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Introduction: Cotton rats are a suitable model for the study of influenza disease symptoms and responses to influenza vaccination. We have previously shown that two immunizations with 15 µg whole inactivated virus (WIV) influenza vaccine could completely protect animals from infection with the H1N1pdm09 virus. Methods: To further explore the cotton rat model, we here investigated the protective potential of a single intramuscular immunization and of prime/boost intramuscular immunizations with a low amount of antigen. Results: A single intramuscular immunization with doses more than or equal to 0.5 µg WIV reliably evoked antibody responses and doses more than or equal to 1 µg protected the animals from virus replication in the lungs and from severe weight loss. However, clinical symptoms like an increased respiration rate were still apparent. Administration of a booster dose significantly increased the humoral immune responses but did not or only moderately improved protection from clinical symptoms. Conclusion: Our data suggest that complete and partial protection by influenza vaccines can be mimicked in cotton rats by using specific vaccination regimens.

Current influenza vaccines mainly induce antibody responses to the variable hemagglutinin proteins of the virus strains included in the vaccine. Instead, a broadly protective influenza vaccine should aim at inducing antibody and/or cell -mediated immunity against conserved viral proteins. Vacc-FLU is a peptide based vaccine combining conserved B and T cell epitopes. Peptide selection was done using a proprietary peptide design platform technology focusing on responses to human leukocyte antigen (HLA)-restricted epitopes. Immunization of wild -type mice and mice transgenic for HLA-A2.1 with the peptide mix successfully induced both humoral and cell mediated immune responses. Partial protection from severe weight loss upon challenge was observed in both mouse strains but was stronger and observed at lower vaccine doses in transgenic mice. Our results show that the Vacc-FLU peptide mix is capable of inducing IFNy-producing T cells and antibody-producing B cells which can protect from severe disease symptoms upon infection.

Adjuvanted whole inactivated virus (WIV) influenza vaccines show promise as broadly protective influenza vaccine candidates. Using WIV as basis we assessed the relative efficacy of different adjuvants by carrying out a head-to-head comparison of the liposome-based adjuvants CAF01 and CAF09 and the protein-based adjuvants CTA1-DD and CTA1-3M2e-DD and evaluated whether one or more of the adjuvants could induce broadly protective immunity. Mice were immunized with WIV prepared from A/Puerto Rico/8/34 (H1N1) virus intramuscularly with or without CAF01 or intranasally with or without CAF09, CTA1-DD, or CTA1-3M2e-DD, followed by challenge with homologous, heterologous or heterosubtypic virus. In general, intranasal immunizations were significantly more effective than intramuscular immunizations in inducing virus-specific serum-IgG, mucosal-IgA, and splenic IFNγ-producing CD4 T cells. Intranasal immunizations with adjuvanted vaccines afforded strong cross-protection with milder clinical symptoms and better control of virus load in lungs. Mechanistic studies indicated that non-neutralizing IgG antibodies and CD4 T cells were responsible for the improved cross-protection while IgA antibodies were dispensable. The role of CD4 T cells was particularly pronounced for CTA1-3M2e-DD adjuvanted vaccine as evidenced by CD4 T cell-dependent reduction of lung virus titers and clinical symptoms. Thus, intranasally administered WIV in combination with effective mucosal adjuvants appears to be a promising broadly protective influenza vaccine candidate.

