Liposome-based Vaccine Research Articles

Effect of Oligomannose-Coated Liposome-Based Vaccine on Rodent Babesiosis

Effect of Oligomannose-Coated Liposome-Based Vaccine on Rodent Babesiosis

Induction of Cytotoxic T-Lymphocytes and Antitumor Activity by a Liposomal Lipopeptide Vaccine

We have previously described a simple yet effective liposome-based therapeutic vaccine, DOTAP/E7, which contains only two molecules, the cationic lipid DOTAP and a peptide antigen derived from the E7 oncoprotein of human papillomavirus (HPV) type 16. In the current report, we have improved the vaccine formulation by incorporation of E7-lipopeptide instead of the water-soluble native E7 peptide into the DOTAP liposome. The lipopeptide consists of an N-terminal alpha- or -palmitoyl lysine connected to the E7 peptide via a dipeptide Ser-Ser linker. The DOTAP/E7-lipopeptide vaccine exhibited an enhanced functional antigen-specific CD8 (+) T lymphocyte response in vivo compared to the previous DOTAP/E7 formulation. More importantly, the cytotoxic T cells induced by the DOTAP/E7-lipopeptide vaccine could efficiently eliminate an existing HPV positive TC-1 tumor. The antitumor activity of lipopeptide formulated in DOTAP liposome was more than twice as potent as that of native E7, likely owing to the increased peptide entrapment efficiency in the liposomal complex. Our results also showed that it is essential to have the dipeptide spacer sequence between E7 peptide and the attached fatty acid to achieve a full immune response. Overall, the improved DOTAP/E7-lipopeptide vaccine described herein showed a significantly enhanced therapeutic effect for the treatment of a cervical cancer model.

Liposomal vaccines with conformation-specific amyloid peptide antigens define immune response and efficacy in APP transgenic mice

We investigated the therapeutic effects of two different versions of Abeta(1-15 (16)) liposome-based vaccines. Inoculation of APP-V717IxPS-1 (APPxPS-1) double-transgenic mice with tetra-palmitoylated amyloid 1-15 peptide (palmAbeta(1-15)), or with amyloid 1-16 peptide (PEG-Abeta(1-16)) linked to a polyethyleneglycol spacer at each end, and embedded within a liposome membrane, elicited fast immune responses with identical binding epitopes. PalmAbeta(1-15) liposomal vaccine elicited an immune response that restored the memory defect of the mice, whereas that of PEG-Abeta(1-16) had no such effect. Immunoglobulins that were generated were predominantly of the IgG class with palmAbeta(1-15), whereas those elicited by PEG-Abeta(1-16) were primarily of the IgM class. The IgG subclasses of the antibodies generated by both vaccines were mostly IgG2b indicating noninflammatory Th2 isotype. CD and NMR revealed predominantly beta-sheet conformation of palmAbeta(1-15) and random coil of PEG-Abeta(1-16). We conclude that the association with liposomes induced a variation of the immunogenic structures and thereby different immunogenicities. This finding supports the hypothesis that Alzheimer's disease is a "conformational" disease, implying that antibodies against amyloid sequences in the beta-sheet conformation are preferred as potential therapeutic agents.

Therapy of established B16-F10 melanoma tumors by a single vaccination of CTL/T helper peptides in VacciMax®

