Chiung-Yi Huang

Emulsified Nanoparticles Containing Inactivated Influenza Virus and CpG Oligodeoxynucleotides Critically Influences the Host Immune Responses in Mice

Background Antigen sparing and cross-protective immunity are regarded as crucial in pandemic influenza vaccine development. Both targets can be achieved by adjuvantation strategy to elicit a robust and broadened immune response. We assessed the immunogenicity of an inactivated H5N1 whole-virion vaccine (A/Vietnam/1194/2004 NIBRG-14, clade 1) formulated with emulsified nanoparticles and investigated whether it can induce cross-clade protecting immunity. Methodology/Principal Findings After formulation with PELC, a proprietary water-in-oil-in-water nanoemulsion comprising of bioresorbable polymer/Span®85/squalene, inactivated virus was intramuscularly administered to mice in either one-dose or two-dose schedule. We found that the antigen-specific serum antibody responses elicited after two doses of non-adjuvanted vaccine were lower than those observed after a single dose of adjuvanted vaccine, PELC and the conventional alum adjuvant as well. Moreover, 5 µg HA of PELC-formulated inactivated virus were capable of inducing higher antibodies than those obtained from alum-adjuvanted vaccine. In single-dose study, we found that encapsulating inactivated virus into emulsified PELC nanoparticles could induce better antibody responses than those formulated with PELC-adsorbed vaccine. However, the potency was rather reduced when the inactivated virus and CpG (an immunostimulatory oligodeoxynucleotide containing unmethylated cytosine-guanosine motifs) were co-encapsulated within the emulsion. Finally, the mice who received PELC/CpG(adsorption)-vaccine could easily and quickly reach 100% of seroprotection against a homologous virus strain and effective cross-protection against a heterologous virus strain (A/Whooper swan/Mongolia/244/2005, clade 2.2). Conclusions/Significance Encapsulating inactivated H5N1 influenza virus and CpG into emulsified nanoparticles critically influences the humoral responses against pandemic influenza. These results demonstrated that the use of PELC could be as antigen-sparing in preparation for a potential shortage of prophylactic vaccines against local infectious diseases, in particular pandemic influenza. Moreover, the cross-clade neutralizing antibody responses data verify the potential of such adjuvanted H5N1 candidate vaccine as an effective tool in pre-pandemic preparedness.

Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates.

Abstract The recombinant nucleocapsid (rN) protein of the coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS) was cloned and expressed in Escherichia coli, extracted from cell lysates containing 6M urea, then purified by Ni2+-affinity chromatography. In animal immunogenicity studies, we found that most anti-rN protein antibodies were IgG2a in BALB/c mice vaccinated with rN emulsified in Montanide ISA-51 containing the synthetic oligodeoxynucleotide, CpG. In contrast, anti-rN protein antibodies of mice immunized with rN protein in PBS were found to mainly be IgG1. These results indicated that ISA-51/CpG-formulated rN protein was dramatically biased toward a Th1 immune response. To identify the B-cell immunodominant epitopes of the rN protein in the mouse and monkey, the reactivities of antisera raised against purified rN proteins formulated in ISA-51/CpG were tested with a panel of overlapping synthetic peptides covering the entire N protein sequence. Three immunodominant linear B-cell epitope regions were mapped to residues 166–180, 356–375, and 396–410 of the rN protein. When the reactivities of these peptides were screened with human sera from five SARS patients, peptides corresponding to residues 156–175 reacted strongly with sera from two of the SARS patients. These results indicated that the region around residues 156–175 of the N protein is immunogenic in the mouse, monkey, and human. We found that peptides corresponding to residues 1–30, 86–100, 306–320, and 351–365 contained murine immunodominant T-cell epitopes. To identify functional CTL epitopes of the N protein, BALB/c mice were immunized with peptides containing the H-2Kd CTL motif emulsified in adjuvant ISA-51/CpG. Using an IFN-γ secretion cell assay and analysis by flow cytometry, peptides containing residues 81–95 were found to be capable of stimulating both CD4+ and CD8+ cell proliferation in vitro. We also only observed that peptides corresponding to residues 336–350 were capable of stimulating IFN-γ production in T-cell cultures derived from peripheral blood mononuclear cells (PBMCs) of macaques immunized with the rN protein emulsified in ISA/CpG adjuvant. Our current results together with those of others suggest that some immunodominant B-cell and T-cell epitopes are conserved in the mouse, monkey, and human. This information is very important for the development SARS diagnostic kits and a vaccine.

