael̈ le Vacher,† Matthias D. Kaeser,‡ Christian Moser,‡ Robert Gurny,† and Gerrit Borchard†,* †

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1211 Geneva, Switzerland Pevion Biotech AG, Ittigen, Switzerland

‡

ABSTRACT: Mucosal immunization offers the promises of eliciting a systemic and mucosal immune response, as well as enhanced patient compliance. Mucosal vaccination using defined antigens such as proteins and peptides requires delivery systems that combine good safety profiles with strong immunogenicity, which may be provided by virus-like particles (VLP). VLP are assembled from viral structural proteins and thus are devoid of any genetic material. They excel by mimicking natural pathogens, therefore providing antigenprotecting particulate nature, inherent immune-cell stimulatory mechanisms, and tissue-specific targeting depending on their parental virus. Nevertheless, despite of promising preclinical results, VLP remain rarely investigated in clinical studies. This review is intended to give an overview of obstacles and promises of VLPbased mucosal immunization as well as to identify strategies to further improve VLP while maintaining a good safety and tolerability profile. KEYWORDS: virus-like particles, virosomes, mucosal immunization, vaccine

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INTRODUCTION The progress of vaccine development from live attenuated vectors to recombinant vaccines is aimed at obtaining safer and more efficient products at competitive production costs.1 Historically, live attenuated vaccines (weakened replication competent pathogens) as well as inactivated vaccines (killed pathogens) have been used as immunizing agents (http://www. historyofvaccines.org/content/articles/different-types-vaccines, accessed on March 18, 2013). Despite the stronger immune response obtained with live attenuated vaccines, obvious safety concerns have directed current strategies in vaccine development toward fully inactivated vaccines, wherever technically possible. Early versions of inactivated vaccines consist of killed, yet whole, pathogens. Subunit vaccines, as a further development of inactivated vaccines, are reduced to a selection of welldefined pathogen-derived components in combination with a delivery system. Thus, their composition is better characterized and adjusted than whole inactivated pathogens. Subunit vaccines are generally safer vaccines having fewer side effects but tend to be less effective due to the lack of immunostimulatory components.2 Mucosal surfaces of the respiratory, intestinal and urogenital tracts are the port-of-entry for most pathogens. Establishing a local first line of defense within the mucosal epithelium to prevent infection has substantial benefits over fighting off a systemically established infection. Administration of vaccines at mucosal surfaces has been shown to elicit an adequate local immune response at the administration (effector) site and at distant mucosal sites, as well as a systemic immune response.3 Mucosal immunization is generally needle-free,4 thus being accompanied by socio-economical advantages, including increase in patient compliance, in some cases decrease in © 2013 American Chemical Society

costs by simplified manufacturing (e.g., no sterilization necessary as for parenteral vaccines), and potentially the avoidance of cold storage conditions, which represents an important issue especially in developing countries. When compared to conventional (i.m./s.c.) injection, mucosal vaccine administration by different routes is facing several obstacles against effective immunization. In particular for nonreplicating vaccines, these are the mechanical barrier (epithelium, mucus) limiting vaccine access to relevant immune cells, an environment that promotes rapid extracellular degradation, and a short retention time at the local site of administration.3 Therefore, antigens must be protected by efficient carrier systems and their activity potentiated by adjuvants improving their targeted delivery to antigenpresenting cells (APC) in mucosal tissues. A broad spectrum of adjuvants, among them alum salts and emulsions, has been developed for intramuscular injection. However, none of these has proven optimal for application at mucosal surfaces.3 Viruslike particles (VLP) and virosomes combine the properties of a particulate carrier system with the presentation of antigenic structures necessary to be recognized and successively processed by the innate immune system. At mucosal surfaces, VLP and virosomes therefore tend to have a superior efficacy/ toxicity ratio as carriers and adjuvants for antigen delivery.5 The aim of this review discusses the in vivo potential of VLP and virosomes as antigen delivery systems for subunit vaccines at mucosal surfaces. Received: Revised: Accepted: Published: 1596

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secretion18 to promote antigen capture and to prevent direct mucosal contact of commensal bacteria or pathogens. Secretory IgA (S-IgA) antibodies provide an effective immunological barrier of the adaptive immune response against subsequent infections with the same pathogen. The importance of S-IgA was clearly demonstrated as higher susceptibility to Salmonella infections was observed in mice deficient of the secretory component.19 Therefore, promoting S-IgA secretion at the local site of pathogen entry is set to prevent pathogen entry and thus infection. A major hurdle for mucosal immunization is the poor responsiveness to soluble or particulate proteins, requiring subunit antigens to be delivered by the use of suitable carrier systems and/or in combination with specific mucosal adjuvants.20 Table 1 provides an overview of adjuvant/VLPcombinations already tested in preclinical or early clinical studies.

