Proteoliposome Research Articles

Immunogenicity of a novel anti-allergic vaccine based on house dust mite purified allergens and a combination adjuvant in a murine prophylactic model.

The outer-membrane-derived proteoliposome (PL) of Neisseria meningitidis has been reported as a potent vaccine adjuvant, inducing a Th1-skewed response. This work aimed to assess the immunogenicity of a novel anti-allergic vaccine candidate based on allergens from Dermatophagoides siboney house dust mite and a combination adjuvant containing PL and Alum. In a preventative experimental setting, BALB/c mice were administered with three doses containing 2 µg of Der s1 and 0.4 µg Der s2 allergen, PL and Alum, at 7 days intervals, by subcutaneous route. Furthermore, mice were subjected to an allergen aerosol challenge for 6 consecutive days. Serum IgE, IgG1, and IgG2a allergen-specific antibodies were assessed by ELISA. Cytokine levels in supernatants of D. siboney stimulated lymphocyte cultures and in bronchoalveolar lavage (BAL) were measured by ELISA. Lung tissues were subjected to histological examination. The vaccine prevented the development of both, systemic (IgE) and local allergic responses (featuring lower IL-4, and IL-5 levels in BAL) upon allergen exposure by the inhalant route. Histological examination showed also a diminished allergic inflammatory response in the lungs. After the allergen challenge, cytokine levels in stimulated lymphocyte cultures showed lower values of IL-13 and augmented IFN-γ and IL-10. The vaccine induced a mixed IgG2a/IgG1 antibody response; although only IgG2a was PL-dependent. Both, IgG1/IgE and IgG2a/IgE ratios, showed significantly greater values in vaccinated mice. The findings support a preventative anti-allergic effect associated with the induction of a Th1-like IFN-γ/IL-10 response. IgG1/IgE and IgG2a/IgE ratios could be useful biomarkers for translation into clinical trials.

Kinetic characterisation and inhibitor sensitivity of Candida albicans and Candida auris recombinant AOX expressed in a self-assembled proteoliposome system

Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality. Candidemia incidence has been rising worldwide following increases in fungicide-resistant pathogens highlighting the need for more effective antifungal agents with novel modes of action. The membrane-bound enzyme alternative oxidase (AOX) promotes fungicide resistance and is absent in humans making it a desirable therapeutic target. However, the lipophilic nature of the AOX substrate (ubiquinol-10) has hindered its kinetic characterisation in physiologically-relevant conditions. Here, we present the purification and expression of recombinant AOXs from C. albicans and C. auris in a self-assembled proteoliposome (PL) system. Kinetic parameters (Km and Vmax) with respect to ubiquinol-10 have been determined. The PL system has also been employed in dose–response assays with novel AOX inhibitors. Such information is critical for the future development of novel treatments for Candidemia.

Safety of a proteoliposome from Neisseria meningitides as adjuvant for a house dust mite allergy vaccine

The proteoliposome (PL) of Neisseria meningitidis serogroup B has been reported as a safe and potent vaccine adjuvant, inducing a TH1-skewed response. The present study describes a pre-clinical safety evaluation of an allergy therapeutic vaccine candidate based on purified allergens from Dermatophagoides siboney house dust mite and PL as adjuvant, both components adsorbed onto aluminum hydroxide gel. Two separate studies of acute toxicity evaluation were performed in mice and rabbits, and two repeat-dose studies were conducted in non-sensitized and allergen-sensitized Balb/c mice, respectively. The study in sensitized mice intends to model a therapeutic setting. Aerosolized allergen challenge was used in both settings to model natural respiratory exposure. In the therapeutic setting, mice were administered with three doses containing 2 μg allergen at weekly intervals [subcutaneous route] and subsequently challenged with aerosolized allergen for 6 consecutive days. Parameters of general toxicity effects were assessed via measures of behavior, body weight, food and water consumption, and macroscopic evaluation of organs. Histological examination of organs and the injection site was performed. Potential immunotoxicity effects at the systemic level were assessed by blood eosinophil counting and serum allergen specific IgE by ELISA The vaccine did not produce general or functional toxic effects of significance, at a dose up to 100 μg allergen per kg body weight. An expected local reaction at the injection site was observed, which could be attributed mostly to the immunological effect of aluminum hydroxide. The models implemented here suggest an acceptable safety profile of this vaccine for testing in clinical trials of allergy immunotherapy.

