Immunopotentiating Reconstituted Influenza Virosomes Research Articles

Development of a Virosomal Malaria Vaccine Candidate: From Synthetic Peptide Design to Clinical Concept Validation

An ideal malaria vaccine would prevent disease and reduce transmission by targeting several developmental stages of human malaria parasites. To be cost-effective, a modular antigen delivery technology is required for the development of such a multivalent subunit vaccine. In this review, we summarize and discuss a strategy to develop synthetic peptidomimetics of key malaria target antigens for inclusion in a multivalent malaria subunit vaccine based on immunopotentiating reconstituted influenza virosomes. Clinical testing of a bivalent virosomal formulation incorporating two structurally optimized peptidomimetics has demonstrated safety, immunogenicity and pilot efficacy. While this clinical validation supports the concept of using peptide-loaded virosomes for vaccination in humans, it is assumed that additional antigens will have to be added to the bivalent formulation to generate a highly effective malaria vaccine.

Immunogenicity of a virosomally-formulated Plasmodium falciparum GLURP-MSP3 chimeric protein-based malaria vaccine candidate in comparison to adjuvanted formulations

BackgroundIn clinical trials, immunopotentiating reconstituted influenza virosomes (IRIVs) have shown great potential as a versatile antigen delivery platform for synthetic peptides derived from Plasmodium falciparum antigens. This study describes the immunogenicity of a virosomally-formulated recombinant fusion protein comprising domains of the two malaria vaccine candidate antigens MSP3 and GLURP.MethodsThe highly purified recombinant protein GMZ2 was coupled to phosphatidylethanolamine and the conjugates incorporated into the membrane of IRIVs. The immunogenicity of this adjuvant-free virosomal formulation was compared to GMZ2 formulated with the adjuvants Montanide ISA 720 and Alum in three mouse strains with different genetic backgrounds.ResultsIntramuscular injections of all three candidate vaccine formulations induced GMZ2-specific antibody responses in all mice tested. In general, the humoral immune response in outbred NMRI mice was stronger than that in inbred BALB/c and C57BL/6 mice. ELISA with the recombinant antigens demonstrated immunodominance of the GLURP component over the MSP3 component. However, compared to the Al(OH)3-adjuvanted formulation the two other formulations elicited in NMRI mice a larger proportion of anti-MSP3 antibodies. Analyses of the induced GMZ2-specific IgG subclass profiles showed for all three formulations a predominance of the IgG1 isotype. Immune sera against all three formulations exhibited cross-reactivity with in vitro cultivated blood-stage parasites. Immunofluorescence and immunoblot competition experiments showed that both components of the hybrid protein induced IgG cross-reactive with the corresponding native proteins.ConclusionA virosomal formulation of the chimeric protein GMZ2 induced P. falciparum blood stage parasite cross-reactive IgG responses specific for both MSP3 and GLURP. GMZ2 thus represents a candidate component suitable for inclusion into a multi-valent virosomal malaria vaccine and influenza virosomes represent a versatile antigen delivery system suitable for adjuvant-free immunization with recombinant proteins.

Design and pre-clinical profiling of a Plasmodium falciparum MSP-3 derived component for a multi-valent virosomal malaria vaccine

