Potent Vaccine Adjuvant Research Articles

Salmonella enterica Serovar Enteritidis Ghosts Carrying the Escherichia coli Heat-Labile Enterotoxin B Subunit Are Capable of Inducing Enhanced Protective Immune Responses

The Escherichia coli heat-labile enterotoxin B subunit (LTB) is a potent vaccine adjuvant. Salmonella enterica serovar Enteritidis ghosts carrying LTB (S. Enteritidis-LTB ghosts) were genetically constructed using a novel plasmid, pJHL187-LTB, designed for the coexpression of the LTB and E lysis proteins. S. Enteritidis-LTB ghosts were characterized using scanning electron microscopy to visualize their transmembrane tunnel structures. The expression of LTB in S. Enteritidis-LTB ghost preparations was confirmed by immunoblot and enzyme-linked immunosorbent assays. The parenteral adjuvant activity of LTB was demonstrated by immunizing chickens with either S. Enteritidis-LTB ghosts or S. Enteritidis ghosts. Chickens were intramuscularly primed at 5 weeks of age and subsequently boosted at 8 weeks of age. In total, 60 chickens were equally divided into three groups (n = 20 for each): group A, nonvaccinated control; group B, immunized with S. Enteritidis-LTB ghosts; and group C, immunized with S. Enteritidis ghosts. Compared with the nonimmunized chickens (group A), the immunized chickens (groups B and C) exhibited increased titers of plasma IgG and intestinal secretory IgA antibodies. The CD3(+) CD4(+) subpopulation of T cells was also significantly increased in both immunized groups. Among the immunized chickens, those in group B exhibited significantly increased titers of specific plasma IgG and intestinal secretory IgA (sIgA) antibodies compared with those in group C, indicating the immunomodulatory effects of the LTB adjuvant. Furthermore, both immunized groups exhibited decreased bacterial loads in their feces and internal organs. These results indicate that parenteral immunization with S. Enteritidis-LTB ghosts can stimulate superior induction of systemic and mucosal immune responses compared to immunization with S. Enteritidis ghosts alone, thus conferring efficient protection against salmonellosis.

Synthesis and functionalization of virus-mimicking cationic block copolymers with pathogen-associated carbohydrates as potential vaccine adjuvants

We report the synthesis of a family of amphiphilic pentablock polymers with different cationic blocks and with controlled architectures as potential vaccine carriers for subunit vaccines. The temperature and pH-dependent micellization and gelation of these pentablock copolymers can provide a depot for sustained protein and gene delivery. The amphiphilic central triblock promotes cellular endocytosis, good gene delivery and has been used effectively as a vaccine adjuvant. The pentablock copolymer outer blocks condense DNA spontaneously as a result of electrostatic interactions for sustained combinational therapy. This family of polymers with different cationic groups was evaluated based on DNA complexation-ability and cytotoxicity to select promising candidates as DNA-based subunit vaccine adjuvants. Modification of other polymer systems with carbohydrates like mannose has been shown to enhance immunogenicity by activating pattern recognition receptors on antigen presenting cells and increasing uptake in these cells. Here, we report the synthesis of a virus-mimicking pentablock copolymer vaccine platform by successful functionalization of these polymers with mannose through an azide–alkyne Huisgen cycloaddition. The synthesis of a mannoside with the alkyne linker was achieved by a recently reported bismuth(V)-mediated activation of a thioglycoside that proved to leave the alkyne intact. The carbohydrate modification was shown not to interfere with the ability of these virus-mimicking block copolymers to complex DNA, thereby making this family of modified materials promising candidates for DNA-based vaccine delivery.

PMA induces vaccine adjuvant activity by the modulation of TLR signaling pathway.