BACKGROUND: Induction of CD8+ cytotoxic T cells (CTLs) to conserved internal influenza antigens, such as nucleoprotein (NP), is a promising strategy for the development of cross-protective influenza vaccines. However, influenza NP protein alone cannot induce CTL immunity due to its low capacity to activate antigen-presenting cells (APCs) and get access to the MHC class I antigen processing pathway.METHODS: To facilitate the generation of NP-specific CTL immunity we developed a novel influenza vaccine consisting of virosomes with the Toll-like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) and the metal-ion-chelating lipid DOGS-NTA-Ni incorporated in the membrane.RESULTS: In vitro, virosomes with incorporated MPLA induced stronger activation of APCs than unadjuvanted virosomes. Virosomes modified with DOGS-NTA-Ni showed high conjugation efficacy for his-tagged proteins and facilitated efficient uptake of conjugated proteins by APCs. Immunization of mice with MPLA-adjuvanted virosomes with attached NP resulted in priming of NP-specific CTLs while MPLA-adjuvanted virosomes with admixed NP were inefficient in priming CTLs. Both vaccines induced equally high titers of NP-specific antibodies. When challenged with heterosubtypic influenza virus, mice immunized with virosomes with attached or admixed NP were protected from severe weight loss. Yet, unexpectedly, they showed more weight loss and more severe disease symptoms than mice immunized with MPLA-virosomes without NP.CONCLUSIONS: Taken together, these results indicate that virosomes with conjugated antigen and adjuvant incorporated in the membrane are effective in priming of CTLs and eliciting antigen-specific antibody responses in vivo. However, for protection from influenza infection NP-specific immunity appeared not to be advantageous.

The skin is an attractive organ for immunization because of the presence of antigen-presenting cells. Intradermal delivery via tattooing has demonstrated superior vaccine immunogenicity of DNA vaccines in comparison to conventional delivery methods. In this study, we explored the efficacy of tattoo injection of a tumor vaccine based on recombinant Semliki Forest virus replicon particles (rSFV) targeting human papillomavirus (HPV). Tattoo injection of rSFV particles resulted in antigen expression in both the skin and draining lymph nodes. In comparison with intramuscular injection, the overall antigen expression determined at the site of administration and draining lymph nodes was 10-fold lower upon tattoo injection. Delivery of SFV particles encoding the E6 and E7 antigens of human papillomavirus type 16 (SFVeE6,7) via tattooing resulted in HPV-specific cytotoxic T cells and in vivo therapeutic antitumor response. Strikingly, despite the observed lower overall transgene expression, SFVeE6,7 delivered via tattoo injection resulted in higher or equal levels of immune responses as compared to intramuscular injection. The intrinsic immunogenic potential of tattooing provides a benefit for immunotherapy based on an alphavirus.

Adjuvants are key components in vaccines, they help in reducing the required antigen dose but also modulate the phenotype of the induced immune response. We previously showed that GPI-0100, a saponin-derived adjuvant, enhances antigen-specific mucosal and systemic antibody responses to influenza subunit and whole inactivated influenza virus (WIV) vaccine administered via the pulmonary route. However, the impact of the GPI-0100 dose on immune stimulation and the immune mechanisms stimulated by GPI-0100 along with antigen are poorly understood. Therefore, in this study we immunized C57BL/6 mice via the pulmonary route with vaccine consisting of WIV combined with increasing amounts of GPI-0100, formulated as a dry powder. Adjuvantation of WIV enhanced influenza-specific mucosal and systemic immune responses, with intermediate doses of 5 and 7.5 μg GPI-0100 being most effective. The predominant antibody subtype induced by GPI-0100-adjuvanted vaccine was IgG1. Compared to non-adjuvanted vaccine, GPI-0100-adjuvanted WIV vaccine gave rise to higher numbers of antigen-specific IgA- but not IgG-producing B cells in the lungs along with better mucosal and systemic memory B cell responses. The GPI-0100 dose was negatively correlated with the number of influenza-specific IFNγ- and IL17-producing T cells and positively correlated with the number of IL4-producing T cells observed after immunization and challenge. Overall, our results show that adjuvantation of pulmonary-delivered WIV with GPI-0100 mostly affects B cell responses and effectively induces B cell memory.

An absolute prerequisite for a therapeutic vaccine against hepatitis C virus (HCV) infection is the potency to induce HCV-specific vigorous and broad-spectrum T-cell responses. Here, we generated three HCV vaccines based on a recombinant Semliki Forest virus (rSFV) vector expressing all-or a part of the conserved nonstructural proteins (nsPs) of HCV. We demonstrated that an rSFV vector was able to encode a transgene as large as 6.1 kb without affecting its vaccine immunogenicity. Prime-boost immunizations of mice with rSFV expressing all nsPs induced strong and long-lasting NS3-specific CD8(+) T-cell responses. The strength and functional heterogeneity of the T-cell response was similar to that induced with rSFV expressing only NS3/4A. Furthermore this leads to a significant growth delay and negative selection of HCV-expressing EL4 tumors in an in vivo mouse model. In general, as broad-spectrum T-cell responses are only seen in patients with resolved HCV infection, this rSFV-based vector, which expresses all nsPs, inducing robust T-cell activity has a potential for the treatment of HCV infections.

