Chitosan hydrogels containing liposomes and cubosomes as particulate sustained release vaccine delivery systems

Sustained release depot systems have been widely investigated for their potential to improve the efficacy of subunit vaccines and reduce the requirement for boosting. The present study aimed to further enhance the immunogenicity of a sustained release vaccine by combining a depot formulation with a particulate antigen delivery system. Sustained release of the model subunit antigen, ovalbumin (OVA), was observed in vivo from chitosan thermogel-based formulations containing cationic, nanosized liposomes loaded with OVA and the immunopotentiator, Quil A (QA). Such formulations demonstrated the ability to induce cluster of differentiation (CD)8⁺ and CD4⁺ T-cell proliferation and interferon (IFN)-γ production, as well as the production of OVA-specific antibody. However, gel-incorporated liposomes showed evidence of instability and similar in vivo immune responses to liposomes in gel formulations were induced by gel-based systems loaded with soluble OVA and QA. The immunogenicity of chitosan thermogels containing cubosomes, a more stable lipidic particulate system, was therefore examined. Similarly, all gel-based formulations produced comparable effector immune responses in experimental mice, irrespective of whether the antigen and immunopotentiator were present in gels within cubosomes or in a soluble form. This work demonstrates the potential for sustained release thermogelling systems and highlights the importance of matching the physicochemical and immunological properties of the particulate system to that of the depot.     

CD8+ CTL priming by exact peptide epitopes in incomplete Freund's adjuvant induces a vanishing CTL response, whereas long peptides induce sustained CTL reactivity

Therapeutic vaccination trials, in which patients with cancer were vaccinated with minimal CTL peptide in oil-in-water formulations, have met with limited success. Many of these studies were based on the promising data of mice studies, showing that vaccination with a short synthetic peptide in IFA results in protective CD8(+) T cell immunity. By use of the highly immunogenic OVA CTL peptide in IFA as a model peptide-based vaccine, we investigated why minimal CTL peptide vaccines in IFA performed so inadequately to allow full optimization of peptide vaccination. Injection of the minimal MHC class I-binding OVA(257-264) peptide in IFA transiently activated CD8(+) effector T cells, which eventually failed to undergo secondary expansion or to kill target cells, as a result of a sustained and systemic presentation of the CTL peptides gradually leaking out of the IFA depot without systemic danger signals. Complementation of this vaccine with the MHC class II-binding Th peptide (OVA(323-339)) restored both secondary expansion and in vivo effector functions of CD8(+) T cells. Simply extending the CTL peptide to a length of 30 aa also preserved these CD8(+) T cell functions, independent of T cell help, because the longer CTL peptide was predominantly presented in the locally inflamed draining lymph node. Importantly, these functional differences were reproduced in two additional model Ag systems. Our data clearly show why priming of CTL with minimal peptide epitopes in IFA is suboptimal, and demonstrate that the use of longer versions of these CTL peptide epitopes ensures the induction of sustained effector CD8(+) T cell reactivity in vivo.     

Effect of liposomal formulations and immunostimulating peptidoglycan monomer (PGM) on the immune reaction to ovalbumin in mice

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDpm(epsilonNH2)-D-Ala-D-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).     

Effect of Liposomal Formulations and Immunostimulating Peptidoglycan Monomer (PGM) on the Immune Reaction to Ovalbumin in Mice

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-l-Ala-d-isoGln-mesoDpm(εNH₂)-d-Ala-d-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).     

Structural characterization of archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) formulations prepared by different protocols and their efficacy upon intranasal immunization of mice

Intranasal administration of ovalbumin (OVA) formulated in an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system prepared by the addition of CaCl2 to small unilamellar archaeosomes (liposomes made from archaeal polar lipids) containing encapsulated OVA, was recently shown to elicit strong and sustained OVA-specific mucosal and systemic immune responses. In this study, we show that the centrifugation/washing and antigen quantization steps required in the standard protocol for obtaining OVA/AMVAD model vaccine formulations can be eliminated by using simpler protocols such as admixing OVA with preformed empty archaeosomes, or by changing the starting ratio (w/w) of archaeal lipid to antigen at the archaeosome preparation stage, prior to the addition of CaCl2 to convert to the AMVAD structures. Irrespective of the vaccine preparation protocol, the AMVAD particle typically comprised of larger spherical structures that had aggregated like a bunch of grapes, and it contained aqueous compartment(s). The anti-OVA IgA antibody responses in vaginal wash, nasal wash, serum, and bile samples, and the anti-OVA IgG antibody responses in sera, in mice intranasally immunized with the OVA/AMVAD formulations prepared by the simplified or the standard protocols, were comparable.     

