A virosomal malaria peptide vaccine elicits a long-lasting sporozoite-inhibitory antibody response in a phase 1a clinical trial.

OBJECTIVES: Peptides delivered on the surface of influenza virosomes have been shown to induce solid humoral immune responses in experimental animals. High titers of peptide-specific antibodies were also induced in a phase 1a clinical trial in volunteers immunized with virosomal formulations of two peptides derived from the circumsporozoite protein (CSP) and the apical membrane antigen 1 (AMA-1) of Plasmodium falciparum. The main objective of this study was to perform a detailed immunological and functional analysis of the CSP-specific antibodies elicited in this phase 1a trial. METHODOLOGY/PRINCIPAL FINDINGS: 46 healthy malaria-naïve adults were immunized with virosomal formulations of two peptide-phosphatidylethanolamine conjugates, one derived from the NANP repeat region of P. falciparum CSP (designated UK-39) the other from P. falciparum AMA-1 (designated AMA49-C1). The two antigens were delivered in two different concentrations, alone and in combination. One group was immunized with empty virosomes as control. In this report we show a detailed analysis of the antibody response against UK-39. Three vaccinations with a 10 microg dose of UK-39 induced high titers of sporozoite-binding antibodies in all volunteers. This IgG response was affinity maturated and long-lived. Co-administration of UK-39 and AMA49-C1 loaded virosomes did not interfere with the immunogenicity of UK-39. Purified total IgG from UK-39 immunized volunteers inhibited sporozoite migration and invasion of hepatocytes in vitro. Sporozoite inhibition closely correlated with titers measured in immunogenicity assays. CONCLUSIONS: Virosomal delivery of a short, conformationally constrained peptide derived from P. falciparum CSP induced a long-lived parasite-inhibitory antibody response in humans. Combination with a second virosomally-formulated peptide derived from P. falciparum AMA-1 did not interfere with the immunogenicity of either peptide, demonstrating the potential of influenza virosomes as a versatile, human-compatible antigen delivery platform for the development of multivalent subunit vaccines. TRIAL REGISTRATION: ClinicalTrials.gov NCT00400101.

A randomized placebo-controlled phase Ia malaria vaccine trial of two virosome-formulated synthetic peptides in healthy adult volunteers.

BACKGROUND AND OBJECTIVES: Influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. The aim of the study was to proof the concept that virosomes can also be used to elicit high titers of antibodies against synthetic peptides. The specific objective was to demonstrate the safety and immunogenicity of two virosome-formulated P. falciparum protein derived synthetic peptide antigens given in two different doses alone or in combination. METHODOLOGY/PRINCIPAL FINDINGS: The design was a single blind, randomized, placebo controlled, dose-escalating study involving 46 healthy Caucasian volunteers aged 18-45 years. Five groups of 8 subjects received virosomal formulations containing 10 microg or 50 microg of AMA 49-CPE, an apical membrane antigen-1 (AMA-1) derived synthetic phospatidylethanolamine (PE)-peptide conjugate or 10 ug or 50 ug of UK39, a circumsporozoite protein (CSP) derived synthetic PE-peptide conjugate or 50 ug of both antigens each. A control group of 6 subjects received unmodified virosomes. Virosomal formulations of the antigens (designated PEV301 and PEV302 for the AMA-1 and the CSP virosomal vaccine, respectively) or unmodified virosomes were injected i. m. on days 0, 60 and 180. In terms of safety, no serious or severe adverse events (AEs) related to the vaccine were observed. 11/46 study participants reported 16 vaccine related local AEs. Of these 16 events, all being pain, 4 occurred after the 1(st), 7 after the 2(nd) and 5 after the 3(rd) vaccination. 6 systemic AEs probably related to the study vaccine were reported after the 1(st) injection, 10 after the 2(nd) and 6 after the 3(rd). Generally, no difference in the distribution of the systemic AEs between either the doses applied (10 respectively 50 microg) or the synthetic antigen vaccines (PEV301 and PEV302) used for immunization was found. In terms of immunogenicity, both PEV301 and PEV302 elicited already after two injections a synthetic peptide-specific antibody response in all volunteers immunized with the appropriate dose. In the case of PEV301 the 50 microg antigen dose was associated with a higher mean antibody titer and seroconversion rate than the 10 microg dose. In contrast, for PEV302 mean titer and seroconversion rate were higher with the lower dose. Combined delivery of PEV301 and PEV302 did not interfere with the development of an antibody response to either of the two antigens. No relevant antibody responses against the two malaria antigens were observed in the control group receiving unmodified virosomes. CONCLUSIONS: The present study demonstrates that three immunizations with the virosomal malaria vaccine components PEV301 or/and PEV302 (containing 10 microg or 50 microg of antigen) are safe and well tolerated. At appropriate antigen doses seroconversion rates of 100% were achieved. Two injections may be sufficient for eliciting an appropriate immune response, at least in individuals with pre-existing anti-malarial immunity. These results justify further development of a final multi-stage virosomal vaccine formulation incorporating additional malaria antigens. TRIAL REGISTRATION: ClinicalTrials.gov NCT00400101.

Influenza virosomes as a combined vaccine carrier and adjuvant system for prophylactic and therapeutic immunizations.

Influenza virosomes are an efficient antigen carrier and adjuvant system that are approved for the use in human vaccines. Structurally, virosomes are spherical vesicles of approximately 150 nm in diameter, composed of a lipid membrane with integrated envelope proteins derived from influenza virus, predominantly hemagglutinin. The particle structure, together with the functions of hemagglutinin--receptor binding, pH-dependent fusion activity and immunostimulation--is responsible for the adjuvant effect of virosomes. In contrast to most other virus-like particles, virosomes are semisynthetic particles reconstituted in vitro from lipids and from virus-derived proteins. The production process has proven to be robust at industrial scale and fully compatible with Good Manufacturing Practice guidelines. At the same time, the formulation procedure is sufficiently flexible to allow modifications of the composition and structure for the intended use, including the positioning of the antigens of interest.