Prophylaxis against influenza could be improved by the development of a stable, easy to deliver, potent mucosal vaccine. In this study, we spray-freeze-dried (SFD) whole inactivated virus influenza vaccine (WIV) alone or supplemented with monophosphoryl lipid A (MPLA) using inulin as a lyoprotectant. Physical characterization revealed that the SFD powder consisted of highly porous particles with a size distribution suitable for pulmonary administration. The receptor-binding properties of WIV and the immunostimulatory properties of MPLA were preserved after spray-freeze-drying as indicated by unchanged hemagglutination titers and a retained ability of the vaccine to activate NFkB after incubation with a reporter cell line, respectively. Pulmonary vaccination of mice with MPLA-adjuvanted liquid or powder WIV resulted in induction of higher mucosal and systemic antibody concentrations than vaccination with non-adjuvanted formulations. When exposed to influenza virus, mice immunized with MPLA-adjuvanted pulmonary vaccine showed similar protection in terms of reduction in lung virus titers and prevention ofweight loss as mice immunized intramuscularly with subunit vaccine. Characterization of the antibody response revealed a balanced IgG2a-to-IgG1 profile along with induction of both memory IgA- and IgG-producing B cells in mice immunized with MPLA-adjuvanted vaccine. These studies suggest that themucosal and systemic immune responses to pulmonary delivered influenza vaccines can be significantly enhanced by using MPLA as adjuvant. MPLA-adjuvanted SFD vaccine was particularly effective implying that delivery of adjuvanted vaccine powder to the lungs can be an attractive way of immunization against influenza. (C) 2013 Elsevier B.V. All rights reserved.

Background Non-replicating respiratory syncytial virus (RSV) vaccine candidates could potentially prime for enhanced respiratory disease (ERD) due to a T-cell-mediated immunopathology, following RSV infection. Vaccines with built-in immune response modifiers, such as Toll-like receptor (TLR) ligands, may avoid such aberrant imprinting of the immune system. Methods We developed reconstituted RSV envelopes (virosomes) with incorporated TLR4 ligand, monophosphoryl lipid A (RSV-MPLA virosomes). Immune responses and lung pathology after vaccination and challenge were investigated in ERD-prone cotton rats and compared with responses induced by live virus and formaldehyde-inactivated vaccine (FI-RSV), a known cause of ERD upon RSV challenge. Results Vaccination with RSV-MPLA virosomes induced higher levels of virus-neutralizing antibodies than FI-RSV or live virus infection and provided protection against infection. FI-RSV, but not RSV-MPLA virosomes, primed for increases in expression of Th2 cytokines IL-4, IL-5, IL-13, and Th1 cytokine IL-1b, 6 hour–5 days after infection. By contrast, RSV-MPLA virosomes induced IFN-γ transcripts to similar levels as induced by live virus. Animals vaccinated with FI-RSV, but not RSV-MPLA virosomes showed alveolitis, with prominent neutrophil influx and peribronchiolar and perivascular infiltrates. Conclusion These results show that RSV-MPLA virosomes represent a safe and immunogenic vaccine candidate that warrants evaluation in a clinical setting.