BackgroundMelanoma tumors are known to express antigens that usually induce weak immune responses of short duration. Expression of both tumor-associated antigens p53 and TRP2 by melanoma cells raises the possibility of simultaneously targeting more than one antigen in a therapeutic vaccine. In this report, we show that VacciMax® (VM), a novel liposome-based vaccine delivery platform, can increase the immunogenicity of melanoma associated antigens, resulting in tumor elimination.MethodsC57BL/6 mice bearing B16-F10 melanoma tumors were vaccinated subcutaneously 6 days post tumor implantation with a mixture of synthetic peptides (modified p53: 232–240, TRP-2: 181–188 and PADRE) and CpG. Tumor growth was monitored and antigen-specific splenocyte responses were assayed by ELISPOT.ResultsVaccine formulated in VM increased the number of both TRP2- and p53-specific IFN-γ producing splenocytes following a single vaccination. Vaccine formulated without VM resulted only in enhanced IFN-γ producing splenocytes to one CTL epitopes (TRP2:180–188), suggesting that VM overcomes antigen dominance and enhances immunogenicity of multiple epitopes. Vaccination of mice bearing 6-day old B16-F10 tumors with both TRP2 and p53-peptides formulated in VM successfully eradicated tumors in all mice. A control vaccine which contained all ingredients except liposomes resulted in eradication of tumors in no more than 20% of mice.ConclusionA single administration of VM is capable of inducing an effective CTL response to multiple tumor-associated antigens. The responses generated were able to reject 6-day old B16-F10 tumors.

Liposomised recombinant ribosomal L7/L12 protein protects BALB/ c mice against Brucella abortus 544 infection

Brucella abortus, a facultative intracellular pathogen, is of tremendous zoonotic importance because of its ability to induce spontaneous abortion in cattle and other livestock. It is also known to cause persistent undulant fever, endocarditis, arthritis, osteomyelitis and meningitis in humans. The available vaccines against this dreadful infection suffer from limitations like short-term immunity, increased risk of hypersensitivity and low prophylactic index in the recipients. In the present study, we have demonstrated that liposomal form of a recombinant ribosomal L7/L12 protein, a B–T cell antigen of B. abortus, activates strong immune response in the host. In contrast, free antigen generates moderate immune response in the immunised animals. The liposomisation of rL7/L12 protein causes tremendous increase in cell-mediated immune response in terms of delayed type hypersensitivity, T-cell proliferation and up-regulation in type I cytokine expression, etc. Moreover, the liposome encapsulated antigen elicited stronger humoral immune response as compared to standard vaccine (S-19) or IFA-L7/L12 combination in the immunised animals. The effectiveness of liposome-based vaccine was also substantiated by better systemic clearance of bacterial load after challenging the animals with B. abortus 544 pathogen. The results of the present study suggest the potential of liposome-based rL7/L12 antigen as prospective and efficient candidate vaccine capable of eliciting both cell mediated as well as humoral immune responses against experimental murine brucellosis.

Protection against H1, H5, H6 and H9 influenza A infection with liposomal matrix 2 epitope vaccines

The recent emergence of multiple avian influenza A subtypes that cause human disease (i.e., H5N1, H9N2 and H7N7), coupled with the fear that one of these strains might precipitate a new pandemic, underscores the need to develop new technological approaches to immunization which elicit protective immune responses against multiple subtypes of influenza A. In response to this demand, several matrix 2 protein ectodomain segments (M2eA) corresponding to the H1N1, H5N1 and H9N2 influenza strains were formulated using a novel liposome-based vaccine technology and evaluated as potential immunogens for developing a “universal” influenza vaccine. Mice immunized with liposomal M2eA survived homologous challenges with H1N1 (100% survival) or H9N2 (80% survival) influenza strains. There were significant reductions in their lung viral load as well as in immunized mice challenged with the H5N1 subtype. The mice vaccinated with an M2eA segment corresponding to the H1N1 and H6N2 (a reassortant influenza A virus carrying the M2eA from PR8/34) strains elicited elevated IgG ELISA antibody titers to this M2eA epitope segment and antiserum from these immunized mice provided passive protection (100% survival) to naïve mice receiving a lethal dose of H6N2 influenza virus. These results provide the first evidence that recombinant M2eA epitopes to multiple subtypes elicited immune protection against a homologous challenge and provides further evidence in favor of the development of a “universal” influenza vaccine based on M2eA.