Mucosal Immunization. In the human body, mucous membranes cover a huge surface area, including the respiratory, digestive, and urogenital tracts, eye conjunctiva, inner ear and the ducts of all exocrine glands,6 representing a total surface area exceeding 400 m2.7 As many pathogens initiate infection at mucosal surfaces, the mucosal immune system represents the first line of defense against infection. The mucosal immune system is subdivided into the innate and adaptive arm of the immune system, which are under the control of several regulatory mechanisms. The mucosal innate defense system itself consists of three main components: first, a mechanical barrier represented by the production and the flow of mucus, as well as a high turnover of epithelial cells; second, an extracellular barrier represented by the secretion of enzymes, scavenger proteins and antimicrobial peptides (e.g., defensins)8 at the mucosal surface; and the third element of epithelial barrier function is represented by the inherent high concentration of cellular effectors of the immune system. Innate control of the adaptive immune response is achieved through pathogen-associated microbial pattern (PAMP) identification by families of highly specialized pathogen-pattern recognition receptors (PRR), such as Toll-like receptors (TLR), expressed by epithelial cells as well as by monocytes, macrophages, neutrophils, dendritic, natural killer, B and T cells.9 In response to innate recognition of PAMPs, local effectors such as antimicrobial peptides, interferons (IFN), proinflammatory cytokines and chemokines are released.10 These provide direct antimicrobial effects or act as mediators to attract and activate innate effector cells (macrophages, polymorphonuclear leukocytes, natural killer cells) and are also essential signals necessary to initiate the processes ultimately culminating in adaptive immunity generation. Approximately 80% of all immunocompetent cells of the human body are present in the mucosa-associated lymphoid tissues (MALT). The goal of mucosal immunization is to prime the adaptive immune response at the mucosal level, thereby preventing the invasion of pathogens via this route. This is achieved by delivering antigens to the mucosa, stimulating their uptake and processing by APC lining the mucosal tissues, namely macrophages and dendritic cells. These migrate to the closest MALT and/or lymph node located in the subepithelial tissue, where they present the processed antigens to T cells.11,12 T cell activation leads to the generation of antigen-specific T cells, which also contribute to the maturation and differentiation of B cells into plasma and memory cells. A mucosal B cell response is characterized by secretion of immunoglobulin A (IgA), which is the only immunoglobulin insensitive to protease degradation13 and therefore capable of resisting the harsh mucosal environment. Human IgA is divided into two subclasses: IgA1 is found mostly in the serum while IgA2 is predominant in secretions.14 Mucosal antibody induction requires mucosal antigen exposure in combination with costimulatory signals, which result in mucosal homing of plasma cells. The class switch recombination (CSR) leading to IgA is activated by local signaling events. The Peyer’s Patches in the gastrointestinal MALT supply a well-adapted environment of cytokines and dendritic cell phenotype that is sufficient to promote an IgAdominated humoral response.13 The constitutive production of retinoic acid by DC is counted among one of the principal drivers of CSR.15 Epithelial cells may also influence the switch by mediators such as BAFF and APRIL, which are secreted upon TLR activation of intestinal epithelial cells,16 lung DC or alveolar macrophages.17 Most of the IgA is destined for local

Table 1. Adjuvants Used in Combination with VLP for Mucosal Immunization adjuvant(s) CT, CTA, CTB

target mucosa

VLP type

Digestive Nasal, pulmonary

NV, HPV, RV HPV, SIV, Qβ, RV HPV HPV, RV, SIV RV SIV RV NV HPV

Flagellin CpG

Vaginal Rectal Nasal Nasal Digestive Nasal Digestive, nasal, vaginal, rectal Nasal Nasal

Imidazoquinolines

Rectal Vaginal Rectal

mLT RANTES ISCOM MPL

Influenza, HIV Coronavirus, SIV, Qβ, RV RV HPV

TLR activated NA

references 21−25 24,26−30

24,31 24,28,32,33

NA NA NA TLR 2 and 439

30,34,35

TLR 541 TLR 945

42−44

36 37,38 40 24

28,36,46,47

28

TLR 7 and 848

24 24

It should be emphasized that the specific properties of a given vaccine antigen determine whether or not a mucosal adjuvant is necessary to induce protective levels of immunity and, if needed, which adjuvant type is most suitable. In general, small, soluble antigens lacking higher structures are poorly immunogenic, in particular when applied via mucosal routes. This type of antigen is likely to require the addition of immunopotentiating compounds in order to achieve protective immune responses. In contrast, larger, particulate forms of antigens such as virus-like particles or synthetic nanoparticles may be sufficiently immunogenic by themselves, or at least require substantially lower doses of additional adjuvants.49 With less or no additional adjuvants involved, the regulatory acceptance tends to be higher. Any additional immunostimulatory compound increases the product complexitiy and slows down the product development process. Bacterial toxins have been known as potent immunostimulators that increase amplitude and duration of immune responses against antigens for a long time, when coadministered via mucosal routes.50 Adjuvants based on saponin derivatives are applied in several parenteral veterinary vaccines but no products for human use have been licensed so far. Beyond bacterial toxins and saponin derivatives, many 1597

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Figure 1. Example of the common steps of the VLP manufacturing process.

Table 2. Current Parenteral VLP Vaccines, Marketed or in Clinical Development VLP type

production system

particle characteristics

development status

manufacturers

Hepatitis B surface (HBs) Hepatitis B core (HBc) HPV Influenza virosome Influenza

Yeast E. coli Yeast Insect cells Egg Insect cells Plant cells Cell culture Insect cells

Micelle VLP Nonenveloped VLP Nonenveloped VLP Enveloped VLP Enveloped VLP

Marketed Phase I Marketed Marketed Phase III

Nonenveloped VLP Enveloped VLP

Phase I Phase I

GSK, Merck Sanofi Merck GSK Crucell, Solvay Novavax Protein sciences Medicago, Fraunhofer Ligocyte Novavax

