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Influenza virosomes as a vaccine adjuvant and
carrier system. Expert Rev Vaccines
ARTICLE in EXPERT REVIEW OF VACCINES · APRIL 2011
Impact Factor: 4.21 · DOI: 10.1586/erv.11.15 · Source: PubMed

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Mario Amacker

Pevion Biotech

Mymetics SA

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Rinaldo Zurbriggen
Lonza
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Available from: Mario Amacker
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only very few are actually applied in licensed human vaccines. UK). This unique feature ensures a tight control of their composition and at the same time provides the flexibility to adapt the particle to various types of antigens. DCs express a wide range of innate sensor molecules and strongly influence the activation of antigen-specific B and T lymphocytes in reaction to innate stimuli [1. The Netherlands. Vaccine adjuvants can come to action at any stage of this process in order to amplify the desired type of immune response [1] . for sustained.6. in particular dendritic cells (DCs). In-depth knowledge of the mode of action of adjuvants provides a rational basis for risk assessments. a thorough safety assessment is also the key issue in regulatory guidelines for clinical testing and market approval of novel adjuvants [13–16] .15 mode of action of established adjuvants [3–8] . Solvay. and for the design of conclusive toxicology studies and clinical trials. differentiate and migrate between the administration site and lymphatic tissue within a time course of several weeks [1. recombinant virus capsids [18. they have a complex mode of action [3. 437–446 (2011) Christian Moser†1 Mario Amacker1 and Rinaldo Zurbriggen2 Pevion Biotech AG. antigen-specific effector and accessory cells.moser@pevion. Therefore.12] . play a central role in this process. London.19] . Structurally and functionally. It should be emphasized that safety concerns are more difficult to address in clinical trials than immuno­ genicity because severe side effects are not acceptable even at very low frequencies [11. potentially harmful or even immune suppressive. The goal of any vaccination is to generate populations of mature. Aside from their individual components. Keywords : adjuvant • antigen delivery • influenza • vaccines • virosomes The need for safe and efficacious vaccines has promoted the use of subunit antigens in order to remove antigenic components that are unrelated to protection. although they are assembled in vitro.11.17] . Belgium).2] .com 1 Influenza virosomes have been used for more than 10  years in commercial vaccines. Basel. Novel insights into the interplay between innate and adaptive immunity have not only led to more rational selection of novel adjuvant candidates. CH-3930 Visp.10] . the reduction in antigen complexity in combination with a high purity frequently impacts negatively on the immunogenicity.com/toc/erv/10/4 For reprint orders. CH-3063 Ittigen. but have also provided explanations for the www. Subunit antigens can be purified from the respective pathogens.com 10. which requires several distinct cell types to interact. AS03 and AS04 (GSK. Antigen-presenting cells (APCs).9. as illustrated by influenza vaccines.Review SPECIAL FOCUS y Adjuvants www. The induction of an adaptive immune response via vaccination is a highly complex process. but it © 2011 Expert Reviews Ltd ISSN 1476-0584 437 . their particle characteristics contribute to their function as adjuvants. vaccine adjuvants were identified empirically by evaluation of their potential to enhance immune responses against coadministered antigens. The technology has been further developed as a carrier and adjuvant system for subunit vaccines. including memory cells. Switzerland).expert-reviews. please contact reprints@expert-reviews. Brussels. long-lasting immunity. aside from aluminum salts. Switzerland † Author for correspondence: Tel. Vaccines 10(4). Historically.: +41 315 504 424 Fax: +41 315 504 445 christian. A wide variety of adjuvants are capable of enhancing the immunogenicity of weak antigens but. VLPs were defined as nonreplicating. in particular for synthetic peptides. virosomes are enveloped virus-like particles. Switzerland 2 Lonza AG.1586/ERV. Virus-like particles (VLPs) represent a distinct class of adjuvants. but recombinant proteins or synthetic peptides are increasingly being used. Leiden. and influenza virosomes (Crucell. Originally.com Influenza virosomes as a vaccine adjuvant and carrier system Expert Rev. such as specific ligands of innate danger and tissue damage sensors [3–5] . However. The mode of action of virosomes is complex and includes carrier as well as immune-stimulatory functions. and thus. The extensive amount of preclinical and clinical data supports the notion that influenza virosomes represent a platform technology that ensures robust and long-lasting immune responses against subunit antigens with an excellent safety profile. namely MF59 (Novartis.expert-reviews. Worblentalstrasse 32.