Design and development of multivesicular liposomal depot delivery system for controlled systemic delivery of acyclovir sodium

The aim of the present study was to design a depot delivery system of acyclovir sodium using multivesicular liposomes (MVLs) to overcome the limitations of conventional therapies and to investigate its in vivo effectiveness for sustained delivery. MVLs of acyclovir were prepared by the reverse phase evaporation method. The loading efficiency of the MVLs (45%–82%) was found to be 3 to 6 times higher than conventional multilamellar vesicles (MLVs). The in vitro release of acyclovir from MVL formulations was found to be in a sustained manner and only 70% of drug was released in 96 hours, whereas conventional MLVs released 80% of drug in 16 hours. Following intradermal administration to Wistar rats, the MVL formulations showed effective plasma concentration for 48 hours compared with MLVs and free drug solution (12–16 hours). C_max values of MVL formulations were significantly less (8.6–11.4 μg/mL) than MLV and free drug solution (12.5 μg/mL). The AUC_0–48 of the MVL formulations was 1.5- and 3-fold higher compared with conventional liposomes and free drug solution, respectively. Overall, formulations containing phosphatidyl glycerol as negatively charged lipid showed better results. The MVL delivery system as an intradermal depot offers the advantage of a very high loading and controlled release of acyclovir for an extended period of time. The increase in AUC and decrease in C_max reflects that the MVL formulations could reduce the toxic complications and limitations of conventional IV and oral therapies. Published: September 20, 2005     

Immunoglobulin immobilized liposomal constructs for transmucosal vaccination through nasal route

The aim of the present investigation was to evaluate the prospective of surface-engineered vesicular carriers for mucosal immunization via the nasal route. IgG antibody was immobilized on the surface of hepatitis B surface antigen (HBsAg) antigen–loaded liposomes. The developed formulations were characterized on the basis of physicochemical parameters, such as morphology, particle size, polydispersity index, entrapment efficiency, and zeta potential. Liposomal formulations were then evaluated for in-process antigen stability and storage stability. In vivo studies were conducted to visualize targeting potential, localization pattern, and immunogenicity. In addition, immune response was compared with alum-HBsAg vaccine injected intramuscularly. The serum anti-HBsAg titer, obtained from the postnasal administration of IgG-coupled liposomes, was significantly higher than plain liposomes. Moreover, IgG-coupled liposomes generated both humoral (i.e., systemic and mucosal) and cellular immune responses upon nasal administration, while the alum-adsorbed antigen displayed neither cellular (cytokine level) nor mucosal (IgA) response. The formulation also displayed enhanced transmucosal transport, improved in vitro stability, and effective immunoadjuvant property. To conclude, IgG antibody-coupled liposomes may serve as novel carriers to augment the secretory immune response of antigen encapsulated in the liposomes, apparently by escalating liposome uptake via M cells, thereby rationalizing their use as a carrier adjuvant for nasal subunit vaccines.     

Interleukin-2/liposomes potentiate immune responses to a soluble protein cancer vaccine in mice