Norovirus RSV

enveloped VLP result from particle budding from the host cell. Enveloped VLP carry membrane host cell proteins in the VLP envelope, thus emphasizing the importance of the VLP expression system. Additionally, and in contrast to expression in bacteria, expression of VLP components in cells derived from higher order organisms may also cause host-dependent posttranslational modifications of the VLP components.65 Classical expression systems for VLP are E. coli, yeast, insect cells or plant-based expression systems.66 These host cells enable the spontaneous self-assembly of viral structural proteins56 of VLP structures. Secreted VLPs can be harvested from the cell culture medium, however, most VLP remain cellassociated, and therefore, the harvest process generally includes cell disruption and subsequent purification67 (Figure 1). The thorough characterization of the purified VLP is crucial for the release as vaccine product and to control the production process. It includes the biochemical content (specific and total protein content, impurities such as nucleic acids and endotoxin), as well as their physical properties (particle size, homogeneity, and density). In particular for enveloped VLP derived from complex expression systems, contaminating host cell proteins and host cell-dependent modifications still limit VLP use.66 VLPs have been derived from a wide variety of viruses56,68 including Hepatitis B,69 Human papillomavirus (HPV),70 Human immunodeficiency virus (HIV) 71 or Norwalk virus.72,73 An overview of VLP types used in marketed vaccines and advanced clinical development is given in Table 2. Influenza Virosomes. Influenza virosomes are VLP assembled in vitro independently of any host cell.62 They are generated from inactivated virus produced in embryo chicken eggs or cell culture, or from recombinantly expressed viral subunitsthe same material used for the production of influenza vaccines. The virus is dissolved in an appropriate detergent, the solubilized viral envelope components are purified by removal of the insoluble core by ultracentrifugation. The unilamellar, spherical virosomes composed of lipids and integrated viral envelope proteins are subsequently reassembled by detergent removal. The addition of additional lipids prior to assembly is

compounds with mucosal adjuvant potential have been described, including an increasing number of ligands of innate sensor molecules with a well-defined mode of action.51 However, the use of adjuvants raises safety concerns and increase the hurdles for clinical applications, in particular after side effects have been observed with bacterial toxins.52,53 An objective comparison between the different adjuvants with regard to their potency is extremely difficult, despite the large number of published preclinical and clinical studies. The bacterial toxins CT and HLT are frequently used as a reference for mucosal adjuvants because they are commercially available and easily applicable. However, it is debatable whether the comparison with a highly potent but unsafe compound is suitable to identify adjuvants acceptable for clinical use. Virus-like Particles (VLP). General Characteristics. Selfassembly of viral capsid proteins has been postulated as early as the 1970s54 and vaccine properties of VLP were first mentioned in the 1980s.55 VLP are compact and highly ordered nanostructures mimicking the genuine viruses in terms of size (22−150 nm) and envelope composition but lacking viral genetic material.56 Due to their virus-like appearance and especially their repetitive surface structures, VLP show high immunogenicity and specific antigenicity56 and can interact with the immune system through similar pathways as the original pathogens do. Therefore, VLP were increasingly studied over the last decades, starting with their use as homologous vaccine57 to their potential as a novel and versatile vaccine platform carrying heterologous payload antigens,58,59 which are thus protected from degradation.60,61 VLP manufacturing processes are based on the ability of the viral structural proteins, when expressed as recombinant proteins, to self-assemble into structures similar to the parental viruses.62 For the manufacturing process, the DNA sequence encoding the viral building block of the VLP63 is engineered into suitable viral or plasmid expression vectors. This sequence can be supplemented (i.e., in case of chimeric VLP) with specific epitopes and include one or multiple proteins. The flexibility is limited by the requirement of self-assembly.64 Nonenveloped single- or multiple-capsid VLP are obtained from self-assembling virus-derived capsid proteins. In contrast, 1598

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Figure 2. Manufacturing process of the IRIV virosomes decorated with exogenous antigens.

presenting cells with antigens directly sampled from the intestinal lumen by transcytosis. Subsequent presentation of the antigens to B, T and memory cells can then initiate the activation cascade of the adaptive immune system. One of the first investigated VLP for oral administration was the Norwalk VLP expressed in insect cells after recombinant baculovirus transfection (rBV NV-VLP). In mice with Cholera toxin (CT) adjuvant21 and in humans (phase I study) in the absence of any adjuvant, oral administration of rBV NV-VLP demonstrated immunogenicity and no related side effects.73,88 After oral administration, NV-VLP-specific serum IgG and mucosal secretory IgA were observed.21 With regard to the cellular immune response after oral immunization, APC, and especially dendritic cells of the mesenteric lymph nodes and PP, may specifically augment the expression of the mucosal homing receptor α4β7 expression on lymphocytes.89 Integrin α4β7 expression in turn then leads to recruitment of the lymphocytes activated by oral immunization to the human GALT endothelium, which expresses the binding partner of α4β7, mucosal vascular addressin cell adhesion molecule 1 (MadCAM-1).90 This receptor-mediated recognition of mucosal addressins provides a molecular mechanism to ensure that lymphocytes activated by antigen presentation after oral administration are recruited to the GALT tissue. The same type of immune response was observed after oral immunization of mice with CT-adjuvanted plant-expressed NVVLP,22,91 unconjugated hepatitis E virus (HEV) VLP92 and unconjugated chimeric HEV-VLP.93 The rationale behind using NV-VLP and HEV-VLP as antigenic epitope carrier systems for oral immunization relies on the fact that both NV and HEV are natural orally transmissible viruses. The nonreplicative particles appear to follow the same pathway as the genuine pathogens, as reported also for rotavirus VLP.94 Nevertheless, in comparison with per os rBV NV-VLP administration in mice, a higher immune response was observed when the NV-VLP vaccine was adjuvanted with CT. However, toxicity concerns limit the use of CT in humans.21,22 Recently, rotavirus (RV) VLP in the absence of any adjuvant demonstrated another interesting feature of oral vaccine administration.94 Even though the ileum is the target tissue of the virus and a large part of the intact particles were absorbed through the mucosa of the small intestine, intact particles as well as incomplete VLP were also taken up by the colon mucosa after oral application in mice. This was shown by incorporation of green fluorescence protein (GFP) as reporter protein into VLP, which was then detected in colon tissue. Likewise, the capsid protein VP2 was found in colon tissue homogenates. Uptake of VLP in colonic tissue was shown in the healthy state as well as under inflammatory conditions,