have been combined successfully with influenza virosomes in order to obtain immunogenic vaccine candidates  [48–56] . All these proteins are also present in influenza virosomes in similar ratios as in the parental virus. Retaining the authentic conformation and functionality of the viral proteins is a key aspect of the virosome formulation process.57] . Second.44. using influenza virosomes as a carrier and adjuvant system for heterologous subunit antigens [35–37] . The addition of charged lipids has proven useful to associate and deliver negatively charged molecules such as nucleic acids [39. detergent removal triggers the formation of empty membrane vesicles. approximately fivefold less neuraminidase tetramers. Common to all VLPs is their particulate structure that mimics a virus combined with their complete inability to replicate. for the purpose of functional studies or as vaccine candidates. the antigen is either formulated to be displayed on the surface or encapsulated in the lumen. For surface display. influenza and hepatitis A virus) and in a considerable number of prophylactic and therapeutic vaccine candidates in clinical development in the field of infectious diseases. the antigen of interest (AoI). In addition. The addition of phospholipids to the solubilized viral envelope fraction has proven essential for a robust. as exemplified by the pH-dependent fusion activity of HA [39. Conventional influenza virosomes have a limited stability unless stored at 2–8°C. These features ensure a high safety level combined with the advantages of a VLP structure enhancing the immune stimulation and the stability of the antigen. First. proteins and carbohydrates. This simple principle of assembling empty envelope particles in vitro is applicable to all enveloped viruses. most recently for HIV and respiratory syncytial virus [25. but their unique versatility soon promoted their use as carrier and adjuvant systems suitable for heterologous antigens. Virosome technology The concept of virosomes and the idea to use them as vaccines dates back to the 1970s [27. Amacker & Zurbriggen seems appropriate to expand the definition and to include viruslike structures derived from enveloped viruses [20. Depending on the preferred type of immunity. The dominance of the influenza virus as the substrate for virosome-based products may be explained by two aspects.45] .34] .21] . VLPs represented just another form of vaccine antigen. the formulation process including lyophilization and combination with other adjuvants. the purified and inactivated virus is dissolved in detergent. Unlike most VLPs. by the biological properties of the viral envelope proteins. since the density is a direct function of the protein:lipid ratio.Review Moser. a thorough quality control of virosomal vaccines goes far beyond content ana­lysis and includes particle characterization. with the virosome particle is a prerequisite for the full immune-enhancing function [49.28] . Notably. namely. influenza virus grown in cell culture is equally suited for the production of virosomes [201] . influenza virosomes remain the only virosome type that is applied in licensed human vaccines [33. the envelope fraction comprising the membrane-associated viral proteins and lipids is separated from the core complex containing the genetic material and internal proteins. As a consequence. including virosomes. In the past few years. The physico-chemical and biological properties of the resulting virosome particles can be modulated not only by the amount. The same material has proven suitable and cost-efficient 438 for the generation of influenza virosomes. covering multiple aspects of use as a vaccine platform and drug delivery system. The addition of lipids minimizes the loss of influenza components during detergent removal and ensures the assembly of a homogenous particle population that renders further purification steps obsolete [32] . and are generally manufactured in recombinant expression systems. immune-potentiating particle structure [3. Virus-like particles can be derived from a variety of nonenveloped and enveloped viruses. and by the availability of virus material. To date. A wide range of antigen types. These formulations are stable and retain their fusion activity in a broad temperature range [46. including peptides. as well as a verification of the fusion activity [40] .59] . it should be noted that the density of the envelope proteins on the virosome surface is reduced in comparison to the parental virus particle. Influenza virus was used in the early studies on virosomes [27. The envelope of influenza virus contains predominantly HA trimers. while influenza-derived VLPs.47] . Virosomes are spherical.29.28] . where they provide a higher.58] .30] . Third. Originally. human papillomavirus. with or without cationic lipids. lyophilization can stabilize labile subunit antigens. The physical association of the payload. The addition of sugars. scalable particle assembly step.40] .44. Influenza virosomes are protected by a considerable number of patents. Vaccines 10(4). In addition. are generally used as stand-alone products. cancer and allergy. antibody or cytotoxic T lymphocytes (CTL). was shown to protect the virosomes from the negative effects of freezing and to generate temperature-insensitive virosomes that can be lyophilized. several clinical trials have been performed. In the following years.31] and established as an industrial scale process compliant with good manufacturing practice (GMP) guidelines [32] . However. VLPs are applied in several licensed vaccines (hepatitis B virus.22–26] . respectively [40. by reconstituting lyophilized empty virosomes with a peptide solution [46] . and are particularly sensitive to freezing. Most VLP-based vaccines are combined with other adjuvant types such as alum salts and Toll-like receptor (TLR) agonists. and enables a novel approach to efficiently encapsulate small antigens like peptides. the production of influenza virosomes was further optimized [22. unilamellar vesicles composed of membrane lipids and integrated viral envelope proteins. and traces of M2e tetramers [38] . either Expert Rev.45. the AoI needs to be anchored in the lipid bilayer. (2011) . GMP-grade influenza virus is produced in embryonated chicken eggs every year for several hundred million doses of influenza vaccines. but also by the type of lipids added [41–43] . virosome particles are not produced by host cells but assembled in vitro in a three-step process from the parental virus. virosomes were shown to function as delivery system for nucleic acids and drugs [39. in particular of influenza hemagglutinin (HA).