One of the advantages of dry influenza vaccines over conventional liquid influenza vaccines is that they can be used for alternative routes of administration. Previous studies showed that spray freeze-drying is an excellent technique to prepare vaccine containing powders for pulmonary delivery U.P. Amorij, V. Saluja, A.H. Petersen, W.L.J. Hinrichs, A. Huckriede, H.W. Frijlink, Pulmonary delivery of an inulin-stabilized influenza subunit vaccine prepared by spray-freeze drying induces systemic, mucosal humoral as well as cell-mediated immune responses in BALB/c mice, Vaccine 25 (2007) 8707-8717; S.A. Audouy, G. van der Schaaf, WIT Hinrichs, H.W. Frijlink, J. Wilschut, A. Huckriede. Development of a dried influenza whole inactivated virus vaccine for pulmonary immunization, Vaccine (2011)). The aim of this study was to investigate the physical and immunogenic stability of spray freeze-dried whole inactivated virus influenza vaccine prepared by using inulin, dextran, and a mixture of dextran and trehalose as protectants. Physical and biochemical characteristics of the vaccine powder' were maintained at temperatures up to 30 degrees C for 3 months. In addition, in vivo data indicate that also, the immunogenic properties of the vaccine were maintained under these storage conditions. On the other hand, in vivo results also revealed that subtle changes in powder characteristics were induced during storage at 30 degrees C. However, laser diffraction measurements showed that problems associated with these subtle changes can be overcome by using dry powder inhalers with an efficient powder dispersing capacity. (C) 2013 Elsevier B.V. All rights reserved.

Respiratory syncytial virus infection remains a serious health problem, not only in infants but also in immunocompromised adults and the elderly. An effective and safe vaccine is not available due to several obstacles: non-replicating RSV vaccines may prime for excess Th2-type responses and enhanced respiratory disease (ERD) upon natural RSV infection of vaccine recipients. We previously found that inclusion of the Toll-like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) in reconstituted RSV membranes (virosomes) potentiates vaccine-induced immunity and skews immune responses toward a Th1-phenotype, without priming for ERD. As mucosal immunization is an attractive approach for induction of RSV-specific systemic and mucosal antibody responses and TLR ligands could potentiate such responses, we explored the efficacy and safety of RSV-MPLA virosomes administered intranasally (IN) to mice and cotton rats. In mice, we found that incorporation of MPLA in IN-administered RSV virosomes increased both systemic IgG and local secretory-IgA (S-IgA) antibody levels and resulted in significantly reduced lung viral titers upon live virus challenge. Also, RSV MPLA virosomes induced more Th1-skewed responses compared to responses induced by FI-RSV. Antibody responses and Th1/Th2-cytokine responses induced by RSV-MPLA virosomes were comparable to those induced by live RSV infection. By comparison, formalin-inactivated RSV (FI-RSV) induced serum IgG that inhibited viral shedding upon challenge, but also induced Th2-skewed responses. In cotton rats, similar effects of incorporation of MPLA in virosomes were observed with respect to induction of systemic antibodies and inhibition of lung viral shedding upon challenge, but mucosal sS-IgA responses were only moderately enhanced. Importantly, IN immunization with RSV-MPLA virosomes, like live virus infection, did not lead to any signs of ERD upon live virus challenge of vaccinated animals, whereas IM immunization with FI-RSV did induce severe lung immunopathology under otherwise comparable conditions. Taken together, these data show that mucosally administered RSV-MPLA virosomes hold promise for a safe and effective vaccine against RSV. (C) 2013 Elsevier Ltd. All rights reserved.

The recombinant Semliki Forest virus (rSFV) encoding human papilloma virus (HPV)-E6,7 tumor antigens induces both strong, longlasting CTL responses in a mouse model of cervical carcinoma and effective eradication of established tumors of HPV-transformed cells. Current therapeutic approaches of cervical cancer patients with advanced disease include radiotherapy combined with chemotherapy. Using a large tissue micro array consisting out of primary tumor tissue of 375 patients with advanced disease we studied the role of tumor infiltrating lymphocytes. Target specific therapies, like immunotherapy, might optimize treatment outcome of these patients. To achieve optimal benefit from immunization protocols, strategies need to be developed that support optimal migration and activity of cytotoxic T lymphocytes (CTLs) into the tumor microenvironment. We developed a method allowing us to assess the trafficking of tumorinduced, adoptively transferred as well as vaccine-induced antigenspecific CTLs into tumors. To obtain antigen-specific CTLs, donor mice were immunized with (rSFV) encoding HPV-E6,7 tumor antigens. In vitro restimulated E6,7-specific CTLs were CFSE-labeled and adoptively transferred into TC-1 (HPV-transfected) tumor-bearing recipient mice four days after local tumor irradiation. Homing of both tumor-induced effector T cells and adoptively transferred E6,7-specific CTLs to TC-1 tumors was analyzed one day after transfer. Local tumor irradiation induced a significant increase in intratumoral levels of adoptively transferred E6,7-specific CTLs. A similar effect was observed in the infiltration of tumor-induced effector T cells and MDSCs (myeloid derived suppressor cells), when compared with nonirradiated tumors. To assess the effect of irradiation on tumor trafficking of vaccine-induced antigen-specific CTLs, TC-1 tumor bearing mice were locally irradiated and one day later intramuscularly vaccinated. Local tumor irradiation caused a drastic increase in intratumoral levels of both tumor- and vaccine-induced specific CTLs. Intratumoral levels of vaccine-induced specific CTLs were 5-fold higher than intratumoral levels of adoptively transferred E6,7-specific CTLs. In summary, we demonstrated that vaccine-induced CTLs home into tumors and that local tumor irradiation increases the tumorhoming efficacy of antigen-specific CTLs. This study indicates that rSFV-based immunotherapy combined with tumor radiotherapy could improve treatment outcome in cervical cancer patients with advanced disease.