BackgroundClinical profiling of two components for a synthetic peptide-based virosomal malaria vaccine has yielded promising results, encouraging the search for additional components for inclusion in a final multi-valent vaccine formulation. This report describes the immunological characterization of linear and cyclized synthetic peptides comprising amino acids 211-237 of Plasmodium falciparum merozoite surface protein (MSP-3).MethodsThese peptides were coupled to phosphatidylethanolamine (PE); the conjugates were intercalated into immunopotentiating reconstituted influenza virosomes (IRIVs) and then used for immunizations in mice to evaluate their capacity to elicit P. falciparum cross-reactive antibodies.ResultsWhile all MSP-3-derived peptides were able to elicit parasite-binding antibodies, stabilization of turn structures by cyclization had no immune-enhancing effect. Therefore, further pre-clinical profiling was focused on FB-12, a PE conjugate of the linear peptide. Consistent with the immunological results obtained in mice, all FB-12 immunized rabbits tested seroconverted and consistently elicited antibodies that interacted with blood stage parasites. It was observed that a dose of 50 μg was superior to a dose of 10 μg and that influenza pre-existing immunity improved the immunogenicity of FB-12 in rabbits. FB-12 production was successfully up-scaled and the immunogenicity of a vaccine formulation, produced according to the rules of Good Manufacturing Practice (GMP), was tested in mice and rabbits. All animals tested developed parasite-binding antibodies. Comparison of ELISA and IFA titers as well as the characterization of a panel of anti-FB-12 monoclonal antibodies indicated that at least the majority of antibodies specific for the virosomally formulated synthetic peptide were parasite cross-reactive.ConclusionThese results reconfirm the suitability of IRIVs as a carrier/adjuvant system for the induction of strong humoral immune responses against a wide range of synthetic peptide antigens. The virosomal formulation of the FB-12 peptidomimetic is suitable for use in humans and represents a candidate component for a virosomal multi-valent malaria subunit vaccine.

Contribution of influenza immunity and virosomal-formulated synthetic peptide to cellular immune responses in a phase I subunit malaria vaccine trial

We have demonstrated recently in a phase Ia clinical trial that synthetic malaria peptides delivered by immuno-potentiating reconstituted influenza virosomes (IRIV) induced long-lived peptide-specific antibody responses in all volunteers. In the current ancillary study to this clinical trial we have investigated the cellular immune responses specific for IRIV and the surface bound synthetic malaria peptides tested. After vaccination, in 50% (8/16) of the volunteers at least one positive lymphoproliferative response specific for the 49mer peptide derived from the Plasmodium falciparum apical membrane antigen-1 (AMA-1) was observed with stimulation indices ranging from 2 to 4.5. All volunteers showed pre-existing IRIV specific cellular immunity assessed by ex vivo IFN-γ ELISpot analysis and lymphoproliferation. The pre-existing influenza specific T cell responses did not interfere negatively with the induction of malaria peptide-specific humoral and cellular immune responses. Our results support the view that IRIV constitute a safe antigen delivery system for induction of peptide-specific immune responses in human populations.

Preclinical profiling of the immunogenicity of a two-component subunit malaria vaccine candidate based on virosome technology

Presentation of synthetic peptides on immunopotentiating reconstituted influenza virosomes is a promising technology for subunit vaccine development. An optimized virosomally delivered peptide representing 5 NPNA repeats of P. falciparum circumsporozoite protein is highly immunogenic in mice. Antibodies against this peptide (UK-39) inhibit sporozoite invasion of human hepatocytes. A second peptide (AMA49-C1) based on domain III of apical membrane antigen 1, induces antibodies that inhibit blood-stage parasite growth in vitro. Here we show a detailed pre-clinical profiling of these virosomally formulated peptides alone and in combination in mice and rabbits. Two immunizations with virosomally formulated UK-39 or AMA49-C1 were enough to elicit high titers of parasite cross-reactive antibodies in both species. A low dose of 10 μg UK-39 was enough to induce maximal titers in rabbits. Higher doses of peptide did not increase antibody titers. In contrast, AMA49-C1 induced higher antibody titers with 25 and 50 μg peptide. Combination of UK-39 and AMA49-C1 on separate virosomes did not have any negative effect on anti-peptide antibody titers in mice or rabbits. No MHC restriction was observed in the development of humoral responses in outbred rabbits with different immunogenetic backgrounds. All vaccine formulations were safe in toxicity studies in rabbits and rats. Taken together, low amounts of synthetic peptides delivered on virosomes induced high antibody titers in mice and rabbits. Moreover, different peptides could be combined without interfering with individual anti-peptide responses, augmenting the value of this system for the development of a multivalent malaria vaccine.