Toll-like receptor (TLR) ligands are being developed for use as vaccine adjuvants and as immunomodulators because of their ability to stimulate innate and adaptive immune responses. Flagellin, a TLR5 ligand, was reported to show potent mucosal vaccine adjuvant activity. To identify ligands that potentiate the adjuvant activity of flagellin, we screened a plant library using HEK293T cells transiently cotransfected with phTLR5 and pNF-κB-SEAP plasmids. The 90% EtOH extract from Croton tiglium showed significant NF-κB transactivation in a TLR5-independent manner along with the increase of a flagellin activity. We have studied to characterize an active component from Croton tiglium and to elucidate the action mechanisms. Phorbol 12-myristate 13-acetate (PMA) was isolated as an active component of Croton tiglium by activity-guided fractionation, column chromatography, HPLC, NMR, and MS. PMA at a range of nM induced PKC-dependent NF-κB activation and IL-8 production in both TLR5− and TLR5+ assay systems. In in vivo mouse vaccination model, PMA induced antigen-specific IgG and IgA antibody responses and increased IL-12 production corresponding to T cell responses in spleen lymphocytes. These results suggest that PMA would serve as an efficacious mucosal vaccine adjuvant.

The Immunobiology of Toll-Like Receptor 4 Agonists

Lipopolysaccharide (LPS, endotoxin) is a structural component of the gram-negative outer membrane. The lipid A moiety of LPS binds to the LPS receptor complex expressed by leukocytes, endothelial cells, and parenchymal cells and is the primary component of gram-negative bacteria that is recognized by the immune system. Activation of the LPS receptor complex by native lipid A induces robust cytokine production, leukocyte activation, and inflammation, which is beneficial for clearing bacterial infections at the local level but can cause severe systemic inflammation and shock at higher challenge doses. Interestingly, prior exposure to LPS renders the host resistant to shock caused by subsequent LPS challenge, a phenomenon known as endotoxin tolerance. Treatment with lipid A has also been shown to augment the host response to infection and to serve as a potent vaccine adjuvant. However, the adverse effects associated with the pronounced inflammatory response limit the use of native lipid A as a clinical immunomodulator. More recently, analogs of lipid A have been developed that possess attenuated proinflammatory activity but retain attractive immunomodulatory properties. The lipid A analog monophosphoryl lipid A exhibits approximately 1/1,000th of the toxicity of native lipid A but retains potent immunoadjuvant activity. As such, monophosphoryl lipid A is currently used as an adjuvant in several human vaccine preparations. Because of the potency of lipid A analogs as immunoadjuvants, numerous laboratories are actively working to identify and develop new lipid A mimetics and to optimize their efficacy and safety. Based on those characteristics, lipid A analogs represent an attractive family of immunomodulators.

Effects of different CpG oligodeoxynucleotides with inactivated avian H5N1 influenza virus on mucosal immunity of chickens

Oligodeoxynucleotide containing unmethylated CpG motifs (CpG-ODN) has been proved to be a potent and safe vaccine adjuvant. However, the application of CpG-ODN in poultry vaccines was limited because of its high cost to benefit ratio. The objective of this study was to identify the CpG-ODN with efficient adjuvant activity and low cost in chickens. Four sequences of CpG-ODN were designed based on CpG-ODN 2006, which was used as a template and positive sequence in our study. In the current study, in vitro observations revealed that the designed CpG-ODN had efficient immunostimulatory effects on chicken splenic lymphocytes. The in vivo results showed that the mRNA expressions of IL-6, IL-12, interferon-γ, and Toll-like receptor (TLR) 21 in upper respiratory tract tissues increased significantly in the early period after intranasal immunization with inactivated avian H5N1 influenza virus (IAIV) and CpG-ODN (P < 0.01). In addition, the avian influenza virus (AIV)-specific secretory IgA antibody level in the lavage fluid of upper respiratory tract increased significantly after intranasal immunization with IAIV and CpG-ODN, so did AIV-specific IgG in serum (P < 0.01). Among all the designed CpG-ODN, CpG-ODN F3 with an addition of poly-guanosine strings at the 3′-end not only had the best enhancement on local mucosal immune response but also showed an effective induction of systemic immune response. Most importantly, the virus challenge study showed that prior administration of IAIV with CpG-ODN F3 could protect chickens effectively against live AIV H5N1 challenge. Additionally, among all the CpG-ODN in our study, the cost of the designed CpG-ODN F3 was the lowest because of the partially phosphorothioate backbone. Therefore, we speculated that CpG-ODN F3 with efficient adjuvant activity and a big cost advantage over CpG-ODN F1 (CpG-ODN 2006) might serve as an efficient and affordable nasal adjuvant for inactivated AIV vaccine in chicken.

Chemokines as Cancer Vaccine Adjuvants.