Respiratory syncytial virus (RSV) causes severe respiratory disease in children and the elderly. There is no registered RSV vaccine. Early experimental non-replicating vaccines have been found to exacerbate RSV symptoms upon infection causing enhanced respiratory disease. Here we show that immunization of mice with reconstituted virosomes produced from RSV envelopes and containing the lipopeptide adjuvant (P3CSK4), induces high-titer virus-neutralizing antibodies, and the secretion of IFN-gamma through both MHC-I and MHC-II presentation of antigen, with a balanced Th1/Th2 profile. Immunization with RSV virosomes provides sterilizing immunity to virus challenge in mice and cotton rats, while not producing symptoms of enhanced disease. Therefore, these virosomes represent a promising candidate inactivated RSV vaccine formulation. (C) 2010 Elsevier Ltd. All rights reserved.

Flaviviruses infect their host cells by a membrane fusion reaction. In this study, we performed a functional analysis of the membrane fusion properties of West Nile virus (WNV) with liposomal target membranes. Membrane fusion was monitored continuously using a lipid mixing assay involving the fluorophore, pyrene. Fusion of WNV with liposomes occurred on the timescale of seconds and was strictly dependent on mildly acidic pH. Optimal fusion kinetics were observed at pH 6.3, the threshold for fusion being pH 6.9. Preincubation of the virus alone at pH 6.3 resulted in a rapid loss of fusion capacity. WNV fusion activity is strongly promoted by the presence of cholesterol in the target membrane. Furthermore, we provide direct evidence that cleavage of prM to M is a requirement for fusion activity of WNV.

Stable vaccines administered to the lungs by inhalation could circumvent many of the problems associated with current immunizations against respiratory infections. We earlier provided proof of concept in mice that pulmonary delivered whole inactivated virus (WIV) influenza vaccine formulated as a stable dry powder effectively elicits influenza-specific antibodies in lung and serum. Yet, mucosal IgA, considered particularly important for protection at the site of virus entry, was poorly induced. Here we investigate the suitability of various Toll-like receptor (TLR) ligands and the saponin-derived compound GPI-0100 to serve as adjuvant for influenza vaccine administered to the lungs as dry powder. The TLR ligands palmitoyl-3-cysteine-serine-lysine-4 (Pam(3)CSK(4)), monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODN) as well as GPI-0100 tolerated the process of spray freeze-drying well. While Pam(3)CSK(4) had no effect on systemic antibody titers, all the other adjuvants significantly increased influenza-specific serum and lung IgG titers. Yet, only GPI-0100 also enhanced mucosal IgA titers. Moreover, only GPI-0100-adjuvanted WIV provided partial protection against heterologous virus challenge. Pulmonary immunization with GPI-0100-adjuvanted vaccine did not induce an overt inflammatory response since influx of neutrophils and production of inflammatory cytokines were moderate and transient and lung histology was normal. Our results indicate that a GPI-0100-adjuvanted dry powder influenza vaccine is a safe and effective alternative to current parenteral vaccines.