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-mediated delivery of tumor antigen to plasmacytoid dendritic cells

Cytotoxic T lymphocytes (CTL) are crucial in viral clearance and tumor growth control. Thus the induction of CTL activity is an important aim in vaccine development. We investigate an innovative delivery system for peptide transfer to the MHC class I processing pathway of APC with the aim to trigger CTL in the context of an antitumoral response. The strategy relies on a novel antigen delivery system termed “chimeric immunopotentiating reconstituted influenza virosomes” (CIRIV) targeting plasmacytoid dendritic cells (PDC). By using virosomes containing encapsulated Melan-A peptide and a PDC line developed in our laboratory, we evaluated the response of Melan-A specific T cells. Virosomes have the capacity to bind PDC and are endocyted within vesicles in the cytosol. This endocytosis is inhibited by neuraminidase, suggesting that it is mediated by sialic acid present on cell surface. Furthermore, PDC loaded with Melan-A virosomes can induce a Melan-A specific T cell activation. Interestingly, they activate T cells with a better efficiency than PDC loaded with a free peptide and when PDC where previously activated by a TLR ligand. These results indicate that virosomes could be a suitable delivery system for tumor peptide in immunotherapy of cancer.

Efficient induction of tumoricidal cytotoxic T lymphocytes by HLA-A0201 restricted, melanoma associated, L 27Melan-A/MART-1 26–35 peptide encapsulated into virosomes in vitro

Cancer immunotherapy requires the induction of HLA class I restricted cytotoxic T lymphocytes (CTL) specific for tumor associated antigens (TAA). While a number of TAA have been identified, there is an urgent need for the development of adjuvants capable of stimulating CTL responsiveness. Previously, we reported the capacity of immunopotentiating reconstituted influenza virosomes (IRIV) to enhance CTL responses specific for synthetic peptides simultaneously added to cultures in soluble form. This effect was based on IRIV mediated activation of CD4 + T cells. Here we investigated the “in vitro” immunogenicity of a novel virosome formulation coupling in a single reagent the adjuvant power of IRIV to the capacity of liposomes to efficiently encapsulate synthetic peptides. As a model epitope we chose L 27Melan-A/Mart-1 26–35 HLA-A0201restricted peptide from a melanoma-associated antigen widely used in tumor immunotherapy. The reagent thus developed induced the proliferation of CD4 + T cells characterized by a T helper 1 cytokine profile and CXCR3 expression. Most importantly, it significantly enhanced the generation of L 27Melan-A/Mart-1 26–35 specific CTL, as compared to soluble peptides, in particular at low nominal epitope concentrations (<1 μg/ml). These effector cells were able to efficiently kill HBL melanoma cells expressing Melan-A/MART-1 and HLA-A0201. The adjuvant effects observed were also detectable in the absence of CD4 + T cells. Taken together our results suggest that this highly immunogenic antigenic formulation might qualify for clinical use in active, antigen-specific, melanoma immunotherapy.

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.

Use of immunopotentiating reconstituted influenza virosomes (IRIV) as vector to deliver antigens into dendritic cells

Dendritic cells (DC) are the most potent antigen presenting cells and therefore represent a promising tool for cancer immuno-therapy. We developed single use cell processors, allowing generation of large numbers of clinical grade monocyte-derived DC in serum free medium containing GM-CSF and IL-13. We have previously demonstrated that these DC cross present antigens to specific CD8 T cells on MHC class I molecules in vitro. In order to improve specific CD8 T cell responses, we tested Immunopotentiating Reconstituted Influenza Virosomes (IRIV) as vehicles to deliver antigens into DC. IRIVs are spherical, unilamellar vesicles with a mean diameter of 150 nm derived from the envelope of the Influenza virus. IRIVs could enter into DC via binding of hemagglutinin flu protein present at the surface of IRIV to sialic acid. It has been shown that antigens encapsulated into IRIVs are delivered to the cytosol and enter the MHC class I pathway inducing a CD8 T cell immune response. In this study, we demonstrated efficient uptake of PKH-26 labeled IRIVs by DC. IRIVs did not affect DC viability and did not induce DC maturation. In addition, IRIVs did not prevent maturation induced by bacterial extract (FMKp) and IFNγ. In order to study the capacity of IRIV associated with antigen to present epitopes at the DC cell surface, we developed a model antigen containing the highly immunogenic mutated Melan-A26–35(27L) peptide encapsulated into IRIVs from Association for Immunotherapy of Cancer: Cancer Immunotherapy – 2nd Annual Meeting Mainz, Germany, 6–7 May 2004