A critical element in improving the potency of cancer vaccines, especially pure protein or peptide antigens, is to develop procedures that can strongly but safely increase their ability to induce immune responses. Here, we describe that encapsulation of a pure protein antigen and interleukin-2 (IL-2) together into liposomes significantly improves immune responses and tumor protection. Groups of C57Bl/6 mice were immunized weekly ×4 with –0.1 mg of ovalbumin (OVA) injected subcutaneously in PBS or encapsulated in liposomes with or without human recombinant IL-2. Control groups included mice immunized to irradiated E.G7-OVA cells (that express ovalbumin), or to PBS. Sera were collected and pooled by immunization group at baseline and at weeks 2 and 4 to measure antibody responses to OVA by ELISA. Splenocytes obtained at week 4 were tested for anti-OVA cellular responses by ELISPOT. Mice were then challenged to a lethal dose of E.G7-OVA cells to measure tumor-protective immunity. IL-2 liposomes caused no detectable toxicity. Antibody, CD8⁺ T cell, and tumor-protective immune responses were markedly enhanced in mice immunized to OVA + IL-2 in liposomes compared to mice immunized to OVA, either alone or encapsulated into liposomes without IL-2. These results indicate that IL-2 liposomes enhance antibody, cellular, and tumor-protective immune responses to immunization with a soluble protein. This may provide a simple, safe, and effective way to enhance the immunogenicity of vaccines that consist of pure protein antigens. Supported by grant CA096804 (DJ)     

Characterization and evaluation of stabilized particulate formulations as therapeutic oral vaccines for allergy

Abstract

Allergic conditions affect more than a quarter of the population in developed countries, but currently available treatments focus more on symptom relief than treating the underlying atopic condition. α-Galactosylceramide (α-GalCer) is a potent immunomodulating compound that has been shown to have a regulatory effect when delivered systemically in nanoparticles. Parenteral delivery is not preferred for chronic conditions, such as allergy, and therefore, the aim of this study was to determine whether a regulatory response could be induced through oral administration in a model of atopy through incorporation of α-GalCer into stable particulate formulations (cationic liposomes, polymerized liposomes and poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs)). The formulations showed only minor changes in particle size, polydispersity index and retention of the model antigen ovalbumin (OVA) during incubation in simulated gastrointestinal (GI) conditions. Oral delivery of α-GalCer in cationic liposomes could induce immunostimulating effects systemically, as seen through increases in serum IgG antibody levels, whereas delivery of α-GalCer in polymerized liposomes and PLGA NPs induced local cytokine changes in the mesenteric lymph nodes (MLNs). The generated responses did not exhibit tolerogenic traits which could be useful for immunoregulation, but the responses generated varied between formulations and suggests that further characterization and optimization could lead to the desired immune response.     

Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help

The unique ether glycerolipids of ARCHAEA: can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8(+) T cell response, as measured by IFN-gamma production and cytolytic activity toward the immunodominant CTL epitope OVA(257-264). In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8(+) T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4(+) T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8(+) T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8(+) T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8(+) T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.     

Oral delivery of antigens in liposomes with some lipid compositions modulates oral tolerance to the antigens

Several liposomes containing ovalbumin (OVA), a model antigen, with different lipid compositions were prepared in order to evaluate their ability to induce oral tolerance. Oral administration of these liposomal OVAs induced suppression of the proliferative responses of popliteal lymph node cells from the treated mice to OVA, suggesting that these treated mice were tolerized. The efficiency of the induction of oral tolerance was affected by the liposome composition. OVA entrapment in these liposomes could modulate the tolerizing dose of OVA itself. These results suggest that some liposomes can be suitable antigen-delivery systems for modulated and/or effective induction of oral tolerance.     

Immunogenicity is unrelated to protective immunity when induced by soluble and particulate antigens from Nocardia brasiliensis in BALB/c mice

Cell-mediated immunity plays a major role in protection against intracellular microbes. Nocardia brasiliensis is a facultative intracellular pathogen that causes chronic actinomycetoma. In this work, we injected BALB/c mice with soluble P24 and particulate antigens from N. brasiliensis. A higher antibody titer and lymphocyte proliferation was induced by the particulate antigen than by the soluble antigen. However, five months after antigen injection, antibody concentration and lymphocyte proliferation were similar. An increase in CD45R and CD4 T cells was unrelated to protective immunity. Active immunization with soluble or particulate antigens induced complete protection during the primary immune response. This protective response was IgM mediated. The higher immunogenicity was not related to protective immunity since the particulate antigen induced protection similar to the soluble antigen. Using particulate antigens for vaccination guarantees a stronger immune response, local and systemic side effects, but not necessarily protection.     

VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph node lymphatics

Tumor expression of the lymphangiogenic factor VEGF-C is correlated with metastasis and poor prognosis, and although VEGF-C enhances transport to the draining lymph node (dLN) and antigen exposure to the adaptive immune system, its role in tumor immunity remains unexplored. Here, we demonstrate that VEGF-C promotes immune tolerance in murine melanoma. In B16 F10 melanomas expressing a foreign antigen (OVA), VEGF-C protected tumors against preexisting antitumor immunity and promoted local deletion of OVA-specific CD8(+) T cells. Naive OVA-specific CD8(+) T cells, transferred into tumor-bearing mice, were dysfunctionally activated and apoptotic. Lymphatic endothelial cells (LECs) in dLNs cross-presented OVA, and naive LECs scavenge and cross-present OVA in vitro. Cross-presenting LECs drove the proliferation and apoptosis of OVA-specific CD8(+) T cells ex vivo. Our findings introduce a tumor-promoting role for lymphatics in the tumor and dLN and suggest that lymphatic endothelium in the local microenvironment may be a target for immunomodulation.     

The Inclusion of Chitosan in Poly-ε-caprolactone Nanoparticles: Impact on the Delivery System Characteristics and on the Adsorbed Ovalbumin Secondary Structure

This report extensively explores the benefits of including chitosan into poly-ε-caprolactone (PCL) nanoparticles (NPs) to obtain an improved protein/antigen delivery system. Blend NPs (PCL/chitosan NPs) showed improved protein adsorption efficacy (84%) in low shear stress and aqueous environment, suggesting that a synergistic effect between PCL hydrophobic nature and the positive charges of chitosan present at the particle surface was responsible for protein interaction. Additionally, thermal analysis suggested the blend NPs were more stable than the isolated polymers and cytotoxicity assays in a primary cell culture revealed chitosan inclusion in PCL NPs reduced the toxicity of the delivery system. A quantitative 6-month stability study showed that the inclusion of chitosan in PCL NPs did not induce a change in adsorbed ovalbumin (OVA) secondary structure characterized by the increase in the unordered conformation (random coil), as it was observed for OVA adsorbed to chitosan NPs. Additionally, the slight conformational changes occurred, are not expected to compromise ovalbumin secondary structure and activity, during a 6-month storage even at high temperatures (45°C). In simulated biological fluids, PCL/chitosan NPs showed an advantageous release profile for oral delivery. Overall, the combination of PCL and chitosan characteristics provide PCL/chitosan NPs valuable features particularly important to the development of vaccines for developing countries, where it is difficult to ensure cold chain transportation and non-parenteral formulations would be preferred.     

Effect of incorporation of the adjuvant Quil A on structure and immune stimulatory capacity of liposomes

Liposomes have been widely used as drug delivery systems for many years. However, they are of limited use as delivery systems for subunit vaccines due to their low immunogenicity. Here we examine the effect of incorporating the adjuvant Quil A into liposomes on the type of particles produced, on the ability of the different particles to incorporate antigen and on the ability of the different particles to stimulate murine bone-marrow-derived dendritic cells (DC) and lymphocytes. The incorporation of increasing amounts of Quil A, from 20% to 70% of the total lipid into liposomes, reduces the size of the particles that form in aqueous dispersion and decreases antigen incorporation and uptake by DC. Interestingly, the particles with 20% Quil A were more toxic to cells in culture than the particles containing 70% Quil A, and the 20% particles were also more immunostimulatory.     

Liposomes with differential lipid components exert differential adjuvanticity in antigen-liposome conjugates via differential recognition by macrophages