620. Liposome-Based Therapeutic Vaccine for Cervical Cancer

Top of pageAbstract Cervical cancer is the second most common cancer among women worldwide. More than 99% of cervical cancers contain human papillomavirus (HPV) DNA while HPV type 16 is the most common subtype and is associated with 50% of cervical cancer cases. Preventive vaccine against HPV infection is already available on market, however, there is not yet a therapeutic vaccine thus far for replacing of the traumatic surgery on patients with cervical carcinoma. The E7 viral protein of HPV16, which is continuously expressed in cervical cancer cells and pre-cancerous lesions, is also a unique antigen for targeting cervical tumor and can be used a potential therapeutic strategy. Previous work in our lab has led to the development of a liposome-based non-viral vaccine delivery system, LPD. The nanoparticle LPD is self-assembled from cationic lipid, protamine and plasmid DNA. We have demonstrated that LPD formulated with a peptide epitope from the HPV16 E7 protein can be taken up by the dendritic cells and the vaccine induces both preventive as well as therapeutic activity against TC-1, a murine cancer cell line which is HPV16-DNA positive and a model for human cervical cancer. In order to study the mechanism how LPD plays a novel adjuvant for stimulating both humoral and cellular immune responses, we have found that DOTAP liposome can stimulate murine dendritic cells (professional antigen presenting cells) toward the expression of costimulatory molecules, i.e. CD80 and CD86. The results were also confirmed in human DC-like cell line, KG-1. Surface markers on KG-1 for identifying DC lineage, i.e. CD11c CD80, CD83 and CD86 were increased via DOTAP liposome stimulation and the amount of the increase was DOTAP dose-dependent. Since high dose of liposome has been reported to cause possible unwanted toxicity, we have tested serum TNF-alpha cytokine secretion from the mice received our LPD/E7 formulation and we found that very low or not detectable amount of TNF-alpha were measured after mice received s.c. injection. In order to optimize our vaccine formulation, we have found that the reduced amount of liposome can also elicit efficient activity and provide therapeutic effect on tumor-bearing mice. All the results obtained from mouse system are being confirmed on human primary DC isolated from human peripheral mononuclear cells (PBMC). Success of these experiments will constitute a strong rationale for a future phase I clinical trial. Supported by Mary Kay Ash Charitable Foundation.

Non PC liposome entrapped promastigote antigens elicit parasite specific CD8 + and CD4 + T-cell immune response and protect hamsters against visceral leishmaniasis

Leishmania donovani promastigote soluble antigens (sLAg) were encapsulated in non-phosphatidylcholine (non-PC) liposomes (escheriosomes) prepared from E. coli lipids. The escheriosome-based vaccine was investigated for its potential to elicit a protective immune response against experimental visceral leishmaniasis. The vaccine administration induced strong humoral as well as cell mediated immune responses both in hamsters and BALB/c mice. Immunization of BALB/c mice with escheriosome entrapped sLAg (EL-sLAg) elicited stronger CD8 + cytotoxic T lymphocyte (CTL) response as compared to sLAg entrapped in egg PC/chol liposome (EPC-sLAg) or sLAg administered with incomplete Freund's adjuvant (IFA-sLAg). EL-sLAg also induced the release of mixed (Th1 and Th2) types of cytokines in the immunized BALB/c mice. In addition, the delivery of sLAg via escheriosomes enhanced the expression of costimulatory signals (CD80 and CD86) as determined in peritoneal macrophages obtained from BALB/c mice. In another set of experiments, the EL-sLAg immunized hamsters were found to be better protected than those immunized with EPC-sLAg. The prophylaxis coincided with increased lymphocyte proliferation as well as high nitric oxide (NO) production by peritoneal macrophages among EL-sLAg immunized hamsters. Escheriosomes thus seem to have potential in delivering the antigen to cytosol of the antigen presenting cells (APCs) and in the development of liposome-based vaccine against leishmaniasis as well as other intracellular infections.