Influenza virosomes enhance class I restricted CTL induction through CD4+ T cell activation

Immunopotentiating reconstituted influenza virosomes (IRIV) are one of the few adjuvants currently licensed for human use. While their adjuvant capacity in the induction of humoral responses is clearly documented, few data exist on their effects on T cell immune response. Here we addressed IRIV adjuvance in the induction of HLA class I restricted cytotoxic T lymphocytes (CTL) in vitro.Lymphocyte stimulation with IM58–66 and IRIV resulted in marked expansion of specific CTL as compared to cultures performed in the presence of either antigen alone or antigen and control liposomes (L). Studies addressing underlying adjuvant mechanisms demonstrated that IRIV activated CD4/CD45RO+ T cells, induced a cytokine profile consistent with T helper 1 (Th1) stimulation and increased the percentage of CD4+ T cells expressing CXCR3. Furthermore, supernatants from IRIV stimulated PBMC cultures promoted dendritic cell maturation. Most importantly, IRIV mediated CTL adjuvance required the presence of live CD4+ T cells. Powerful adjuvant effects of IRIV were also observed in the induction of CTL specific for the melanoma associated Melan-A/MART-127–35, HLA-A0201 restricted epitope.Taken together these findings indicate that IRIV are endowed with a high adjuvant capacity for HLA class I restricted CTL induction, largely attributable to their ability to antigenically stimulate CD4+ T cells.

Induction of parasite growth-inhibitory antibodies by a virosomal formulation of a peptidomimetic of loop I from domain III of Plasmodium falciparum apical membrane antigen 1.

Apical membrane antigen 1 (AMA-1) of Plasmodium falciparum is a leading candidate antigen for inclusion in a malaria subunit vaccine. Its ectodomain can be divided into three subdomains, each with disulfide bond-stabilized structures. Since the majority of antibodies raised against the ectodomain appear to recognize strain-specific epitopes in domain I, we attempted to develop a vaccine formulation which directs the immune response to a region that contains more conserved epitopes. Here we demonstrate that a virosomal formulation of a peptide that mimics the semiconserved loop I of domain III elicits parasite growth-inhibitory antibodies. A synthetic peptide comprising residues 446 to 490 of AMA-1 (AMA-1(446-490)) was conjugated through the N terminus to a derivative of phosphatidylethanolamine and the phosphatidylethanolamine-peptide conjugate was incorporated into immunopotentiating reconstituted influenza virosomes as a human-compatible antigen delivery system. Both cyclized and linear versions of the peptide antigen elicited antibodies which specifically bound to parasite-expressed AMA-1 in Western blotting with parasite lysates as well as in immunofluorescence assays with blood stage parasites. All 11 peptidomimetic-specific monoclonal antibodies generated were cross-reactive with parasite-expressed AMA-1. Antigen binding assays with a library of overlapping cyclic peptides covering the target sequence revealed differences in the fine specificity of these monoclonal antibodies and provided evidence that at least some of them recognized discontinuous epitopes. The two immunodominant epitopes comprised the conserved linear sequences K(459)RIKLN(464) and D(467)DEGNKKII(475). A key feature of the synthetic vaccine formulation proposed here is the display of the peptide antigen in a native-like state on the surface of the virosome.

In vivo study of the GC90/IRIV vaccine for immune response and autoimmunity into a novel humanised transgenic mouse.