key to a robust, industrial scale production process, which generates virosomes called immunopotentiating reconstituted influenza virosomes (IRIV, Figure 2).62,74 Virosomes can act as carrier systems for various antigenic molecules.75,76 Lipophilic payload antigens are added before the reconstitution of the particles, and thus spontaneously cointegrated along with the viral envelope proteins. Along with the payload antigens, the reconstituted viral envelopes present the same surface proteins at identical ratios as the parental virus, hemagglutinin (HA) and neuraminidase (NA).62 Thus, virosomes show the same pH-dependent fusion activity and are able to deliver their contents into the cytosol of the targeted cell.77 Their mode of action is based on the same previously described property of presenting multiple copies of surface antigenic protein and size comparable to the parental virus. For efficient heterologous vaccination, the antigen must be physically associated with the particle membrane.78 In addition, influenza-based virosomal vaccines benefit from a generalized pre-existing anti-influenza immunity among the human population, resulting in enhanced immune response directed against the displayed antigen.62 The production of influenza virosomes on an industrial scale under good manufacturing practice (GMP) conditions62 is fully established and, so far, it remains the only approved virosome type for use in humans.79,80 This success is based on influenza virus availability, costs of production, as well as specific properties of HA and NA. The suitability of the virosomal platform as an efficient adjuvant and carrier system in parenteral vaccine formulations is illustrated by the successful and continued commercialization of two products (Epaxal and Inflexal). Other promising clinical studies are ongoing and may allow virosome-based vaccines to become a key platform to address pathologies as diverse as breast cancer,81 HIV (Mymetics homepage http://www.mymetics.com/ accessed on October 25, 2012), malaria (phase I),82,83 and recurrent vulvovaginal candidiasis (phase I, clinical trial NCT01067131, http://www. pevion.com/ accessed on October 25, 2012). Mucosal Immunization using VLP. Digestive Tract Immunization. Immunization targeting the digestive tract is orally administered and, historically, was the first investigated needle-free route, used especially with live attenuated pathogens such as poliomyelitis, Cholera and Salmonella.84−86 As mentioned below, the major challenges in antigenic subunit vaccination using the oral route are enzymatic digestion and degradation by the low gastric pH. Therefore, a protective and efficient delivery system is needed to enhance safe antigen delivery to the GALT through the gastro-intestinal tract. After passage through the stomach, antigens arriving in the gut are processed by the cells in the Peyer’s Patches (PP).87 In the PP, microfold cells (M-cells) supply the underlying antigen1599

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using a TNBS-induced colitis model. Therefore, orally administered vaccines may reach the terminal digestive tract and deliver colonic disease-relevant antigens to the affected mucosal site. Due to the ease of administration and the patients’ higher compliance, an orally administered RV VLP is therefore considered more promising than a comparable vaccine applied rectally. Finally, one of the most potent strategies to increase vaccine efficacy is the use of the prime-boost regimen. The impact of varying the mucosal route and the protocol of VLP administration was especially investigated in intranasal and oral vaccination using 2/6 VLP in gnotobiotic pigs. 2/6 VLP designates an expressed RV VLP containing VP2, which is the most abundant and important protein of the wild type RV for particle formation, and VP6, which is the highly antigenic major inner capsid protein of RV.95 In gnotobiotic pigs, immunogenicity but not protection was elicited after intranasal administration of 2/6 VLP with mutant LT (mLT).34 The same results were observed after three oral administrations of 2/6 VLP with ISCOM (immuno-stimulating complex).37 By contrast, pigs receiving either three oral doses of attenuated RV or oral attenuated RV priming followed by an intranasal 2/6 VLP with mLT boost or oral attenuated RV priming followed by oral 2/6 VLP with ISCOM boost demonstrated similar partial protection.37 The rationale behind the use of live attenuated vaccines as the priming vaccine agent is based on their replication-dependent induction of pro-inflammatory cytokines, as well as expression of antigenic proteins that induce high rates of antibodies after oral administration. In comparison with intranasal priming, the higher efficacy of oral priming may be attributed to the priming of the inductive sites at the natural targeted mucosa.35 Taken together, the strategy of combining oral priming using attenuated RV with two intranasal boosts using 2/6 VLP bound to ISCOM showed the most promising results. Whether ISCOM-adjuvanted vaccines will obtain regulatory approval for use in man remains to be seen; currently, no product is approved by the authorities so far even though particulate formulations of the ISCOM type have shown potential, especially for the nasal route.96 Finally, in forthcoming studies, the safety concern of using live attenuated virus could be addressed using a recombinant 2/6/4/7 RV VLP to replace the entire nonreplicative RV. Indeed, even though protection was obtained using RV VLP lacking VP4 and VP7, these proteins are known to induce virus-neutralization antibodies.97 The prime-boost strategy to enhance VLP vaccine immunogenicity after mucosal immunization was also suggested for HIV-VLP oral administration in mice without any adjuvant (Table 3).98 Immunization studies indicated that nonreplicative orally administered VLP derived from naturally gut infecting pathogens could ensure proper delivery of the antigenic epitope to the intestinal mucosa in the absence of an additional adjuvant, with variable outcomes probably depending on the type of VLP used. Indeed, the tissue specificity of the parental virus can facilitate appropriate delivery, since VLP often maintain their natural tropism. Additional stimulation of the immune response can be achieved by the oral administration of adjuvanted VLP.57,104 VLP dosage resulting in comparable response could be further decreased by intranasal administration, probably due to the less harsh mucosal environment. Additionally, in contrast to application at the digestive mucosa, intranasal application was far less associated with unintended tolerogenic responses.105