Specific and cross-reactive immune response against Mycobacterium tuberculosis antigens in mice immunized with proteoliposomes from Mycobacterium bovis BCG

ObjectiveTo characterize the immunogenicity and the induction of cross-reactive responses against Mycobacterium tuberculosis (M. tuberculosis) of a proteoliposome (PL) from Mycobacterium bovis Bacillus Calmette–Guérin (BCG) with and without alum hydroxide (AL) as adjuvant (PLBCG-AL and PLBCG, respectively) in BALB/c mice. MethodsBALB/c mice were inoculated with phosphate buffer solution, BCG, PLBCG and PLBCG-AL. The humoral immunogenicity was determined by ELISA [immunoglobulin G (IgG), IgG1 and IgG2a] and the cellular immunogenicity was evaluated in vivo by delayed type hypersensitivity. The humoral cross-reactive response against M. tuberculosis was determined by Western blot. ResultsSera from animals immunized with PLBCG-AL and PLBCG showed significant increase in specific total IgG and IgG1 antibodies and the presence of cross-reactive antibodies against M. tuberculosis antigens, which were more intense with the use of alum as adjuvant. Mice immunized with PLBCG and PLBCG-AL also showed a specific cellular response in vivo. ConclusionsThe cellular and humoral immunogenicity of PLBCG and the capacity to induce cross-reactive responses against M. tuberculosis is in agreement with the protective capacity previously demonstrated by this vaccine candidate and supports the continuation of its evaluation in further stages.

Mucosal adjuvants

Mucosal adjuvants

The Application of a Proteoliposome Adjuvant System in the Development of a Campylobacter Jejuni Vaccine

The high incidence of Campylobacter jejuni-associated diarrhoea, the increase in the frequency of drug resistance, and the correlation between infection and Guillain-Barré syndrome, has heightened the need to develop effective anti-Campylobacter vaccines. Due to the risk of auto-immunity, vaccinations using whole-cells or attenuated cells are not clinical candidates. Subunit vaccines are viable alternatives and have been shown to be effective against other enteric pathogens. This research tested the efficacy of the catalase (KatA) from C. jejuni as a potential vaccine candidate against the pathogen. The protein was found to be immunogenic following an intramuscular murine immunization. A balanced IgG1/IgG2a response was generated when KatA was co-administered with monophosphoryl lipid A and alum. The antibodies elicited reduced the adhesion and invasion of C. jejuni to human epithelial cells (IECs) in vitro. The next phase of testing will be in combination with a proteoliposome (PL) derived from the outer membrane of V. cholerae. PL nano-vesicles were generated with an average size of ≈70nm and had a zeta potential of ≈-40mV.

プロテオリポソーム工学：膜タンパク質組込みリポソーム構築のためのシャペロン科学

プロテオリポソーム工学：膜タンパク質組込みリポソーム構築のためのシャペロン科学

Proteoliposome and Polysaccharide-Based Meningococcal Vaccine Are Immunogenic in Infants and Toddlers and Primes for Memory against Serogroup C Polysaccharide