both in vitro and in vivo [58. demonstrating that nanoparticles up to 200 nm in size and nonenveloped VLPs are detectable in APCs resident in the lymph node. Pre-existing immunity against influenza affects the carrier as well as the adjuvant functions.68] . TNF-a and GM-CSF were secreted at high levels rapidly after exposure to virosomes. AoI on the virosome surface are processed within the late endosome and presented in the context of MHC class II to generate T-helper cells. in vitro stimulation with virosomes [62] . The VLP structure facilitates a rapid uptake by APCs.48] . After uptake by APCs. Further investigations are needed to clarify whether virosomes can directly activate DCs.64] . cell type present in PBMC preparations. although this cell line is highly responsive to both DNA and RNA. Nucleic acids are degraded by treatment with b-propiolactone.65] . VLP: Virus-like particle. Schematic representation of the overlapping functions of virosomes to highlight the central role of the VLP structure. This feature is of particular www. Antibodies interact directly with the particle structure. Notably. Therefore. efficient encapsulation is achieved by a specifically designed formulation process  [46. The carrier functions result predominantly from the particle structure. Carrier functions The carrier function is related to the particle structure and plays a key role in the early events after vaccine administration.49] . the encapsulated AoI is released into the cytoplasm.expert-reviews. By contrast. pre-existing immunity significantly impacts on the adjuvant function of virosomes [40. After uptake by APCs. and quench the immune response against the AoI [75–77] .Influenza virosomes as a vaccine adjuvant & carrier system via a hydrophobic protein domain in the AoI. Within the lymph node. and proliferation of influenza-specific memory T cells in PBMCs obtained from adult donors but not from cord blood [61. but larger particles were found only in macrophages originating from the injection site [66] . Virosomes are rapidly taken up by cells. As a result. However. Virosomes have been shown to stimulate human peripheral blood mononuclear cells (PBMCs) in vitro. GM-CSF. IFN-g and IL-2 but not IL-4) consistent with a Th1 profile. Therefore. no nucleic acids are detectable in virosomes. and if so. Antigens of interest displayed on the outer surface of the virosomes in a repetitive array are optimal to prime B lymphocytes for antibody production. The adjuvant functions relate to both the particle structure and the individual virosome components with stimulatory effects and synergistic potential. virosomes are thought to interact with B cells as intact particles. Adjuvant functions Role of pre-existing immunity The adjuvant effect of virosomes relates to their ability to enhance antigen processing and presentation by APCs. the pH-dependent fusion activity of HA enables virosomes to act as cytoplasmatic delivery vehicles. suggesting that these cytokines at least do not originate from the proliferating memory cells but from a more abundant. recombinant influenza HA was reported to activate both murine and human DCs in vitro  [71–73] .17. PAMP: Pathogen-associated molecular pattern. as it has been shown for other viruses and particulate antigens [67.60–62] . In addition. so far unidentified. For AoI encapsulated in the lumen. resulting in cytokine secretion (TNFa.com 439 .74] . while cellular immunity expands the pool of T-helper cells. Accordingly.62. When administered via intramuscular injection. the repetitive display of the AoI on the virosome surface acts as a strong activation signal for antigen-specific B cells via cross-linking of immuno­globulin receptors [69. By contrast. Carrier Review Adjuvant Delivery Antigen protection Repetitive antigen display PAMP signals Costimulation VLP structure APC targeting CD4+ help Pre-existing immunity Antibodies T-cell memory Figure 1. which is performed to inactivate the influenza virus prior to virosome assembly.70] . by which mechanisms.48. Direct activation of DCs by virosomes was demonstrated in the murine system  [63] . CTL induction critically depends on the fusion activity of HA [63] . the acidification within the late endosome triggers HA-mediated fusion between the virosomal and endosomal membrane.23. the immune potentiating effect of influenza virosomes is enhanced by pre-existing immunity against influenza. The free draining hypothesis is supported by a recent report. or in association with migrating cells. virosomes can access the draining lymph node via free lymph drainage. Mode of action Virus-like particles including