Table 3. Preclinical VLP Vaccination Studies Targeting the Digestive Mucosa parental virus NV RV

animal model Mice Mice Gnotobiotic minipigs

HPV

HEV HIV

Mice

Mice Mice

adjuvant

references

CT Without CT PCPP ISCOM Without mLT CT Without Without

21,22,91 94 25,99,100 57 37,38,101,102 103 104 23,24 92,93 98

Intranasal and Respiratory Tract Immunization. The ease of vaccine application, the high vascularisation and the low local proteolytic activity together render the intranasal route very appealing for mucosal immunization. The human nasopharynx, which is the principal inductive tissue of the NALT, is the major target of intranasal vaccines.106 In direct comparisons, VLP were more immunogenic than administration of the individual soluble viral antigenic proteins administered by the same route.26,36 This confirms the importance of the particulate nature of VLP for vaccination, which is in line with in vitro investigations, suggesting that incomplete VLP (i.e., VLP lacking some capsid proteins in comparison with the genuine virus) lose their capability to efficiently target dendritic cells.107 Even though NALT in rodents is considered equivalent to the Waldeyer’s ring formed by the human tonsils in humans,108 species-specific differences in the NALT may impact on the evaluation of vaccines. Despite these differences, phase I clinical trials with BV-expressed NV VLP, containing chitosan as mucoadhesive and MPL (monophosphoryl lipid A, a Toll-like receptor 4 agonist) as adjuvant, were shown to be safe and immunogenic after nasal immunization in healthy volunteers.109 Consequently, various strategies such as the use of adjuvants, formulations design and variation of the administration site were developed to increase intranasal VLP immunogenicity. Mucosal adjuvants enhance immunogenicity but need to be safe and compatible with the VLP structure. Bacterial toxin adjuvants, for example, CT or Heat-labile toxin (HLT), were shown to be highly effective in enhancing immunogenicity but exhibited severe toxic side effects or are the cause for considerable safety concerns,52,53 which were also observed after intranasal administration. In mice, conjugation of adjuvant to different VLP was reported to elicit higher humoral and cellular immune response than mere coadministration, as described for B subunit of CT with SIV VLP26 and flagellin with chimeric HIV VLP.44 The higher efficacy of the bound flagellin was attributed to the codelivery of antigen and flagellin to the same immune cells.42 On the other hand, other adjuvants such as cytosine-phosphate-guanosine (CpG) intranasally coadministered in mice with severe acute respiratory syndrome (SARS) coronavirus VLP46 or SIV-VLP36 and MPL intranasally coadministered with NV VLP in humans 40 were also demonstrated to be efficiently increasing protection in animals. Aside from incorporating adjuvants together with the vaccine in the same formulation, other strategies were investigated to increase VLP immunogenicity. One was to increase the retention time of NV VLP at the nasal mucosa of mice by incorporating NV VLP in an in situ gelling powder.110 In 1600

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comparison with liquid formulation, higher specific immune response was obtained both locally and systematically. Another approach aimed at the deeper administration of the antigen into the respiratory tract using an aerosol, reported to increase immunogenicity of HPV VLP vaccine. In mice, higher titers of neutralizing antibodies were detected when the antigen loaded onto HPV VLP was deposited at the lung than in the upper part of the respiratory tract.23 The same observation was established with Qβ-EC1 VLP in rats27 and with HPV 16 VLP administered in human volunteers.111 This is consistent with previous work assigning the lungs and in particular the mediastinal lymph nodes located in between the lungs as the major immune inductive sites of the lower respiratory tract.112 Besides its well-documented efficacy, intranasal vaccination offers other interesting features. In the classical scheme of mucosal immunization, the most effective local immune response is generated close to the vaccination site. However, especially if the genital tract is the targeted tissue for VLP immunization, stimulation of the lower respiratory tract also appears to be an asset.23 Indeed, after intranasal immunization, even though the specific mechanisms determining precursor lymphocyte homing at the vaginal mucosa are not clearly understood, a high level of specific B cells was established.27 Previous studies report specific IgA and IgG response in serum and cervical secretions113 after intranasal vaccine administration. Nardelli-Haefliger and collaborators extensively explored this property of vaginal immune response after intranasal immunization with HPV type 16 VLP23,103,112,114,115 and extended the intranasal immunization concept to airway vaccination. After intranasal immunization, vaccination-induced antibodies were found to efficiently neutralize the pathogen when it was introduced into the genital tract of immunized mice (Table 4).