We are witnessing a new era of immune-mediated cancer therapies and vaccine development. As the field of cancer vaccines advances into clinical trials, overcoming low immunogenicity is a limiting step in achieving full success of this therapeutic approach. Recent discoveries in the many biological roles of chemokines in tumor immunology allow their exploitation in enhancing recruitment of antigen presenting cells (APCs) and effector cells to appropriate anatomical sites. This knowledge, combined with advances in gene therapy and virology, allows researchers to employ chemokines as potential vaccine adjuvants. This review will focus on recent murine and human studies that use chemokines as therapeutic anti-cancer vaccine adjuvants.

Escherichia coli‐based cell free production of flagellin and ordered flagellin display on virus‐like particles

Bacterial flagellin has been explored as a potential vaccine adjuvant for enhancing immune responses. In this article, we describe Escherichia coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce soluble phase 1 flagellin (FliC) protein from Salmonella typhimurium. The yield was about 300 µg/mL and the product had much higher affinity for the TLR5 receptor (EC50 = 2.4 ± 1.4 pM) than previously reported. The flagellin coding sequence was first optimized for cell-free expression. We then found that the D0 domain at the C-terminus of flagellin was susceptible to proteolytic degradation in the CFPS system. Proteolysis was reduced by protease inhibitors, the use of protease-deficient cell extracts or deletion of the flagellin D0 domain. A human Toll-Like Receptor 5 (hTLR5)-specific bioactivity analysis of purified flagellin demonstrated that, although the D0 domain is far from the TLR5 recognition region, it is important for flagellin bioactivity. We next incorporated a non-natural amino acid displaying an alkyne moiety into flagellin using the CFPS system and attached flagellin to hepatitis B core virus-like particles (VLPs) using bioorthogonal azide-alkyne cycloaddition reactions. The ordered and oriented VLP display of flagellin increased its specific TLR5 stimulation activity by approximately 10-fold.

CpG oligodeoxynucleotide ligand potentiates the activity of the pVAX1-Sj26GST

Schistosomiasis is considered one of the most important neglected tropical diseases and remains a major public health problem in endemic countries. Toll-like receptor (TLR) ligands have been investigated as potential vaccine adjuvants for tumor and virus immunotherapy. However, few TLR ligands affecting schistosoma vaccines have been characterized. In this study, we evaluated a TLR9 ligand (CpG oligodeoxynucleotide 1826, CpG) as an adjuvant for a partially protective DNA vaccine encoding a 26-kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST). Vaccination with pVAX1-Sj26GST in combination with CpG inhibited Treg immunosuppressive function, upregulated the production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-10, IL-2 and IL-6, and decreased CD4+CD8+Foxp3+ expression in vitro, which may contribute to the escape from Treg-mediated suppression during vaccination, allowing expansion of antigen-specific T cells against pathogens. In conclusion, our data demonstrated that selective TLR ligand combination may increase protective efficacy against schistosomiasis, which may synergistically antagonize Treg-mediated suppression.

Development of Soluble Inulin Microparticles as a Potent and Safe Vaccine Adjuvant and Delivery System

The goal of the present study is to develop a potent and safe vaccine adjuvant that can also stabilize vaccine formulations during lyophilization and storage. Inulin is a safe plant polysaccharide, and in its water soluble isoform, it is known to stabilize protein formulations during storage. However, soluble inulins have never been shown to stimulate the immune system. In this study, for the first time, we showed that water soluble inulins could be developed into vaccine adjuvants by formulating as antigen encapsulated microparticles. A method was developed to prepare soluble inulin microparticles (sIMs) with high encapsulation efficiency (∼75%) and loading (∼75 μg/mg) of the antigen. When immunized in mice, sIMs have generated robust Th2-type antibody titers (IgG1: 500,000) compared to unadjuvanted antigens (IgG1: 17,500) or alum adjuvanted antigens (IgG1: 80,000). In vitro assays showed that a higher proportion of antigen presenting cells (APC's) have taken up the antigen when presented in sIMs versus in solution (99 % vs 22 %). In addition, the amount of antigen taken up per cell has also been enhanced by more than 25 times when antigen was presented in sIMs. Efficient uptake of the antigen by APCs through sIMS was attributed to the observed enhancement in the immune response by antigen loaded sIMs. The sIMs neither caused any granuloma/tissue damage at the injection site in mice nor were they toxic to the APC's in cell culture. In conclusion, the current study has developed a safe, soluble inulin based vaccine adjuvant and delivery system.