The high efficacy of therapeutic cancer vaccines in preclinical studies has yet to be fully achieved in clinical trials. Tumor immune suppression is a critical factor that hampers the desired antitumor effect. Here, we analyzed the combined effect of a cancer vaccine and the receptor tyrosine kinase inhibitor sunitinib. Sunitinib was administered intraperitoneally, alone or in combination with intramuscular immunization using a viral vector based cancer vaccine composed of Semliki Forest virus replicon particles and encoding the oncoproteins E6 and E7 (SFVeE6,7) of human papilloma virus (HPV). We first demonstrated that treatment of tumor-bearing mice with sunitinib alone dose-dependently depleted myeloid-derived suppressor cells (MDSCs) in the tumor, spleen and in circulation. Concomitantly, the number of CD8(+) T cells increased 2-fold and, on the basis of CD69 expression, their activation status was greatly enhanced. The intrinsic immunosuppressive activity of residual MDSCs after sunitinib treatment was not changed in a dose-dependent fashion. We next combined sunitinib treatment with SFVeE6,7 immunization. This combined treatment resulted in a 1.5- and 3-fold increase of E7-specific cytotoxic T lymphocytes (CTLs) present within the circulation and tumor, respectively, as compared to immunization only. The ratio of E7-specific CTLs to MDSCs in blood thereby increased 10- to 20-fold and in tumors up to 12.5-fold. As a result, the combined treatment strongly enhanced the antitumor effect of the cancer vaccine. This study demonstrates that sunitinib creates a favorable microenvironment depleted of MDSCs and acts synergistically with a cancer vaccine resulting in enhanced levels of active tumor-antigen specific CTLs, thus changing the balance in favor of antitumor immunity.

Meeting abstracts Cancer vaccines aim at inducing tumour-specific immune responses. However, in clinical studies so far these approaches have limited antitumour effect. Evidence is accumulating that MDSC (myeloid-derived suppressor cells) can suppress the antitumour immune response. Reversal of

Background Development of influenza vaccines capable of inducing broad protection against different virus subtypes is necessary given the ever‐changing viral genetic landscape. Previously, we showed that vaccination with whole inactivated virus (WIV) induces heterosubtypic protection against lethal virus infection in mice. Whole inactivated virus‐induced cross‐protection was found to be mediated primarily by flu‐specific CD8+ T cells. Objectives As it has been demonstrated that the route of vaccine administration strongly influences both the quantity and quality of vaccine‐induced immunity, in this study, we determined which route of WIV administration induces optimal heterosubtypic cross‐protection. Methods We compared the magnitude of the immune response and heterosubtypic protection against lethal A/PR/8/34 (H1N1) infection after subcutaneous (SC), intramuscular (IM), and intranasal (IN) vaccination with A/NIBRG‐14 (H5N1) WIV. Results Subcutaneous and IM administration was superior to IN administration of influenza WIV in terms of flu‐specific CD8+ T‐cell induction and protection of mice against lethal heterosubtypic challenge. Surprisingly, despite the very low flu‐specific CD8+ T‐cell responses detected in IN‐vaccinated mice, these animals were partially protected, most likely due to cross‐reactive IgA antibodies. Conclusion The results of this study show that the magnitude of WIV‐induced flu‐specific CD8+ T‐cell activity depends on the applied vaccination route. We conclude that parenteral administration of WIV vaccine, in particular IM injection, is superior to IN vaccine delivery for the induction of heterosubtypic cross‐protection and generally appears to elicit stronger immune responses than mucosal vaccination with WIV.