Enhancement of potent antibody and T-cell responses by a single-dose, novel nanoemulsion-formulated pandemic influenza vaccine.

Vaccine shortages are a major obstacle to influenza pandemic preparedness. Increasing vaccine efficiency provides a potentially effective way to overcome this problem. Specifically, using single-dose immunization to induce protective immunity is an attractive approach to emergency/massive vaccination. In this report, we propose a novel nanoemulsion comprised of the bioresorbable polymer, Span 85, and squalene forming a ready-to-use adjuvant, called PELC. After formulation with PELC, inactivated H5N1 virus was intramuscularly administered to mice via a single injection. The data demonstrate that inactivated virus containing 0.5microg hemagglutinin (HA) and formulated with PELC induced more potent antigen-specific antibodies, hemagglutination inhibition, and virus neutralization than non-adjuvanted inactivated virus containing 5microg HA. In addition, T-cell proliferative responses, as well as interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) secretion were significantly enhanced after immunization with PELC-adjuvanted inactivated virus. These results indicate that PELC can be used for effective single-dose immunization and could thus play an important role in influenza pandemic preparedness.

Formulation and immunological evaluation of novel vaccine delivery systems based on bioresorbable poly(ethylene glycol)-block-poly(lactide-co-ε-caprolactone)

Novel emulsion-type vaccine delivery systems based on the amphiphilic bioresorbable polymer poly(ethylene glycol)-block-poly(lactide-co-epsilon-caprolactone) (PEG-b-PLACL) and selected oils were developed here. Physicochemical characterizations such as stability, a droplet test, microscopic aspects, and in vitro release showed that PEG-b-PLACL-emulsified formulations have several advantages over traditional vaccine adjuvants in that they are stable, reproducible, and homogeneous fine particles with an appropriate size to facilitate the induction of potent immune responses. Different dispersion-type emulsions have provided different release profiles using ovalbumin in model studies. Immunogenicity studies in mice have shown that antigen-specific antibody titers and T-cell proliferative responses, as well as the secretion of IFN-gamma, were significantly enhanced for ovalbumin after formulation with PEG-b-PLACL-based emulsions. These features are of great interest for applications in delivery systems of prophylactic and therapeutic vaccine candidates.

Design and Development of Immunomodulatory Antigen Delivery Systems Based on Peptide/PEG–PLA Conjugate for Tuning Immunity

Cancer vaccines are considered to be a promising tool for cancer immunotherapy. However, a well-designed cancer vaccine should combine a tumor-associated antigen (TAA) with the most effective immunomodulatory agents and/or delivery system to provoke intense immune responses against the TAA. In the present study, we introduced a new approach by conjugating the immunomodulatory molecule LD-indolicidin to the hydrophilic chain end of the polymeric emulsifier poly(ethylene glycol)-polylactide (PEG-PLA), allowing the molecule to be located close to the surface of the resulting emulsion. A peptide/polymer conjugate, named LD-indolicidin-PEG-PLA, was synthesized by conjugation of the amine end-group of LD-indolicidin to the N-hydroxysuccinimide-activated carboxyl end-group of PEG. As an adjuvant for cancer immunotherapeutic use, TAA vaccine candidate formulated with the LD-indolicidin-PEG-PLA-stabilized squalene-in-water emulsion could effectively help to elicit a T helper (Th)1-dominant antigen-specific immune response as well as antitumor ability, using ovalbumin (OVA) protein/EG7 cells as a TAA/tumor cell model. Taken together, these results open up a new approach to the development of immunomodulatory antigen delivery systems for vaccine adjuvants and cancer immunotherapy technologies.

Enzymatic Stability and Immunoregulatory Efficacy of a Synthetic Indolicidin Analogue with Regular Enantiomeric Sequence

Cell-mediated immunity plays a major role in protecting the host from viral infections and tumor challenge. Here, we report the enzymatic stability and adjuvanticity of a peptiomimetic stereoisomer of the bovine neutrophil peptide indolicidin. The analogue, dubbed ld-indolicidin, contains the regular enantiomeric sequence of indolicidin and is synthesized by general stepwise solid-phase strategy. ld-Indolicidin possesses high resistance to enzymatic degradation and shows tolerance in mice. As vaccine adjuvant, ld-indolicidin is better able than the native form of indolicidin to enhance cell-mediated immune responses, using inactivated H5N1 virus as a model antigen. Taken together, these results open up a new approach to the development of vaccine adjuvants and immunotherapy technologies.