virosomes exert their effects on APCs and on antigen-specific lymphocytes via their overall particle structure and their individual components [3. but does not require integration of the AoI into the lipid membrane. The embedding of the AoI in the virosome particle can also protect it from premature degradation by extracellular proteases. thereby providing access to MHC class I presentation and CTL induction [46. virosomes represent an excellent example for a multifunctional carrier and adjuvant system (Figure 1). APC: Antigen-presenting cell. while a human plasmacytoid DC cell line did not increase expression of activation markers upon Pre-existing immunity against the carrier proteins can impair the function of viral vectors and VLPs. Multifunctional mode of action of virosomes. or via a synthetic phospholipid conjuguated to the AoI.

80] . Four vaccines based on the virosome technology are licensed for commercial use. the antibodies need neither virus neutralizing nor hemagglutination inhibiting capacity. In the presence of pre-existing immunity. is considered the main cause for cell-mediated immune enhancement. By contrast. Without truly naive subjects available as negative controls. (2011) . influenza-specific CD4 + memory cells can significantly contribute to the differentiation and proliferation of AoI-specific effector cells. ELISA titer importance since pre-existing immunity against influenza is widespread among the human population and can be detected in nearly every individual [74. At day 21 and day 42.78. respectively (anti-malaria d42 and anti-malaria d43). Anti-influenza antibodies are thought to target virosomes to Fc receptors of APCs.Review Moser. before the first immunization with the virosomal peptide vaccine (anti-Virosome A d21) and. Further preclinical and clinical studies are necessary to dissect the contributions 10 of the different elements and to determine None Influenza A Influenza B Virosome A whether the positive effect directly correlates Pre-immunization with the magnitude of pre-existing immunity or rather requires threshold levels of Figure 2. A speculative explanation for the lack of enhanced CTL responses is interference of antibodies with the HA function necessary for the cytoplasmatic delivery. Amacker & Zurbriggen Reactivation of influenza-specific memory cells. including the influenza proteins. these cells process and present all virosome components in the context of MHC class II. since immunity against influenza B had the same positive effect as the homologous influenza A/H1N1 strain used to assemble the virosomes (Figure 2) . the animals were immunized with a virosomal vaccine composed of a malaria‑derived peptide and virosomes assembled from influenza A. 440 Clinical experience with virosomes Over the last two decades.000 peptides formulated on virosomes [79] . Positive effect of pre-existing immunity against influenza is strain pre-existing humoral and cellular immunity. influenza B virus or virosomes made from homologous influenza A. Preclinical studies in mice have clearly demonstrated that pre-existing immunity against influenza enhances the antibody response against unrelated antigens administered subsequently in the context of virosomes [49] . as it has been clearly demonstrated in animal 100 models [49] . For that reason. Mere binding to any of the influenza proteins present on virosomes is sufficient. a considerable amount of clinical data has been generated with influenza virosomes.79] . while over 70 million doses of Crucell’s influenza and hepatitis A vaccines have been distributed Expert Rev. it appears extremely difficult to clinically verify the positive cor1000 relation between baseline anti-influenza immunity and response to the AoI. independent. However. second. The hemagglutination inhibition (HI) titer against the virus strain used in virosomes A/Singapore/6/86 (H1N1) was measured in several clinical trials.000 was detectable between either pre-existing Anti-malaria d42 cellular immunity against influenza and Anti-malaria d63 the response against the malaria-derived 10. thereby excluding at least a negative effect [33. BALB/c mice were pre-immunized at day 0 with either influenza A/H1N1 virus. The enhancer effect is strain independent. these cells can obtain their signal 2 not only from antigen-specific but also from influenza-specific CD4 + T-helper cells. which might be sufficient to compensate for the positive effects of improved APC targeting. Data provided by Pevion Biotech AG (Ittigen. against influenza A virosomes at day 21. B cells specific for the AoI are activated upon contact with the