Current influenza vaccines fail to induce protection against antigenically distinct virus strains. Accordingly, there is a need\ud for the development of cross-protective vaccines. Previously, we and others have shown that vaccination with whole\ud inactivated virus (WIV) induces cross-protective cellular immunity in mice. To probe the mechanistic basis for this finding,\ud we investigated the role of TLR7, a receptor for single-stranded RNA, in induction of cross-protection. Vaccination of TLR72/\ud 2 mice with influenza WIV failed to protect against a lethal heterosubtypic challenge; in contrast, wild-type mice were fully\ud protected. The lack of protection in TLR72/2 mice was associated with high viral load and a relative paucity of influenzaspecific\ud CD8+ cytotoxic T lymphocyte (CTL) responses. Dendritic cells (DCs) from TLR72/2 mice were unable to crosspresent\ud WIV-derived antigen to influenza-specific CTLs in vitro. Similarly, TLR72/2 DCs failed to mature and become\ud activated in response to WIV, as determined by the assessment of surface marker expression and cytokine production.\ud Plasmacytoid DCs (pDCs) derived from wild-type mice responded directly to WIV while purified conventional DCs (cDCs) did\ud not respond to WIV in isolation, but were responsive in mixed pDC/cDC cultures. Depletion of pDCs prior to and during WIV\ud immunization resulted in reduced numbers of influenza-specific CTLs and impaired protection from heterosubtypic\ud challenge. Thus, TLR7 plays a critical role in the induction of cross-protective immunity upon vaccination with WIV. The\ud initial target cells for WIV appear to be pDCs which by direct or indirect mechanisms promote activation of robust CTL\ud responses against conserved influenza epitopes.

Introduction: RSV infection remains a serious threat to newborns and the elderly. Currently, there is no vaccine available to prevent RSV infection. A mucosal RSV vaccine would be attractive as it could induce mucosal as well as systemic antibodies, capable of protecting both the upper and lower respiratory tract. Previously, we reported on a virosomal RSV vaccine for intramuscular injection with intrinsic adjuvant properties mediated by an incorporated lipophilic Toll-like receptor 2 (TLR2) ligand. However, it has not been investigated whether this virosomal RSV vaccine candidate would be suitable for use in mucosal immunization strategies and if additional incorporation of other innate receptor ligands, like NOD2-ligand, could further enhance the immunogenicity and protective efficacy of the vaccine.Objective: To explore if intranasal (IN) immunization with a virosomal RSV vaccine, supplemented with TLR2 and/or NOD2-ligands, is an effective strategy to induce RSV-specific immunity.Methods: We produced RSV-virosomes carrying TLR2 (Pam(3)CSK(4)) and/or NOD2 (L18-MDP) ligands. We tested the immunopotentiating properties of these virosomes in vitro, using TLR2- and/or NOD2-ligand-responsive murine and human cell lines, and in vivo by assessing induction of protective antibody and cellular responses upon IN immunization of BALB/c mice.Results: Incorporation of Pam(3)CSK(4) and/or L18-MDP potentiates the capacity of virosomes to activate (antigen-presenting) cells in vitro, as demonstrated by NF-kappa B induction. In vivo, incorporation of Pam(3)CSK(4) in virosomes boosted serum IgG antibody responses and mucosal antibody responses after IN immunization. While L18-MDP alone was ineffective, incorporation of L18-MDP in Pam(3)CSK(4)-carrying virosomes further boosted mucosal antibody responses. Finally, IN immunization with adjuvanted virosomes, particularly Pam(3)CSK(4)/L18-MDP-adjuvanted-virosomes, protected mice against infection with RSV, without priming for enhanced disease.Conclusion: Mucosal immunization with RSV-virosomes, supplemented with incorporated TLR2- and/or NOD2-ligands, represents a promising approach to induce effective and safe RSV-specific immunity.

Respiratory Syncytial Virus (RSV) is a major cause of viral brochiolitis in infants and young children and is also a significant problem in elderly and immuno-compromised adults. To date there is no efficacious and safe RSV vaccine, partially because of the outcome of a clinical trial in the 1960s with a formalin-inactivated RSV vaccine (FI-RSV). This vaccine caused enhanced respiratory disease upon exposure to the live virus, leading to increased morbidity and the death of two children. Subsequent analyses of this incident showed that FI-RSV induces a Th2-skewed immune response together with poorly neutralizing antibodies. As a new approach, we used reconstituted RSV viral envelopes, i.e. virosomes, with incorporated monophosphoryl lipid A (MPLA) adjuvant to enhance immunogenicity and to skew the immune response towards a Th1 phenotype. Incorporation of MPLA stimulated the overall immunogenicity of the virosomes compared to non-adjuvanted virosomes in mice. Intramuscular administration of the vaccine led to the induction of RSV-specific IgG2a levels similar to those induced by inoculation of the animals with live RSV. These antibodies were able to neutralize RSV in vitro. Furthermore, MPLA-adjuvanted RSV virosomes induced high amounts of IFN gamma and low amounts of IL5 in both spleens and lungs of immunized and subsequently challenged animals, compared to levels of these cytokines in animals vaccinated with FI-RSV, indicating a Th1-skewed response. Mice vaccinated with RSV-MPLA virosomes were protected from live RSV challenge, clearing the inoculated virus without showing signs of lung pathology. Taken together, these data demonstrate that RSV-MPLA virosomes represent a safe and efficacious vaccine candidate which warrants further evaluation.