Degradable emulsion as vaccine adjuvant reshapes antigen-specific immunity and thereby ameliorates vaccine efficacy

This study describes the feasibility and adjuvant mechanism of a degradable emulsion for tuning adaptive immune responses to a vaccine antigen. We featured a mouse model with ovalbumin (OVA) as the antigen to deepen our understanding of the properties of a degradable emulsion-based adjuvant, dubbed PELC, interacting with immune cells and to elucidate their roles in vaccine immunogenicity in vivo. First, we demonstrated that the emulsion, which is stabilized by an amphiphilic bioresorbable polymer, shows degradation in mimic human body conditions and considerable tolerance in vivo. Then, we confirmed the model protein could be loaded into the emulsion and released from the matrix in a sustained manner, subsequently driving the production of antigen-specific antibodies. We also comprehended that PELC not only recruits antigen-presenting cells (APCs) to the injection site but also induces the activation of the recruited APCs and migration to the draining lymph nodes. As an adjuvant for cancer immunotherapy, PELC-formulated OVA could strongly enhance antigen-specific T-cell responses as well as anti-tumor ability with respected to non-formulated OVA, using OVA protein/EG7 cells as a tumor antigen/tumor cell model. Accordingly, our data paved the way for the clinical application of degradable emulsions based on amphiphilic bioresorbable polymers as vaccine adjuvants.

Unsaturated Squalene Content in Emulsion Vaccine Adjuvants Plays a Crucial Role in ROS-Mediated Antigen Uptake and Cellular Immunity

Emulsion-based adjuvants have been demonstrated to be an effective tool in increasing vaccine efficacy. Here, we aimed to launch a mechanistic study on how emulsion adjuvants interact with immune cells and to elucidate the roles of the core oil in vaccine immunogenicity. Our results showed that treatment of dendritic cells (DCs) and splenocytes with a squalene-based emulsion (referred as SqE) induced reactive oxidative species (ROS) production and resulted in an increase in apoptotic and necrotic cells in a concentration- and time-dependent manner. Furthermore, DCs cocultured with cellular debris of SqE-pretreated splenocytes resulted in a higher level of ovalbumin (OVA) antigen uptake by DCs than those cocultured with untreated splenocytes. Interestingly, the potency was rather attenuated when splenocytes were pretreated with N-acetyl-cysteine, an antioxidant. Notably, SqE possesses a high impact on eliciting ROS-mediated antigen uptake compared with a squalane-based emulsion (SqA). Concordantly, immunogenicity studies have shown that SqE is better able than SqA to activate antigen-presenting cells, and to enhance antigen-specific T-cell immunity. Taken together, our results show that unsaturated squalene oil cored within emulsions plays a crucial role in ROS-mediated antigen uptake and cellular immunity, providing a basis for the design and development of vaccine adjuvant.

Polysorbasome: A Colloidal Vesicle Contoured by Polymeric Bioresorbable Amphiphiles as an Immunogenic Depot for Vaccine Delivery

To accomplish an innovative vaccine design, there are two key challenges: developing formulations that avoid cold chain shipment and finding a delivery vehicle that is absorbable in vivo. Here, we explored the design and performance of a colloidal vesicle that enabled us to consider both challenges. We used polymeric bioresorbable amphiphiles as surface-active agents for stabilizing oily/aqueous interfaces and formed a colloidal vehicle named polysorbasome (PSS, polymeric absorbable vesicle), without using conventional emulsifiers such as sorbitan esters or their ethoxylates. Homogenizing the oil/water compartments forms a colloid containing an aqueous solution in its core and provides an oily barrier that isolates the encapsulated material from external materials. In this form, the PSS serves as a depot for sustained delivery of vaccine antigens. Following vaccination, the antigen-specific antibodies and the cell-mediated immunity can be manipulated after the antigen being formulated with PSS particles. Then, the degradability intrinsic to the polymeric bioresorbable amphiphiles complies with the destruction and further absorbance of the vehicles in vivo. The structural features of these versatile vesicles based on bioresorbable amphiphilic engineering may provide new insights into vaccine delivery.