We previously reported that liposomes having differential lipid components displayed differential adjuvant effects when antigen was coupled with liposomes via glutaraldehyde. In the present study, antigen-liposome conjugates prepared using liposomes having differential lipid components were added to the macrophage culture, and phagocytosis and the antigen digest of liposome-coupled antigen by macrophages were then investigated. Antigen presentation by macrophages to an antigen-specific T-cell clone was further investigated using the same conjugates. Antigen-liposome conjugates which induced higher levels of antibody production in vivo were recognized more often, and the liposome-coupled antigen was digested to a greater degree by macrophages than antigen-liposome conjugates which induced lower levels of antibody production. These results correlated closely with those regarding antigen presentation by macrophages; when antigen was coupled to liposomes showing higher adjuvant effect, macrophages cocultured with antigen-liposome conjugates activated antigen-specific T-cells at a higher degree. The concentration of OVA in the macrophage culture added as antigen-liposome conjugates was approximately 32 microg/mL. However, the extent of T-cell activation was almost equal to that when 800 microg/mL of soluble OVA was added to the culture. The results of the present study demonstrated that the adjuvant activity of liposomes observed primary in vivo correlated closely with the recognition of antigen-liposome conjugates and antigen presentation of liposome-coupled antigen by macrophages, suggesting that the adjuvant effects of liposomes are exerted at the beginning of the immune response, i.e., recognition of antigen by antigen-presenting cells.     

Preparation and characterization of iron-containing liposomes: their effect on soluble iron uptake by Caco-2 cells

The aim of this work was to study the iron uptake of Caco-2 cells incubated with five different formulations of liposomes containing iron. The vesicles were also characterized before, during, and after in vitro digestion. Caco-2 cells were incubated with digested and nondigested liposomes, and soluble iron uptake was determined. Nondigested liposomes made with chitosan (CHI) or the cationic lipid, DC-Cholesterol (DC-CHOL), generated the highest iron uptake. However, these two formulations were highly unstable under in vitro digestion, resulting in nonmeasurable iron uptake. Digested conventional liposomes composed of soybean phosphatidylcholine (SPC), hydrogentated phosphatidylcholine (HSPC), or HSPC and cholesterol (CHOL) presented the highest iron-uptake values. These liposomal formulations protected iron from oxidation and improved iron uptake from intestinal cells, compared to an aqueous solution of ferrous sulphate.     

Antigen delivery to mucosa-associated lymphoid tissues using liposomes as a carrier

Oral vaccination requires an antigen delivery vehicle to protect the antigen and to enhance translocation of the antigen to the mucosa-associated lymphoid tissue. A variety of antigen delivery vehicles including liposomes have been studied for mucosal immunization. The advantages of liposome formulations are their particulate form and the ability to accommodate immunomodulators and targeting molecules in the same package. Many conventional liposomes are variably unstable in acids, pancreatic juice and bile. Nevertheless, carefully designed liposomes have demonstrated an impressive efficacy in inducing mucosal IgA responses, compared to free antigens and other delivery vehicles. However, liposomes as an oral vaccine vehicle are not yet optimized. To design liposomes that are stable in the harsh intestinal environment and are efficiently taken up by the M cells remains a challenge. This review summarizes recent research efforts using liposomes as an antigen carrier for oral vaccines with practical attention to liposome designs and interaction with the M cells.     

A soluble lymphocyte activation gene-3 molecule used as a vaccine adjuvant elicits greater humoral and cellular immune responses to both particulate and soluble antigens

The lymphocyte activation gene-3 (LAG-3) product is a MHC class II ligand that has been used in vivo to stimulate MHC class II+ APCs to increase tumor-specific immune responses. We investigated whether LAG-3 could also play an adjuvant role in vivo for the induction of humoral and CD4 or CD8 cell-mediated immune responses when immunizing mice with a particulate (hepatitis B surface Ag) or soluble (OVA) Ag. In both cases, coadministration of 1 microg of a soluble fusion protein between murine LAG-3 and the Fc fraction of a murine IgG2a mAb (mLAG-3Ig) as a vaccine adjuvant induced or increased CTL responses to the corresponding MHC class I-restricted peptide. In addition, splenocytes of mice vaccinated with either the particulate or soluble Ag plus mLAG-3Ig exhibited a significantly greater proliferative response than did splenocytes of mice immunized with Ag and a control Ig molecule. Similarly, these splenocytes had a greater Th1- but not Th2-type cytokine response. Finally, mice immunized with Ag plus mLAG-3Ig produced higher titers of Abs than mice immunized with Ag and a control Ig molecule. Thus, these data provide evidence of a novel means of improving the immunogenicity of subunit vaccines.     

Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses

Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.