Multivalent liposome-based vaccines containing different serosubtypes of PorA protein induce cross-protective bactericidal immune responses against Neisseria meningitidis

Four serosubtypes (P1.7, 16, P1.7-2, 4, P1.19, 15 and P1.5-1, 10-4) of the PorA outer-membrane protein of Neisseria meningitidis were purified as recombinant proteins and incorporated into liposomes to investigate their immunogenicity. Each serosubtype induced high levels of bactericidal activity against the homologous strain. In addition, liposome preparations containing multiple serosubtypes induced high levels of bactericidal activity against each of the four strains. Significantly, antisera raised against monovalent and multivalent liposomes also showed cross-reactive bactericidal activity against heterologous strains. These data demonstrate that multivalent liposome vaccines, containing multiple PorA serosubtypes, have the potential to provide protection against a broad range of meningococcal strains.

The covalent coupling of HAV-VP3 (110–121) synthetic peptide to liposomes: physicochemical studies

In this work we have studied the conjugation of the immunogenic peptide sequence (110–121) belonging to the VP3 capsid protein of hepatitis A virus to the surface of preformed liposomes by means of an amide bond between the vesicles and the synthetic peptide. The surface activity of the conjugate at air/water interface was determined. Moreover, the interaction of the conjugate with lipids was also studied recording the pressure increases produced after the injection of the liposome–peptide preparation under dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and stearylamine (SA) monolayers at different initial surface pressures. As expected, due to the negative net charge of the liposome–peptide complex, the higher interaction was found with positive charge monolayers (SA). However, the conjugate was also able to incorporate to zwitterionic and anionic lipids. This behaviour was also confirmed performing compression isotherms of monolayers of these lipids spread on subphases containing the conjugate. These results suggest that the coupling of VP3 (110–121) to liposomes does not influence its ability to interact with membrane lipids such as DPPC and DPPG. Then it can be assumed that its immunogenicity will be preserved or even increased after this modification. All these results are also useful in the preparation of liposome-based synthetic peptide vaccines.

A liposome-based therapeutic vaccine against beta -amyloid plaques on the pancreas of transgenic NORBA mice.

Immune tolerance to beta-amyloid (Abeta) was broken in NORBA transgenic mice presenting Abeta plaques on their pancreases. Vaccination of Black C57, BALB/c, and NORBA mice with the synthetic Abeta(1-16) sequence modified by covalently attaching two palmitoyl residues at each end of the peptide, subsequently reconstituted in liposomes-Lipid A elicited titers of 1:5,000 of anti-Abeta(1-16) antibodies within 10 weeks after the first inoculation. On direct interaction, sera with antibody titers of 1:5,000 solubilized in vitro up to 80% of preformed Abeta(1-42) aggregates. Cryosections of pancreases of unvaccinated NORBA mice show, on staining with Thioflavin T, extensive areas of high-intensity fluorescence in the acinar cell fields. Quantitation of the average fluorescence intensity in each section indicated that: (i) whereas nonvaccinated NORBA mice develop plaques within 45-60 days after birth, vaccinated 8-week-old NORBA mice did not develop amyloid plaques on their pancreases over a period of 7 months; (ii) cryosections from pancreases of 9- and 15-month-old vaccinated NORBA mice showed less than 50% of the fluorescence shown by cryosections from unvaccinated animals of the same age. The results indicate that palmitoylated Abeta peptides, reconstituted in liposomes-lipid A, are highly immunogenic, eliciting "therapeutic" antibody titers within 3 months of the first inoculation and preventing amyloid plaque formation in young animals or significantly reducing existing plaques in older transgenic mice. Possible implications for the therapy of Alzheimer's disease are discussed.