Parathyroid hormone-related protein (PTH-rP), a secreted protein produced by prostate carcinoma and other epithelial cancers, is considered a key agent for the development of bone metastases. We investigated the construct GC90/IRIV, composed of immunopotentiating reconstituted influenza virosomes (IRIV) containing PTH-rP gene plasmids (GC90), as a potential tool for human anticancer immunotherapy into humanised mice transgenic for HLA-A(*)02.01, the human-β2 microglobulin, and the human CD8α molecule. Intranasal administration of GC90/IRIV resulted in the induction of a PTH-rP-specific multiepitope cytotoxic T-cell (CTL) response. Cytotoxic T cells derived from vaccinated mice were capable of lysing in vitro syngenic murine PTH-rP transfectants and human HLA-A(*)02.01+/PTH-rP+ prostate carcinoma LNCaP cells as well. The immune response capacity and the absence of any sign of toxicity and/or autoimmunity in vivo suggest the GC90/IRIV vaccine as a valid tool for active specific immunotherapy of human cancers and metastases overexpressing PTH-rP.

Novel approaches in the development of immunopotentiating reconstituted influenza virosomes as efficient antigen carrier systems

One of the most recent major advances in vaccinology, and currently the only licensed alternative to the traditional alum-based adjuvants, are immunopotentiating reconstituted influenza virosomes. The flexibility of this novel antigen delivery system enables the safe and effective delivery of modern vaccine developments, such as viral glycoproteins, bacterial toxoids, inactivated virus, recombinant proteins, synthetic peptides and DNA-plasmids or polynucleotides. These novel vaccine antigens are being incorporated into immunopotentiating reconstituted influenza virosomes in an attempt to develop a variety of new viral, bacterial and parasitic vaccines. In addition, immunopotentiating reconstituted influenza virosomes are showing future potential in the targeting of specific cells, such as tumor cells, and novel routes of vaccine administration.

New technology platforms in the development of vaccines for the future

The desire for improved quality of life in both industrialised and under-developed nations has led to the quest for greater understanding and subsequent prevention and treatment of diseases. Here we discuss some of the latest of modern medicine’s approaches to vaccination and disease treatment. Our main subject of discussion being the novel antigen delivery systems termed immunopotentiating reconstituted influenza virosomes (IRIVs) and their use as vaccines. Particular attention is paid to the currently licensed Epaxal and Inflexal V, good examples of the improvements being made in vaccinology. Alternative uses of virosomes such as peptide delivery, cytosolic drug delivery and gene delivery are also considered, highlighting the flexibility of the IRIV formulation and method of action. The paper concludes with consideration of alternative novel approaches to vaccinology including bacterial carriers for DNA vaccines, recombinant MV vaccines and polysaccharide–protein conjugates.

In vitro studies of core peptide‐bearing immunopotentiating reconstituted influenza virosomes as a non‐live prototype vaccine against hepatitis C virus

Evidence from both animal and human viral diseases indicate that cytotoxic T lymphocytes (CTL) are crucial in antiviral defense. However, a major problem to generate cytotoxic immunity is that in vivo exogenous antigens are usually presented via MHC class II pathway and normally fail to induce CTL. The aim of this study is to describe a novel non-live prototype vaccine based on immunopotentiating reconstituted influenza virosomes (IRIV) as vehicles to deliver HLA-A*0201-restricted hepatitis C virus (HCV) peptides (core 35-44 and 131-140) into the cytoplasm of at least three different target cell types [including T2, a transporter associated with antigen processing (TAP)-deficient cell line] resulting in MHC class I peptide presentation and lysis by peptide-specific CTL lines. Comparison of kinetics and analysis of the influence of peptide-stripping and Brefeldin A (BFA) reveal that there exists an endogenous, TAP-independent and BFA-sensitive pathway for virosomally delivered peptides. Moreover, virosomes containing influenza matrix peptide 58-66 can efficiently re-stimulate in vivo primed CTL and, importantly, IRIV containing HCV core peptides can even prime CTL from peripheral blood mononuclear cells of HCV(-) healthy blood donors in vitro. The fact that in vitro primed CTL are also able to specifically lyse target cells infected with recombinant vaccinia virus encoding the HCV core protein is of great importance for future studies based on in vivo mouse models. One of the most evident advantages of the virosomes in vivo will be their capability to protect the incorporated peptide from a large variety of degrading proteases.