Background\ud \ud The inability of seasonal influenza vaccines to effectively protect against infection with antigenically drifted viruses or newly emerging pandemic viruses underlines the need for development of cross-reactive influenza vaccines that induce immunity against a variety of virus subtypes. Therefore, potential cross-protective vaccines, e.g., whole inactivated virus (WIV) vaccine, that can target conserved internal antigens such as the nucleoprotein (NP) and/or matrix protein (M1) need to be explored.\ud Methodology/Principal Findings\ud \ud In the current study we show that a WIV vaccine, through induction of cross-protective cytotoxic T lymphocytes (CTLs), protects mice from heterosubtypic infection. This protection was abrogated after depletion of CD8+ cells in vaccinated mice, indicating that CTLs were the primary mediators of protection. Previously, we have shown that different procedures used for virus inactivation influence optimal activation of CTLs by WIV, most likely by affecting the membrane fusion properties of the virus. Specifically, inactivation with formalin (FA) severely compromises fusion activity of the virus, while inactivation with β-propiolactone (BPL) preserves fusion activity. Here, we demonstrate that vaccination of mice with BPL-inactivated H5N1 WIV vaccine induces solid protection from lethal heterosubtypic H1N1 challenge. By contrast, vaccination with FA-inactivated WIV, while preventing death after lethal challenge, failed to protect against development of disease and severe body weight loss. Vaccination with BPL-inactivated WIV, compared to FA-inactivated WIV, induced higher levels of specific CD8+ T cells in blood, spleen and lungs, and a higher production of granzyme B in the lungs upon H1N1 virus challenge.\ud Conclusion/Significance\ud \ud The results underline the potential use of WIV as a cross-protective influenza vaccine candidate. However, careful choice of the virus inactivation procedure is important to retain membrane fusion activity and full immunogenicity of the vaccine.

Induction of cytotoxic T lymphocyte (CTL) activity against conserved influenza antigens, e.g. nucleoprotein (NP) could be a step towards cross-protective influenza vaccine. The major challenge for non-replicating influenza vaccines aiming for activation of CTLs is targeting of antigen to the MHC class I processing and presentation pathway of professional antigen presenting cells, in particular dendritic cells (DCs). Intrinsic fusogenic properties of the vaccine particle itself can enable direct cytosolic delivery of the antigen by enhancing release of the antigen from the endosome to the cytosol. Alternatively, the vaccine particle would need to possess the capacity to activate DCs thereby triggering cell-intrinsic mechanisms of cross-presentation, processes that do not require fusion. Here, using fusion-active and fusion-inactive whole inactivated virus (WIV) as a vaccine model, we studied the relative contribution of these two pathways on priming and reactivation of influenza NP-specific CTLs in a murine model. We show that activation of bone marrow-derived DCs by WIV, as well as reactivation of NP-specific CTLs in vitro and in vivo were not affected by inactivation of membrane fusion of the WIV particles. However, in vivo priming of naive CTLs was optimal only upon vaccination with fusion-active WIV. Thus, DC-intrinsic mechanisms of cross-presentation are involved in the activation of CTLs upon vaccination with WIV. However, for optimal priming of naive CTLs these mechanisms should be complemented by delivery of antigen to the cytosol mediated by the membrane fusion capacity of the WIV particles. (C) 2010 Elsevier Ltd. All rights reserved.