Enhanced immune protection by a liposome-encapsulated recombinant respiratory syncytial virus (RSV) vaccine using immunogenic lipids from Deinococcus radiodurans

The radiation-resistant bacterium, Deinococcus radiodurans contains a variety of phospho-, glyco- and phosphoglycolipids, the structures of which appear to be largely unique in nature. We show here that such lipids are immunogenic when administered as liposomes intranasally in mice, as evidenced by the induction of serum antibodies which recognize D. radiodurans lipids but not other lipids by thin layer chromatographic immunostaining. By modifying a liposomal vaccine against respiratory syncytial virus (RSV) we find that vaccine efficacy is markedly enhanced by the inclusion of lipids isolated from D. radiodurans. Using dioleoylphosphatidylcholine (DOPC) or D. radiodurans lipids, liposomes were prepared which encapsulated a soluble fragment of the RSV G protein (G 128–188) fused with a portion of the bacterial thioredoxin (Trx) protein. Mice immunized intranasally with D. radiodurans liposomes showed markedly greater protection against RSV challenge over mice immunized with DOPC liposomes. Enhanced vaccine efficacy was achieved using liposomes prepared from either whole D. radiodurans lipids or from a single isolated phosphoglycolipid previously identified as α-galactosylphosphatidylglyceroylalkylamine (lipid 7). Mice immunized and protected against RSV challenge were free of pulmonary eosinophilic infiltration, an undesirable consequence of many RSV vaccines. The results provide further support for liposome-based vaccines for RSV and underline the importance of lipid composition in liposome formulations.

Synthetic lipopeptides incorporated in liposomes: In vitro stimulation of the proliferation of murine splenocytes and in vivo induction of an immune response against a peptide antigen

Amphiphilic lipopeptides, such as Pam 3CysAlaGly and Pam 3CysSerSer, were synthesized and incorporated into liposomes, and their ability to induce the proliferation of BALB/c mouse splenocyte was tested in vitro. When compared to monophosphoryl lipid A (MPL) the following potency order was found: liposomal lipopeptides > liposomal MPL > free (emulsified) lipopeptides. These results strongly depend on the size of the vesicles used: a mitogenic effect was observed only with lipopeptides incorporated within vesicles of diameter ≤ 100 nm while lipopeptides in larger vesicles (diameter ≈ 300 nm) gave no response. This may be related to the necessity for the liposome-associated lipopeptides to be endocytosed to reach putative intracellular targets. As immunoadjuvanticity seems to be linked to B-lymphocyte activation, the lipopeptides represent attractive alternatives to MPL for the realization of completely synthetic liposome-based peptide vaccine formulations. This was borne out by showing that Pam 3CysAlaGly and Pam 3CysSerSer, when incorporated in small unilamellar vesicles carrying a covalently conjugated synthetic peptide of sequence IRGERA, corresponding to an epitope of the C-terminal region of histone H3, were able to induce a potent and long-lasting immune response.

Liposomal malaria vaccine in humans: a safe and potent adjuvant strategy.

This study describes the safety and immunogenicity of a liposome-based vaccine injected into human subjects. Thirty healthy adult male volunteers were immunized with a liposome-encapsulated recombinant protein (R32NS181) containing epitopes from the repeat region of the circumsporozoite protein of Plasmodium falciparum. This antigen had previously been found to be poorly immunogenic in humans when it was adsorbed with Al(OH)3. In the present study, R32NS181 was encapsulated in liposomes containing monophosphoryl lipid A that were subsequently adsorbed to Al(OH)3. Increasing doses of liposomes containing antigen and monophosphoryl lipid A were used, but the liposomes were always adsorbed to the same dose of Al(OH)3. R32-specific serum IgG antibody responses to liposome-encapsulated R32NS181 were much higher than levels attained previously in humans with R32NS181 adsorbed to Al(OH)3. Geometric mean specific IgG levels after three doses ranged from 14 to 33 micrograms/ml. Sera from volunteers receiving the two highest doses inhibited P. falciparum sporozoite invasion of cultured hepatoma cells by an average of 92%, a result that was again superior to previously reported vaccines. Moderate but acceptable transient local reactogenicity was noted at high doses of the vaccine formulation, but little or no systemic toxicity was seen despite liposomal monophosphoryl lipid A doses up to 2200 micrograms. We conclude that encapsulation of poorly immunogenic circumsporozoite protein repeat peptides in monophosphoryl lipid A-containing liposomes is a successful adjuvant strategy in humans for inducing high levels of specific antibody production.