antigen displayed on the surface of virosomes. influenza-specific CD4 + T cells are more abundant and faster activated than naive CD4 + T cells reactive to the AoI. Vaccines 10(4). no conclusive clinical data are available on the positive effect of pre-existing immunity on the response against a heterologous AoI applied in the context of virosomes.48] . no significant correlation was found between the response against hepatitis A virus and the baseline HI titer against influenza. Therefore. Therefore. the magnitude of CTL responses against virosome-encapsulated peptides was not significantly increased by pre-existing immunity against influenza [46. In a situation of pre-existing immunity. the value of these data is limited because HI titers account for neither humoral immunity against heterologous influenza strains nor for any cellular immunity. Subsequently. as demonstrated in human PBMCs [74] . Antibody titers were determined by ELISA: first. Consistently. No correlation Anti-virosome A d21 100. thereby accelerating and enhancing uptake by APCs. Both humoral and cellular elements of pre-existing immunity against influenza are thought to contribute to the immuneenhancing effects. against the malaria peptide 3 weeks after the first and second immunization with the virosomal peptide vaccine. Switzerland). For this purpose. a single immunization induces antipeptide titers comparable to those achieved with two immunizations in naive animals. Multiple infections by constantly drifting virus strains result in a complex immunity against influenza in the human population. This hypothesis is consistent with the observed strain-type independence.

92] . five novel product candidates have been tested in clinical trials or are part of ongoing studies. when compared with alum-adju. A(H3N2) and B [34] The hepatitis A vaccine. Therefore. Belgium) A(H1N1).101. the basic building blocks of virosomes. Hepatitis A vaccines Review Table 1.Biotech.100] [Pevion Biotech AG. namely the influenza proteins themselves. Ref. Switzerland) Epaxal and the follow-up product Epaxal Junior were character. Inflexal®V (Crucell) is a triva+ A(H1N1) virosomes Unpublished Data] lent seasonal influenza vaccine successfully [99. Epaxal (Crucell). unclear [88] . Epaxal® (Crucell. Notably. Since 2005. influenza strain A/Singapore/6/86 (H1N1) Brussels. the product was only commercially available during the When the virosome concept is applied to influenza vaccines. Leiden. Product Vaccine composition Launched (year) Ref. + A(H1N1) virosomes safe but insufficiently that the virosome formulation did not sig. immunoge. but did [35] improve tolerability [34. the assembly Malaria of virosomes provides the antigens a VLP structure and native Based on two synthetic peptides.302] Candida One recombinant Phase I initiated 2010 marketed since 1997 [34] .Vaccines in clinical development tional. The Epaxal® Junior (Crucell) A(H1N1) virosomes + inactivated [33.92] Virosomes from three influenza strains 2004. The product is albicans protein + A(H1N1) a blend of three different virosome parvirosomes ticles. A(H1N1).96] role of virosome is limited to providing Malaria Two peptides Three Phase I/II trials with bivalent + A(H1N1) virosomes vaccine completed: the antigen a particle structure. A(H3N2) and B + heat labile withdrawn 2001 toxin adjuvant enza virosome technology. A(H3N2) and B not on the market since 2005 and coated after assembly with the inactivated hepatitis A virus as the AoI. but vanted competitor products. The Netherlands) A(H1N1) virosomes + inactivated hepatitis A virus 1994 [33. when applied to influenza vaccines. In 2004. influenza strain [32] . the Target Vaccine composition Status [36. an experimental presence of influenza proteins. Nasalflu® (Berna Biotech.com 441 . and induced text of influenza vaccines. represent the AoI.80] 2008 same virus strain has been used for all subhepatitis A virus sequent applications of influenza virosomes as carrier and adjuvant system for heterologous antigens. and a clear trend towards improved immunogenicity [80. Influenza vaccines However.95. Solvay launched Invivac®. such as stimulatory signals and challenge study and a Phase I trial in an endemic area (Tanzania). The virosomes Switzerland) in this product are generated from the Invivac® (Solvay.79. Both aspects are expected to improve the immuno­ cine was developed to induce protective antibodies against sporozological properties  [59. which ized in numerous clinical studies with regard to safety. The vaccine has been tested in somes for heterologous antigens.94] . contain the same components. all of them based on