Neisseria meningitidis capsular polysaccharides are the main target of the protective immune response against bacterial meningitis. They are thymus-independent type 2 (TI-2) antigens that are poorly immunogenic and not protective in young children, and their administration may impair subsequent challenge with the same polysaccharide. These problems have been addressed using three different vaccines consisting of 1) polysaccharide alone, 2) polysaccharide covalently conjugated to a carrier protein, and 3) polysaccharide with Proteoliposome (PL) adsorbed onto Al(OH)3. VA-MENGOC-BC ® is one of the third types of vaccine. It contains PL (detergent-extracted external membrane proteins forming vesicles) and polysaccharide (Ps) from N. meningitidis serogroups B and C (PsC), respectively. Nevertheless, there is a concern that to overcome the TI-2 nature of Ps the covalently conjugation to a carrier is mandatory. Therefore, we evaluated the immune response induced by VA-MENGOC-BC ® in infants and toddlers in order to determine whether it stimulates the response against the PsC. High IgG anti PsC and anti PL responses were seen following the administration of two doses in infants and toddlers, after a 3 rd dose in pre-teenagers, and after confirmed carrier stages in young adults. The anti PL IgG response persisted longer than anti PsC IgG response and IgM response against both antigens was maintained. An IgG1 anti PL response predominated, as well as IgG4 > IgG3 > IgG1 anti PsC responses. These results suggest that non-covalent incorporation of PsC onto Al(OH)3 containing PL as adjuvant is immunogenic, primes for memory, and induces long-lasting specific antibody response. The improved PsC immunogenicity of this vaccine may be due to the preferential and potent Th1 response induced in mice and human by the PL as adjuvant.

Immunotherapy – 2068. Immunomodulatory and safety profile of a novel anti-allergic vaccine based on allergens from dermatophagoides siboney and a combination adjuvant, in sensitized mice

This article was originally published online on 23 April 2013 Th1/Tr1 promoting adjuvants, in allergen-specific vaccines, could be a valuable alternative to the current allergen immunotherapy. It is now the pro-Th1 adjuvant effect of Neisseria meningitidis proteoliposome (PL). Previous studies in a prophylactic murine model of respiratory allergy the vaccine showed its immunogenicity and safety. To assess the immunogenicity and non-clinical safety profile of a novel anti-allergic vaccine candidate based on purified allergens from Dermatophagoides siboney and a combination adjuvant containing PL and Alum, in sensitized mice. In therapeutic experimental setting C57/Bl6 mice were first sensitized administering D. siboney allergen by IP route and exposing mice to allergen aerosols. Later, allergic mice were treated with 3 doses of the adjuvanted vaccine (2µg of Der s1) by subcutaneous route. Further, mice were subjected to inhalation allergen challenge. IgE, IgG1, and IgG2a allergen-specific antibody response was measured by ELISA. The specific cellular response (IL-13, IL-10 and INF-g), were evaluated in supernatants of Lymphocyte cultures stimulated with the allergen, by eBioscience kit for FACS. Systemic toxicity was assessed measuring body weight and macroscopic and histological examination of several organs and injection site. The experimental model reproduces the allergic condition with allergen-specific IgE production; increased blood eosinophils and Histology of lung tissues show all the characteristic of allergy inflammation. As in the previously tested preventive model, the vaccine induces a strong IgG and IgG1 antibody responses significantly higher than control groups, and moderate levels of IFN-g, although together with Tr1 (IL-10) and Th2 (Il-13) cytokines. After challenge, it was noted a significant increase (p<0.05) of the IgG/IgE ratio, with a decrease in bloods eosinophils and allergic inflammation in lung tissues as compared to placebo. No significant differences between body weights and differential leukocyte count in treated mice and placebo group was observed. Normal histology was observed. Local tolerance at the injection site suggests that granulomes in vaccinated subjects are caused by alum. The adjuvanted vaccine does not exacerbate the allergic response nor promote Th1 inflammation, supporting a satisfactory safety profile for further clinical trials in humans. This immunomodulatory effect suggests clinical benefits both in cellular and blocking antibody responses.

A proteoliposome containing apolipoprotein A-I mutant (V156K) enhances rapid tumor regression activity of human origin oncolytic adenovirus in tumor-bearing zebrafish and mice.