In summary, intranasal immunization with VLP appears to be a convenient and efficient route of vaccination. Especially due to the less harsh environment (physiological pH and low enzymatic activity) and the efficient antigen uptake, lower quantities of antigen are required to elicit an efficient immune response by intranasal vaccination than by vaccination targeting the digestive mucosa. However, the close association of the upper respiratory tract with the olfactory organs and the central nervous system increases the risk for side effects, in particular if adjuvants are used.52,53 In addition, the use of intranasal vaccination may be hampered by widespread pathologies of the upper respiratory tract, such as congested nose or other harmless infections. These factors must be weighed against the advantages offered by intranasal vaccination. Urogenital Tract Immunization. The vaginal mucosal tissue is not as extensively investigated as target site for mucosal vaccination as the gut or the respiratory tracts, and the mechanisms involved in staging an immune response at this mucosal site need to be further examined. In comparison with the previously described mucosal tissues, the cervico-vaginal mucosa is characterized by a nonkeratinised stratified multilayer epithelium lacking specialized M-cells. Furthermore, vaginal vaccination has to address the obstacles presented by the local environment, which is characterized by a slight acidic pH under normal conditions, a daily low liquid volume, the presence of enzymes and antimicrobial peptides (lysozyme, lactoferrin, serine leukocyte protease inhibitor, alpha- and beta-defensins) as well as commensal microorganisms in the local secretions. Moreover, the specific local hormonal responsiveness during the oestrus cycle, whose varying progesterone levels are known to influence antigen uptake as well as bias of immune response.124 After intravaginal immunization of mice using chimeric HPVHIV VLP alone, local immune response was found to be very low, whereas the same VLP administered intramuscularly elicited efficient vaginal immunity.125 The low efficacy of local immunization after vaccination in the genital tract was previously reported and mostly attributed to the lack of MadCAM-1 under normal conditions in the vagina.124 In humans, cervical and vaginal tissues lacking these addressin protein ligands are unable to attract lymphocytes expressing α4β7 integrins. However, MadCAM-1 was reported to be expressed during inflammation of the genital tract in mice. As previously discussed, the preferred strategy to increase VLP immunogenicity is the coadministration of VLP and an adjuvant. Nevertheless, CT demonstrated low efficacy in increasing immune response against HPV 16 VLP after intravaginal application.31 In contrast, another study specified that CT or CTB conjugation with human gamma globulin as model protein antigen leads to a strong humoral response in the genital tract.126 Thus, one can suggest that the discrepancy of these results could be due to the adjuvant/antigen interference in the case of the VLP, to the already described difference of adjuvant efficacy depending on the antigen they are administrated with127 or, in a more general manner, due to hormonal influence during the oestrous cycle in animals.124 In addition to CT, two imidazoquinolines and MPL were tested as vaginal mucosal adjuvants,24 however, without successfully increasing antibody response to HPV 16 VLP. Besides adjuvant strategy, chemical disruption of the pluristratified epithelium was investigated in mice to improve immune response after HPV 16 VLP administration.24 Before

Table 4. Preclinical Vaccination Studies using VLP Targeting the Respiratory Tract parental virus SIV

RV

animal model Mice

Mice

Rabbits Gnotobiotic minipigs Qβ bacteriophage HIV HPV

Mice Rats guinea pigs Mice, macaques Mice

Influenza virus

Mice

NV

Ferrets Mice, rats, guinea pigs Mice

SARS-CoV

adjuvant(s)

references

CTB RANTES, CpG CT CT CpG mLT CT mLT ISCOM Without CTB Flagellin L3, N3 Eurocine Without CT CpG, HLT Resiquimod, imiquimod, MPL Without Flagellin Without Gardiquimod

26

CpG

46

36 116 99,100,117 28 30,118 100 34 101,102 47 27 44 98,119,120 103,113,121 23,24 122 24

123 42,43 123 110

1601

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shown to benefit from VLP-adjuvant combinations. IgA response observed with HPV L1 VLP alone was not significantly different from the response observed in mockimmunized mice, in contrast to HPV L1 VLP adjuvanted with CT, LT, CpG or resiquimod, respectively.24 The highest level of specific IgA was measured in feces of mice receiving CT adjuvant. The serological IgG response in mice immunized with adjuvanted HPV L1 VLP indicated that particles were presented to specialized cells and passed through the lymph node system to induce peripheral immunity. Rectal immunization using SIV p27:Ty-VLP linked to CTB, however, was unable to stimulate the induction IgA or IgG antibodies.138 Immunogenicity of this vaccine could be increased by additional oral administration, which correlated with an additional proliferative CD4+ T cell response.32 These observations underline again the need for adjusting the interplay between mucosal route, adjuvant, immunization regimen and VLP type for efficient immunization (Table 6).

vaccination, animals were pretreated with Depo-Provera (medroxyprogesterone acetate, a synthetic form of progesterone), which thins the epithelium, and with the licensed spermicide nononxynol-9. Despite the positive effect of the epithelium disruption on the local and systemic immunogenicity of the VLP, this method is not clinically acceptable, especially for repetitive administrations as the epithelial disruption leads to an increase of patient susceptibility to sexually transmitted diseases.128,129 A recently described method to increase immune response using Qβ bacteriophage VLP in the absence of an adjuvant is the deeper application into the vaginal tract.130 The rationale behind aerosol vaccine administration into the genital tract is consistent with targeting the cervical transformation zone (TZ), the junction between the pluristratified ectocervix and the single-layer endocervix. TZ is the major inductive and effector site for cell mediated immunity and lymphoid aggregates were identified in the surrounding cervical tissue.131 Aerosol-based vaginal vaccination may be especially interesting with HPV 16 VLP, as TZ has been reported as the main target of HPV.132 However, in the TZ and cervical tissue, the menstrual cycle influences the presence of lymphoid aggregates formed by a B cell core surrounded by T cells and an outer layer of monocytes and macrophages,133 and thus in consequence, the TZmediated induction of an immune response (Table 5).134

Table 6. Preclinical Vaccination Studies Targeting the Rectal Mucosa parental virus RV SIV

Table 5. Preclinical Vaccination Studies Targeting the Vaginal Mucosa parental virus HPV

SIV Qβ bacteriophage PP7 bacteriophage

animal model Mice

Macaques Macaques Mice Mice

adjuvant(s) Without CT Resiquimod, imiquimod, MPL Without CTB, killed cholera vibrios Without Without

PV

animal model Mice Macaques Mice

adjuvant(s)

references

CT, CpG CT, mHLT, CpG, resiquimod CTB CTB, killed cholera vibrios Without CT, resiquimod, imiquimod, MPL

28 139 32 33 125 24

references 125

Mucosal Immunization using Virosomes. Virosomes have been tested in various forms for mucosal application over the past fifteen years (Table 7). Sublingual immunization, designating vaccination on the floor of the mouth, is described to be primed by antigen uptake by subepithelial resident and migratory APC. Successful sublingual immunization with virosomes was demonstrated by