Silica nanorattle with enhanced protein loading: A potential vaccine adjuvant

Nanoparticles are excellent carriers for drug and protein, and have the potential to be used in vaccine delivery system. Here, we prepared different structures silica nanoparticles such as silica nanorattles (SNs), mesoporous silica nanoparticles (MSNs) and solid silica nanoparticles (SSNs), and chosen ovalbumin (OVA) as model protein to study the potential application of silica nanoparticles in protein vaccine delivery system. The results showed that silica nanoparticles were efficient in protein loading and dependent on structure, size and incubation medium. According to the three structure particles, SNs were favorable to be used as protein carriers. Furthermore, we proved low cytotoxicity of silica nanorattle on RAW 264.7 cell line and biocompatibility in vivo. In addition, SNs was capable to up-regulate the humoral immunity reaction when mice were vaccinated with SNs–OVA formulation. Taken together, SNs was excellent carriers for protein vaccine and has the potential to be used as adjuvant.

Highly efficient enzymatic preparation of c-di-AMP using the diadenylate cyclase DisA from Bacillus thuringiensis

Cyclic 3′,5′-diadenosine monophosphate (c-di-AMP) is a newly recognized bacterial nucleotide second messenger molecule. In addition, it has been shown to be a potential vaccine adjuvant. Although multiple methods are available for c-di-AMP synthesis, the yields are low and the purification procedures are laborious. Here, we report an enzymatic method for more efficient and economical c-di-AMP synthesis using a diadenylate cyclase DisA from Bacillus thuringiensis BMB 171 (btDisA). After overexpression and purification of btDisA, the enzyme-catalyzed reaction conditions were further investigated. Under the optimum conditions, in which 100mM CHES (pH 9.5) containing 2μM btDisA, 10mM ATP, and 10mM MgCl2 was incubated at 50°C for 4h, a high conversion rate of c-di-AMP was obtained. Coupling this process with HPLC purification and lyophilization yielded 100mg of highly pure c-di-AMP that was harvested in white powder form from a 50mL enzyme-catalyzed reaction system. The protocol is not only directly applicable for preparing abundant amounts of c-di-AMP for extensive biochemical and immunological use, but can also be scaled up to meet the requirements for medical applications.

Sweeten PAMPs: role of sugar complexed PAMPs in innate immunity and vaccine biology

Innate sensors play a critical role in the early innate immune responses to invading pathogens through sensing of diverse biochemical signatures also known as pathogen associated molecular patterns (PAMPs). These biochemical signatures primarily consist of a major family of biomolecules such as proteins, lipids, nitrogen bases, and sugar and its complexes, which are distinct from host molecules and exclusively expressed in pathogens and essential to their survival. The family of sensors known as pattern recognition receptors (PRRs) are germ-line encoded, evolutionarily conserved molecules, and consist of Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin-like receptors (CLRs), and DNA sensors. Sensing of PAMP by PRR initiates the cascade of signaling leading to the activation of transcription factors, such as NF-κB and interferon regulatory factors (IRFs), resulting in a variety of cellular responses, including the production of interferons (IFNs) and pro-inflammatory cytokines. In this review, we discuss sensing of different types of glycosylated PAMPs such as β-glucan (a polymeric sugar) or lipopolysaccharides, nucleic acid, and so on (sugar complex PAMPs) by different families of sensors, its role in pathogenesis, and its application in development of potential vaccine and vaccine adjuvants.

Chitosan stimulates lymphocyte proliferation during the muscle phase of Trichinella spiralis infection in mice