We recently reported that the efficiency of adenoviral gene delivery and virus stability are significantly enhanced when a proteoliposome (PL) containing apolipoprotein (apo) A-I is used in an animal model. In the current study, we tested tumor removal activity of oncolytic adenovirus (Ad) using PL-containing wildtype (WT) or V156K. Oncolytic Ad with or without PL was injected into tumors of zebrafish and nude mice as a Hep3B tumor xenograft model. The V156K-PL-Ad-injected zebrafish, group showed the lowest tumor tissue volume and nucleic acids in the tumor area, whereas injection of Ad alone did not result in adequate removal of tumor activity. Reactive oxygen species (ROS) contents increased two-fold in tumor-bearing zebrafish; however, the V156K-PL-Ad injected group showed a 40% decrease in ROS levels compared to that in normal zebrafish. After reducing the tumor volume with the V156K-PL-Ad injection, the swimming pattern of the zebrafish changed to be more active and energetic. The oncolytic effect of PL-Ad containing either V156K or WT was about two-fold more enhanced in mice than that of Ad alone 34 days after the injection. Immunohistochemical analysis revealed that the PL-Ad-injected groups showed enhanced efficiency of viral delivery with elevated Ad-E1A staining and a diminished number of proliferating tumor cells. Thus, the antitumor effect of oncolytic Ad was strongly enhanced by a PL-containing apoA-I and its mutant (V156K) without causing side effects in mice and zebrafish models.

106 Neisseria Meningitidis Derived Proteoliposome as an Adjuvant for Allergen Vaccines

BackgroundIn recent years one important trend of Allergen-specific immunotherapy is to investigate new adjuvants with immunomodulatory properties. The outer membrane vesicle or proteoliposome (PL) from Neisseria meningitidis serogroup B has been reported as a potent adjuvant inducing a Th1-skewed response. The aim of this work was to assess the immunogenicity of a novel anti-allergic vaccine candidate based on purified allergens from Dermatophagoides siboney mite and PL as adjuvant, both components adsorbed onto Aluminum hydroxide.MethodsIn a preventative experimental setting BALB/c mice were administered with 3 doses containing 5 μg of Der s 1 allergen at one week intervals by subcutaneous route. Further, mice were subjected to allergen challenge by aerosol inhalation. In another experiment, mice were administered first with 2 doses of PL + Alum and later with the whole vaccines formulation, including the allergen. The allergen-specific antibody response was assessed determining serum levels of IgE, IgG1, and IgG2a by ELISA. The local allergic inflammatory response was evaluated by measuring cytokine levels (IL-4, IL-5, IFNg and IL-10) in broncho-alveolar lavage (BAL) by ELISA.ResultsThe formulation consistently induced IgG2a, as well as IgG1 antibodies with a potential anti-IgE blocking effect. The induction of IgG2a was clearly PL dependent while IgG1 was dependent mostly of Alum. Prior administration of the proteoliposome with alum without allergen showed to enhance this allergen-specific immunogenic effect. The vaccine prevented the development of systemic (IgE) and local allergic response in mice subjected to allergen exposure by inhalant route. Vaccinated mice showed lower levels of serum IgE, Th2 cytokines (IL-4, IL-5) in BAL and lower eosinophil counting in blood as compared to controls. Histological examination of lungs showed also a diminished allergic inflammatory response in vaccinated mice in contrast with mice which were administered with the conventional formulation of Alum-adsorbed allergen.ConclusionsThe antiallergic protective effect was proven in a preventative setting, showing to decrease the inflammatory response in the lungs of mice exposed to allergen aerosol, as well as, a Th2-antagonistic immune response with few injections.

A proteoliposome based formulation administered by the nasal route produces vibriocidal antibodies against El Tor Ogawa Vibrio cholerae O1 in BALB/c mice