subunit antigens in the form of synthetic peptides or recombinant proteins (Table 2) . safe and immunogenic Comparative clinical studies with conventional subunit vaccines demonstrated Hepatitis Three peptides Phase I completed: [46.mucosal adjuvant  [89] . as the first vaccine on the basis of the influ.82] .expert-reviews.was a virosomal influenza vaccine for intranasal application. By contrast to the application of viro. do not come into play in the con. including a Phase I safety trial. Bern.90] Virosomes from three influenza strains 2000. a trivalent seasonal influenza vaccine for intramuscular injection based on the virosome concept [91. was approved for commercial use in 1994 Nasalflu® (Berna [89. each assembled separately from one ® www. nonparticulate subunit influenza vaccines. season 2004/2005.Influenza virosomes as a vaccine adjuvant & carrier system so far (Table 1) . Licensed vaccines based on virosomes.37. the virosomal products demonstrated withdrawn from the market due to an association between heat in several independent studies a significantly better tolerability [82–85] labile toxin adjuvanted vaccination and Bell’s palsy [90] .87] .86] . Virosomes and conventional subunit vaccines essentially Table 2. [91. a bivalent malaria virosomal vacpresentation. The product was launched in 2000.contained heat labile toxin from Escherichia coli as an additional nicity and efficacy [33.81] . Whether the lat.81] Inflexal® V (Crucell) Virosomes from three influenza strains 1997 A(H1N1). expanded CD4 + T-cell help. Compared to conven.ite and blood-stage parasites [93.C virus Unpublished Data] immunogenic nificantly increase immunogenicity.The vaccine proved to be safe and immunogenic. Virosome-based vaccines in clinical development. the positive effects related to the three clinical trials. Bern.Breast Three peptides Phase I completed: + A(H1N1) virosomes safe and immunogenic ter results from the particle structure or from cancer the higher purity of the product remains HIV One peptide Phase I initiated 2010 [301] [Pevion Biotech AG.

but there is sufficient data to declare them multifunctional. which saves time and increases reproducibility. a virulence factor of Candida albicans  [99] . In the Phase I clinical trial the vaccine was well tolerated and safe but failed to induce satisfactory levels of T-cell responses [Pevion Biotech. this response was achieved with only 10 µg of each peptide. illustrated by lower rates of parasite growth and by appearance of morphologically abnormal parasites in vaccinated volunteers. Unlike most VLPs. Candida The Candida vaccine aims to prevent recurrent vulvovaginal candidiasis. Hepatitis C virus The virosomal hepatitis C virus vaccine was designed as a therapeutic T-cell vaccine to clear chronic hepatitis C virus infection. the mode of action of adjuvants has become a central theme in vaccinology. which in turn raise safety issues on their own. The third study demonstrates the safety and immunogenicity results for the lyophilized vaccine formulation. Antibodies were induced even in elderly breast cancer patients. However. and both routes are currently being tested with a monovalent peptide vaccine in a Phase I clinical trial for safety and immunogenicity [301] . truncated form of the Sap2 protein. Expert Rev. or indicates a limitation of the virosome technology. Encouraging preclinical results with a virosome formulation containing three synthetic peptides warranted a Phase I clinical trial. Breast cancer The B-cell vaccine was designed to induce antibodies against Her2. The clinical proof of concept for virosomal subunit vaccines represents the next level of validation. and the impact of pre-existing immunity. recognizing not only the peptides in the vaccine but also the fulllength Her2/neu protein. It remains unclear whether this shortcoming is due to the peptide design. The mode of action of adjuvants will remain a key topic in vaccinology. Novel antigens and targets for the virosome technology are continuously being evaluated at the preclinical stage and are likely to enter the clinical development phase in the near future. The vaccine presentation in a capsule for intravaginal application represents a novelty with advantages regarding delivery and patient compliance. and a peptide acting as a CD4 epitope displayed on the virosome surface. driven by increasingly detailed insights into the underlying biological mechanisms. owing to their generally poor immunogenicity. Modifications of the particle structure or inclusion of additional compounds can be achieved independently of a host cell system.37. thereby expanding