24,31 24

135 32,33 130 130

Table 7. Overview of Mucosally Administered Virosomebased Vaccines

Due to the local physical and immunological characteristics, the formulation of a VLP-based vaccine inducing efficient vaginal immunization appears to be more challenging than for other mucosal routes. Successful vaginal immunization may require the combination of some of the previously described strategies to increase VLP-based vaccine efficacy. Nevertheless, despite the apparent low immune response elicited, the vaginal route remains an interesting strategy for specific immunization against local pathogens. Rectal Immunization. Rectal vaccine administration has clear advantages over oral administration, such as the bypass of antigen destruction in the stomach or the dilution into the high liquid volume of the gut. However, due to the lack of patient compliance, immunization targeting the rectal mucosa is rarely considered, except for children and animals. In mice and humans, the large bowel lymphoid tissues contain isolated lymphoid follicles in abundance, which are immunologically active as inductive tissues, similar to the lympho-epithelial structures present in the GALT.136 In principal, antigen transport and recognition through the rectal lympho-epithelial system is expected to be similar to the gut due to the local presence of organized lymphoid tissue with M-cells and germinal centers.137 The rectal immunization route was

administration route Oral/ sublingual Intranasal

virosome type IRIV IRIV

targeted antigen H5N1 influenza Influenza

RSV Gp41

Intravaginal

1602

Newcastle disease virus IRIV

Newcastle disease rtSap2

type of study

references

Preclinical in mice Preclinical in mice Clinical (phase I) Clinical (phase II) Licensed and withdrawn Preclinical in mice Preclinical in macaques Preclinical in chicken

140

Preclinical in rabbits, minipigs Clinical (phase I)

147,148

141

5

142

52,143

144

145

146

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induction kinetics than intramuscular immunization, requiring one additional dose. Since no local or systemic adverse events were observed, rtSap2-containing virosomes are currently investigated as first-in-class mucosally applied RVVC vaccine in women (phase I clinical trial, www.pevion.com). Taken together, the potential as well as the considerable risks of mucosal adjuvants is evident, also when combined with the virosome carrier system. However, even without any specific mucosal adjuvants, virosomes have been shown to induce relevant immune responses if delivered in an optimal formulation and in a combined regimen including intramuscular injection.

eliciting an immune response resulting in protection against challenge in mice.140 The study reported that the adjuvanticity of the virosome itself in sublingual immunization was not sufficient and required the new mucosal adjuvant cyclic diguanylate (c-di-GMP) to elicit both local and systemic humoral and cellular immune responses. As expected from the differential outcome as measured by protection against antigen challenge, differences in the elicited T-cell immune responses were observed after IRIV alone (Th2 response, reported to be potentially detrimental149) or adjuvanted IRIV administration (Th1 response, described as important for viral infection recovery150), thereby providing a mechanistic explanation for the better performance of the vaccine in the presence of the adjuvant.140 The respiratory tract, and especially the intranasal route, has been most extensively investigated for virosome-based vaccines. Especially Maria Cusi and co-workers extended the HLTadjuvanted influenza virosome concept to several intranasal vaccine application studies. The first study involved the intranasal HLT-adjuvanted influenza virosome immunization carried out in mice,141 in healthy volunteers in clinical phase I trial,5 and on a larger scale in humans in a phase II trial.142 The intranasal influenza HLT-adjuvanted virosome-based vaccine was able to elicit specific neutralizing antibodies without indications of severe related side effects. Furthermore, subjects that were vaccinated with the HLT-adjuvanted vaccine presented an S-IgA response rate twice that of subjects receiving the nonadjuvanted vaccine, and three times higher than those receiving the vaccine intramuscularly. HLTadjuvanted IRIV vaccine was launched in the year 2000 as a commercial influenza vaccine143 (Nasalflu, Berna Biotech). It had to be withdrawn from the market in 2001 due to increased occurrence of Bell’s Palsy.52 This clearly illustrated the importance to address the balance between efficacy and toxicity of the adjuvant used for mucosal virosome-based vaccines. In a second application, the immune response obtained after intranasal administration of the HLT-adjuvanted IRIV carrying the respiratory syncytial virus-F protein (RSV-F) in mice144 was investigated. HLT-adjuvanted RSV-F/IRIV vaccine was also able to efficiently deliver RSV-F protein and induce specific humoral and cell-mediated immunity. A synergetic effect of HLT and HA was observed, triggering increased cytokine secretion. Likewise, protection in an animal model was achieved using an RSV-F virosome adjuvanted with a TLR2 activator.151 More recently, an intranasally administered HIV vaccine based on an unadjuvanted gp41-containing influenza virosome formulation proved protective in the nonhuman primate SHIV (simian-HIV) model.145 It exploited the previously discussed specific combinations of chemokine receptors and mucosal addressins between the respiratory and the vaginal tracts. Interestingly, only a prime-boost regimen combining intramuscular prime followed by nasal administration resulted in full protection.145 This vaccine candidate was tested in a phase I clinical trial, notably in the absence of any safety concerns reported (http://www.mymetics.com/). Direct intravaginal immunization with virosome-based vaccines has been explored in the context of a vaccine against RVVC (recurrent vulvovaginal candidiasis).147,148 A novel formulation based on lyophilized rtSap2 IRIV was tested for vaginal administration. Four intravaginal administrations in minipigs led to a significant serum antibody response against the recombinant antigen, despite the lack of vaginal M-cells.147 Vaginal vaccination, however, resulted in slower antibody