A vaccine candidate against the enteric pathogen Vibrio cholerae was developed based on a proteoliposome (PL) formulation using a wild type strain C7258, V. cholerae O1, El Tor Ogawa as part of strategy to develop a combined formulation against enteric diseases preventable by the stimulation of the mucosal immune system. A detergent extraction method was applied to obtain the PL. Scanning electron microscopy and molecular exclusion chromatography showed the presence of two PL populations. Photon correlation spectroscopy studies were then carried out to evaluate the size (169.27 ± 3.85 nm), polydispersity (0.410) and zeta potential (−23.28 ± 1.21 mV) of the PL. SDS-PAGE and Western blot analysis revealed the presence of lipopolysaccharide (LPS), mannose-sensitive haemagglutinin (MSHA) and a range of outer membrane proteins, including OmpU. BALB/c mice were immunized intranasally with two doses of PL containing 25 μg of LPS each 28 days apart. The mice showed high anti-LPS IgG titres (3.36 ± 0.235) and vibriocidal antibodies (3.70 ± 0.23) after two weeks from last dose. These results show for the first time that PL can be obtained from V. cholerae O1 and when administer by intranasal route has the potential to protect against this pathogen.

Immunological evaluation of bacterial derived Cochleate and Proteoliposome as mucosal adjuvants

We evaluated the potential of two bacterial derived compounds, Cochleate and Proteoliposome (PL), administrated to mice by nasal or oral routes on induction of antibody and cytokine responses. Anti PL IgG and IgA responses were measured by ELISA in saliva, sera or vaginal fluids of immunized mice. Productions of γIFN and IL-5 were determined in spleen cells of immunized mice following a recall in vitro with Cochleate or PL. Intranasal administration elicited a higher anti PL IgA response in both saliva and vaginal fluids as compared with oral route. Mice immunized with Cochleate or PL via intranasal or oral route-induced anti PL IgG and IgG2a antibody responses in their sera and vaginal fluids. Spleen cells from these immunized mice produced γIFN, but not IL-5, after a recall in vitro with Cochleate or PL. These results show that Cochleate and PL are capable of inducing both systemic and mucosal antibody responses as well as a Th1 type of immunity as evidenced by high γIFN and IgG2a antibody responses.

Proteoliposome derived cochleate as novel adjuvant

Cochleate structures (CS) consist in a highly stable lipid structures that have been reported to be a good antigen delivery system. The incorporation of pathogen associated molecular pattern (PAMP) from bacterial membranes into CS became in a promising approach to develop adjuvants, particularly mucosal adjuvants. Therefore, we prepare CS from proteoliposome (PL) obtained from Neisseria meningitidis B (PLCS) and evaluated it for its capability to stimulate the immune system as well as the adjuvant activity. The ability of PLCS to induce Th1 polarization was also explored. The results and the easy capability for new antigen incorporation on CS support its use as adjuvant for immunization with a large variety of pathogen derived antigens and different routes of immunization.

Bacterial derived proteoliposome as ideal delivery system and cellular adjuvant

We explored the potential of a proteoliposome (PL) from the outer membrane of N. meningitidis B, as an immunopotentiator and as a vector for antigen delivery to dendritic cells (DC). DC were incubated with PL resulting in up-regulation of MHC-II, CD40, CD80, and CD86 expression and production of TNFα and IL12(p70). Ovoalbumin (OVA) was incorporated within PL (PL-OVA). PL-OVA presented OVA-specific peptides to CD4 + and CD8 + OVA-specific T-cell hybridomas. PL exerts an immunomodulatory effect on DC and is a general system to deliver antigens for presentation to CD4 + and CD8 + T-cells possibly implicated in the induction CD8 + cytotoxic T lymphocytes (CTLs) responses.

Proteliposome-derived Cochleate as an immunomodulator for nasal vaccine

Proteoliposome (PL) has been recently used as a protective intramuscular (i.m.) anti-meningococcal BC vaccine. It induces a preferential Th1 type of immune response. Nevertheless, mucosal protection is mainly mediated by IgA antibody response, which is not usually induced by i.m. vaccination route. IgA antibody production needs the stimulation of Th3 subpopulation, which is also related to the induction of small dose tolerance. We hypothesized that PL-derived Cochleate can induce a specific mucosal IgA and systemic IgG antibody responses. We could show that mice immunized with two or three intranasal doses of PL-derived Cochleate developed significantly increased levels of local anti PL IgA and systemic IgG antibody responses. Thus, our results suggest that PL-derived Cochleate can be used as a promising immunomodulator and delivery system for the development of mucosal, particularly nasal vaccines.