A new and versatile virosomal antigen delivery system to induce cellular and humoral immune responses.

The purpose of a vaccine is the induction of effective cellular and/or humoral immune responses against antigens. Because defined antigens are often poor immunogens when administered alone, an adjuvant is required to potentiate the immune response. Most of these adjuvants are designed to induce humoral immune responses, including immunopotentiating reconstituted influenza virosomes (IRIVs). IRIVs are one of the few adjuvants currently licensed for human use with the advantage of an excellent safety profile. To induce a potent cytotoxic T lymphocyte (CTL) immune response CTL epitopes have to be encapsulated into IRIVs. However, the existing encapsulation methods are inefficient or rather laborious. We have developed and characterised a new generation of influenza virosomes (TIRIVs) that induced both, strong CTL and antibody responses against specific antigens of choice. In addition, these virosomes were stabilised and offer the possibility of lyophilisation while retaining all their structural, functional and immunogenic properties after reconstitution. TIRIVs induce strong cellular and humoral immune responses and are a versatile and efficient carrier system with adjuvant properties for a variety of antigens. TIRIVs are not only stabilised but also allow easy formulation of new and/or labile T cell and B cell antigens. Considering their immunogenic properties, their flexibility and their superior storage characteristics TIRIVs provide a versatile technology platform for any vaccination strategy.

Virosome-based active immunization targets soluble amyloid species rather than plaques in a transgenic mouse model of Alzheimer's disease.

Active vaccination with amyloid peptides shows promise for the treatment and prevention of Alzheimer's disease (AD). Several studies in transgenic mouse models of AD have revealed the potency of vaccination to prevent or even clear amyloid plaques from mouse brain. However, the idea that soluble oligomeric species of beta-amyloid (Abeta), rather than plaques, trigger the disease has gained momentum, and current active vaccination strategies affect the levels of total or soluble brain Abeta little or not at all. We describe an active vaccination method based on Abeta1-16 presented on the surface of virosomes, which triggered a dramatic decrease in both soluble Abeta40 (75% reduction; p=0.01) and soluble Abeta42 (62% reduction; p=0.03) in a double transgenic mouse model of AD. Whereas Abeta40 and Abeta42 levels in the insoluble fraction tended to be reduced (by 30% and 27%, respectively), the number of thioflavine-S-positive amyloid plaques was not affected. The high specific antibody responses, obtained without eliciting T-cell reactivity, demonstrate that immunostimulating reconstituted influenza virosomes are a promising antigen carrier system against the neuropathology of AD.

Peptide-loaded chimeric influenza virosomes for efficient in vivo induction of cytotoxic T cells.

Virus-specific CD8(+) T cells are thought to play an important role in resolving acute hepatitis C virus (HCV) infection as viral clearance has been associated with a strong and sustained CD8(+) T cell response. During the chronic state of HCV infection virus-specific T cells have a low frequency and a reduced responsiveness. Based on this, a therapeutic vaccine increasing the frequency of specific T cells is a promising alternative for the treatment of chronic HCV infection. We improved an existing vaccine platform based on immunopotentiating reconstituted influenza virosomes (IRIVs) for efficient delivery of peptide epitopes to the MHC class I antigen presentation pathway. IRIVs are proteoliposomes composed of phospholipids and influenza surface glycoproteins. Due to their fusogenic activity, IRIVs are able to deliver encapsulated macromolecules, e.g. peptides to immunocompetent cells. We developed a novel method based on chimeric virosomes [chimeric immunopotentiating reconstituted influenza virosomes (CIRIVs)] combining the high peptide-encapsulation capacity of liposomes and the fusion activity of virosomes. This new approach resulted in a 30-fold increase of the amount of incorporated soluble peptide compared with current preparation methods. To study the immunogenicity of chimeric virosomes HLA-A2.1 transgenic mice were immunized with CIRIVs containing the HCV Core132 peptide. Core132-CIRIVs efficiently induced specific cytotoxic and IFNgamma-producing T cells already with low peptide doses. Vaccine formulations, which include combinations of different HCV-derived CTL epitopes could be used to induce not only a strong but also a multi-specific CTL response, making them potential candidates for therapeutic and maybe prophylactic T cell vaccines in humans.

Vaccine-induced CD8+ T cells eliminate tumors by a two-staged attack.

Data presented here demonstrate that vaccine-induced CD8(+) T cells can eliminate their specific tumor-target with a two-staged attack. First, they release interferon-gamma that results in growth arrest of the tumor cells via induction of antiangiogenic mediators. Then, during the latter stages of the immune response, CD8(+) effector T cells eradicate the remaining tumor cells through perforin-mediated lysis. A combination of these two mechanisms is highly effective in the described model, while either pathway alone fails to completely achieve tumor rejection.

Rabies vaccines: from the past to the 21st century.

Since the first development of a rabies vaccine by Pasteur in the late 19th century, second- and third-generation vaccines with improved efficacy and less reactogenicity have been developed for use in humans and animals. Despite the availability of safe but rather expensive vaccines based on inactivated virus propagated in diploid cell cultures, much of the human vaccinations worldwide are still carried out with nerve tissue-containing vaccines, which have various side effects. A number of experimental vaccines are under development that may provide alternative safe and potent but less expensive vaccine options. These include DNA vaccines, recombinant viral vaccines, and recombinant protein vaccines. Further testing is needed to determine if and which one of these novel vaccines will make their way into mass production and application in the future.