the antigen spectrum from synthetic peptides to larger recombinant proteins. The vaccine included two peptides representing CTL epitopes. Unpublished Data] . Amacker & Zurbriggen long-lasting functional antibody titers [36. Over the past 5 years. (2011) . HIV The concept of the prophylactic HIV vaccine is to induce a protective mucosal immunity that has been shown to efficiently prevent infection [98] . in particular when based on synthetic peptides and recombinant proteins. which emphasizes the safety and efficacy of the technology [33. the vaccine induced antibodies in eight out of ten breast cancer patients [35] . Preclinical models demonstrated induction of both peptide-specific CTL and CD4 + lymphocytes [46] . Candida and HIV). Five-year view A further validation of the virosome technology can be expected from ongoing clinical studies with new targets (e. notably against low dosages of peptides derived from self-antigen [35] . Notably. Lyophilized virosome formulations for intramuscular and intravaginal application were developed with the intention to induce mucosal immunity via a systemic prime/mucosal boost regime. Beyond meeting the safety end points. The liquid formulation was designed for intramuscular as well as mucosal administration. Regarding virosomes.95] . resulting in the reconstitution of the lyophilized vaccine in vivo.g. However.Review Moser. Subunit vaccines meet these requirements. efficient. a target validated by the established passive immuno­therapy with Trastuzumab [97] . specifically the demonstration of protective efficacy in humans. and novel insights can be expected on a regular basis. encapsulated in virosomes. 15 years of clinical experience and 70 million doses distributed to date. as it mimics the broad range of stimulatory signals mediated by a natural infection. The vaccine is based on a recombinant.34] . a solid clinical track record for virosomes has been established. subunit vaccines frequently need to be combined with adjuvants.36. this complexity might be the key to a balanced.. comparable to the previous studies performed with the liquid.95] . Thus. virosomes are assembled in vitro in a tightly controlled process. Although difficult to investigate. In addition. The vaccine is currently being tested in a Phase I clinical trial for safety and immunogenicity [302] . The capsule is designed to dissolve upon administration. the T-cell vaccine approach for hepatitis C virus failed to induce robust responses in humans despite encouraging preclinical data [46] . temperature sensitive form of the vaccine [96] .79. with a strong focus on safety and product definition. With three products on the market. but well-tolerated immune stimulation. 442 Expert commentary The regulatory hurdles for clinical testing and market licensure of novel vaccines have become increasingly stringent. The knowledge of the mode of action of influenza virosomes remains fragmented. early stage clinical studies have demonstrated that the same virosome technology is a safe and potent adjuvant system for synthetic peptides designed as B-cell vaccines against malaria and breast cancer [35. The successful development of a temperature-insensitive. The challenge study showed evidence of blood-stage efficacy [37] . directly on the mucosal surface. both approaches include a mucosal route of application and may thus provide further information on the potential of virosomes as a mucosal adjuvant system. lyophilized form of virosomes illustrates the flexible application of the system [46] . The candidate vaccine is composed of subunit antigens derived from HIV gp41 displayed by virosomes. Vaccines 10(4). further investigations on the mode of action are needed to better define the carrier and adjuvant functions.

Vaccine delivery: a matter of size. 492–503 (2010). 537–545 (2007). Valiante NM. Anderson DA. 18 Jennings GT. Curr. Comprehensive review of various classes of vaccine adjuvants and their mode of action. 16 The European Agency for the Evaluation of Medicinal Products. 4th meeting on novel adjuvants currently in/close to human 443 . S2–S7 (2006). Just M. Influenza virus-like particle vaccines. Ulmer JB. Immunol. Mosca F. Springer. 20 Haynes JR. In: Vaccine Adjuvants. composed of purified or synthetic phospholipids and the envelope fraction of influenza virus. 23–25 June 2003. kinetics and molecular patterns. Cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome. Nat. EMA. Kieny MP. Papers of special note have been highlighted as: • of interest •• of considerable interest 1 • 2 3 Ribeiro CMS. 60–65 (2006). 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