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CONCLUSIONS Various strategies have been developed to enable the use of VLP and virosomes for mucosal vaccine administration. Their efficacy as a carrier system with or without additional adjuvant is illustrated by the various in vivo examples mentioned here. VLP vaccines are able to overcome natural barriers of mucosal surfaces, and feature various advantages and disadvantages (Table 8). For the same route of immunization, VLP vaccine Table 8. Advantages and Disadvantages of VLP as a Platform for Mucosal Vaccines advantages Generally good safety and tolerability profile Antigen protection and proper presentation to mammalian local immune system Local and systemic immune response, both by cellular and humoral effectors Inherent adjuvant effect

disadvantages Comparably weak immune response, often requiring additional mucosal adjuvant and/ or higher doses than injectable forms Locally induced immune response difficult to quantify Prime/boost regimen with parenteral injection may be required

efficiency will depend on individual host characteristics such as the local environment or the genetically based differences in immune response. Considering the number of successful studies in animals, VLP-based vaccines constitute a promising and challenging emergent platform for mucosal vaccination from both socioeconomical and immunological standpoints. Nevertheless, clinical mucosal administration of VLP remains rare (Table 9). This might be due to the absence of sufficiently predictive animal models in the preclinical phase, the weak immunogenicity of VLP vaccines in humans and the safety concern associated with the use of mucosal adjuvants, as illustrated by the HLT adjuvanted nasal influenza vaccines. Furthermore, there are significant regulatory hurdles to overcome. VLP vaccines assembled by expression systems require extensive purification to deplete host cell components.152 This is particularly challenging for enveloped VLP, where the envelope itself may contain host-derived impurities.153 Additional concerns include the stability of host cellexpressed VLP vaccines and batch-to-batch consistency, in particular regarding size-dependent immunogenicity.152 Due to the better assembly control and consistency, as well as simplified analytics, use of in vitro assembly systems based on purified components can address most of these concerns. The versatility of the VLP carrier system platform allows for the use of a broad spectrum of antigens, and is particularly suited for vaccination against mucosal pathogens. Clinical 1603

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Hemagglutinin; HBs: Hepatitis B surface; HIV: Human immunodeficiency virus; HLT: Heat-labile toxin; HPV: Human papillomavirus; IFN: Interferon; IgA: Immunoglobulin A; IL: Interleukin; i.m: Intramuscular; i.n: Intranasal; IRIV: Immunopotentiating reconstituted influenza virus; ISCOM: Immunostimulating complex; MadCAM-1: Mucosal addressin cell adhesion molecule 1; MALT: Mucosa-associated lymphoid tissues; M cells: Microfold cells; mLT: Mutant labile toxin; MPL: Monophosphoryl lipid A; NA: Neuraminidase; NALT: Nasal-associated lymphoid tissues; NV: Norovirus; PAMP: Pathogen-associated microbial pattern; PCPP: poly[di(carboxylatophenoxy) phosphazene]; pIgR: Polymeric immunoglobulin receptor; PP: Peyer’s patch; PRR: Pattern recognition receptor; PV: Parvovirus; RANTES: Regulated upon Activation, Normal T-cell Expressed, and Secreted; RSV: Respiratory syncytial virus; RV: Rotavirus; RVVC: Recurrent vulvo-vaginal candidiasis; SC: Membrane secretory component; s.c: Subcutaneous; S-IgA: Secretory immunoglobulin A; SHIV: Simian-HIV; SIV: Simian immunodeficiency Virus; TGF: Transforming growth factor; TIRIV: Tc-chol immunopotentiating reconstituted influenza virus; TLR: Toll-like receptor; TNBS: 2,4,6-trinitrobenzene sulfonic acid; VLP: Virus-like particles.

Table 9. Clinical Mucosal Vaccination Studies Involving VLP or Virosomes, According to the Administration Route administration route Oral

parental virus NV

Intranasal/bronchial/ pulmonary

Intrarectal

references 88,154,155

Clinical trial: NCT00806962 Clinical trial: NCT00001053

HIV

Clinical phase I

HPV NV

Clinical phase I Clinical phase I Clinical phase II

111

Launched and withdrawn Clinical phase I

143

IRIV Intravaginal

type of study Clinical phase I

rSap2 IRIV HIV VLP

Clinical phase I

40

Clinical trial: NCT00973284

Clinical trial: NCT01067131 Clinical trial: NCT00001053

acceptable strategies to potentiate mucosal VLP vaccine are built around the following factors: • Tropism defined by the parental virus: often, a higher VLP efficacy is observed when the targeted tissue is the natural portal of entry of the parental virus. • Formulation to prolong contact between VLP and the mucosa due to mucoadhesion seems to enhance the immune response. • Combination of accurately selected immunopotentiators or mucosal adjuvants with VLP in the same formulation. • Antigen dose as the initial amount of antigen presented at the VLP surface may play a role in the efficacy of the prime immunization. • Proximity of vaccine administration to the major inductive site. • Administration regimens of the vaccine having impact on the magnitude and the type of the VLP-induced immune response. Over the past two decades, VLP and nanoparticle-based technologies for injected vaccines have gained increasing interest in the field of vaccines. At the same time, technical and regulatory challenges have limited the progress of mucosal vaccination, despite of continued efforts and a convincing scientific rationale supporting this approach. VLP technologies may prove instrumental in the development of safe as well as efficacious mucosal vaccines, and the best way to achieve this goal may be to combine several previously described strategies.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*University of Geneva, 30, Quai Ernest Ansermet, 1211 Geneva, Switzerland. Tel.: +41 22 379 3802. Fax: +41 22 379 6567. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS APC: Antigen presenting cells; BV: Baculovirus; c-di-GMP: Cyclic diguanylate; CpG: Cytosine-phosphate-guanosine; CR: Chemokine receptor; CT: Cholera toxin; CTL: Cytotoxic lymphocyte T; DC: Dendritic cells; DNA: Deoxyribonucleic acid; GALT: Gut-associated lymphoid tissues; GFP: Green Fluorescent Protein; GMP: Good manufacturing practice; HA: 1604

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