Cover Picture

The cover picture shows a constrained peptidomimetic of the Asn-Pro-Asn-Ala-Asn motif that is found in the major surface protein of Plasmodium falciparum sporozoites. The mimetic (bottom) is coupled through a succinate linker to a regioisomer of phosphatidylethanolamine to afford a conjugate (top; the loop on the left represents the mimetic) that can be incorporated into immunopotentiating reconstituted influenza virosomes (IRIVs). When presented to the immune system in this form, the mimetic elicits an efficient parasite cross-reactive antibody response. The background shows immunofluorescence labeling of P. falciparum sporozoites (bright green rods). IRIVs are a form of antigen delivery that is already licensed for human clinical use so this approach may have great potential for the design and delivery of synthetic vaccines. For more details, see the article by Robinson, Pluschke, and co-workers on p. 838 ff.

Exploiting conformationally constrained peptidomimetics and an efficient human-compatible delivery system in synthetic vaccine design.

Peptide and protein mimetics are potentially of great value in synthetic vaccine design. The mimetics should function by stimulating the immune system to produce antibodies that recognize the intact parasite. Also the mimetics should be presented to the immune system in a way that leads to efficient antibody production. Here we investigate the application of cyclic peptidomimetics presented on immunopotentiating reconstituted influenza virosomes (IRIVs), a form of antigen delivery that is licensed already for human clinical use, in synthetic vaccine design. We focus on the central (NPNA)(n) repeat region of the circumsporozoite (CS) protein of the malaria parasite Plasmodium falciparum as a model system. Cyclic peptidomimetics of the NPNA repeats were incorporated into both an IRIV and (for comparison) a multiple-antigen peptide (MAP). Both IRIV and MAP delivery forms induced mimetic-specific humoral immune responses in mice, but only with the mimetic-IRIV preparations did a significant fraction of the elicited antibodies cross-react with sporozoites. The results demonstrate that IRIVs are a delivery system suitable for the efficient induction of antibody responses against conformational epitopes by use of cyclic template-bound peptidomimetics. Combined with combinatorial chemistry, this approach may have great potential for the rapid optimization of molecularly defined synthetic vaccine candidates against a wide variety of infectious agents.

Immunogenicity of IRIV- versus alum-adjuvanted diphtheria and tetanus toxoid vaccines in influenza primed mice

The immunogenicity and protectivity of two different toxoid vaccines were compared in mice. In one formulation, toxoids (diphtheria or tetanus) were adsorbed to alumoxid, whereas in the other formulation the toxoids were crosslinked to immunopotentiating reconstituted influenza virosomes (IRIVs). A preimmunization with influenza antigens is necessary for a good anti-toxoid antibody response when the IRIV formulation was administered. After two immunizations with the IRIV- or alum-based vaccines, the IRIV-based formulation induced a higher humoral immune response than toxoids adsorbed to alum. Using an in vitro test, diphtheria toxin neutralizing antibodies were tested. Di-IRIV induced a significantly ( p=0.002) higher titer of diphtheria toxin neutralizing antibodies than Di-alum. Tetanus challenge experiments showed, that the IRIV-based tetanus vaccine induced a threefold higher titer of protective antibodies than the tetanus toxoid adsorbed to alum. Therefore, the IRIV-based formulations appeared to be superior to the alum-based vaccines in terms of immunogenicity and protectivity.

Adjuvant activity of immunopotentiating reconstituted influenza virosomes (IRIVs)

Using immunopotentiating, reconstituted influenza virosomes (IRIV) as a delivery vehicle, a number of vaccines have been developed. In humans, IRIV-based vaccines containing hepatitis A and influenza antigens have been found to possess enhanced immunogenicity compared to alum-adsorbed vaccine for hepatitis A or commercial subunits or whole virion influenza vaccines. These vaccines were safe and did not engender any antiphospholipid antibodies against the liposome components of the IRIV. Hepatitis B, tetanus toxoid and diphtheria toxoid, and nucleic acids have also been incorporated into IRIVs. These vaccines are now undergoing clinical phase I testing. IRIVs are also being evaluated in phase I trials for their ability to deliver antigens by the intranasal route.