Event Abstract Back to Event Chitosan stimulates lymphocyte proliferation during the muscle phase of Trichinella spiralis infection in mice Klaudia Brodaczewska1, Natalia Wolaniuk1, Katarzyna Donskow-Lysoniewska1 and Maria Doligalska1* 1 University of Warsaw, Department of Parasitology, Faculty of Biology, Poland Chitosan is intensively studied as a potential vaccine adjuvant that can combine high efficacy with low toxicity. Yet how the polysaccharide affects immunity is unclear. Antibody titers were raised when antigen was administered with chitosan but it is not clear what mechanisms are involved. As parasitic infection down-regulates the immune response, modification of suppressed reactions may be promising for anti-parasitic protection. The aim of this study was to evaluate the effect of chitosan administration on the lymphocyte proliferative response in mice infected with T. spiralis. C57Bl6 mice were intra-peritoneally injected with 500 μg of chitosan (> 375 kDa) dissolved in adipic acid, every day, from 5 days before till 9 days after infection with 400 L1 T. spiralis. In the muscle phase of infection, mesenteric lymph node cells were cultured without or with TCR stimulation (anti-CD3 and -CD28 antibodies); levels of proliferation and cytokines were measured. Chitosan treatment restored cell responsiveness; they proliferated intensively and were again responsive to TCR stimulation. Also cytokine secretion was altered. Nonetheless the cells regained reactivity, the level of infection was intensively raised after chitosan treatment; over two times more muscle larvae were recovered from mice that obtained polysaccharide injections. In conclusion, although chitosan stimulated the lymphocyte response, the effect of treatment was not protective. Acknowledgements Research was supported by Faculty of Biology, University of Warsaw Intramural Grant (BW 501/86-102307). Keywords: Mesenteric lymph node, Lymphocytes, Trichinella spiralis, Chitosan, Cytokines Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Brodaczewska K, Wolaniuk N, Donskow-Lysoniewska K and Doligalska M (2013). Chitosan stimulates lymphocyte proliferation during the muscle phase of Trichinella spiralis infection in mice. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00714 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 14 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Maria Doligalska, University of Warsaw, Department of Parasitology, Faculty of Biology, Warsaw, Poland, m.doligalska@biol.uw.edu.pl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Klaudia Brodaczewska Natalia Wolaniuk Katarzyna Donskow-Lysoniewska Maria Doligalska Google Klaudia Brodaczewska Natalia Wolaniuk Katarzyna Donskow-Lysoniewska Maria Doligalska Google Scholar Klaudia Brodaczewska Natalia Wolaniuk Katarzyna Donskow-Lysoniewska Maria Doligalska PubMed Klaudia Brodaczewska Natalia Wolaniuk Katarzyna Donskow-Lysoniewska Maria Doligalska Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Safety and immunogenicity of bacterial and tobacco plant cell line derived recombinant native and mutant Escherichia coli heat-labile toxin in chickens

The safety and immunogenicity of the mammalian mucosal adjuvants, Escherichia coli wild-type heat-labile holotoxin (LT) and E. coli mutant LT (LTA-K63/LTB), were examined in 1-day-old chicks and 10-day-old to 21-day-old broilers. Biologically active, E. coli recombinant wild-type LT and recombinant LTA-K63/LTB produced in a transgenic Nicotiana tabacum (NT-1) tobacco cell line (SLT102) were tested for safety and antigenicity following various routes of administration. Safety was assessed by clinical signs, body weight gain, gross organ pathology and wet organ weight, and histopathology. Antigenicity was assessed using LT-B-specific serum IgG enzyme-linked immunosorbent assay. Parenteral administration of E. coli recombinant wild-type LT did not have any discernible effect on bird health and was well tolerated at levels up to 400 µg per dose. Recombinant, SLT102-derived mutant LT derived from SLT102 cells retained in vitro ganglioside binding and was safe and antigenic following repeated mucosal administration to birds. The highest systemic LT-B-specific IgG titres were detected in birds that received three on-feed doses of SLT102-derived mutant LT. Among the various SLT102-derived mutant LT preparations tested, whole, wet cells or whole cell lysates were the most antigenic. These results demonstrate for the first time that E. coli-derived recombinant, wild-type LT holotoxin is well tolerated following multiple administrations to young birds at body weight doses previously reported to be enteropathogenic and toxic in mammalian species. Moreover, these data also demonstrate the feasibility of using recombinant wild-type and mutant LT produced in transgenic NT-1 tobacco cells as safe and potent vaccine adjuvants in poultry.

Pentamers Not Found in the Universal Proteome Can Enhance Antigen Specific Immune Responses and Adjuvant Vaccines

Certain short peptides do not occur in humans and are rare or non-existent in the universal proteome. Antigens that contain rare amino acid sequences are in general highly immunogenic and may activate different arms of the immune system. We first generated a list of rare, semi-common, and common 5-mer peptides using bioinformatics tools to analyze the UniProtKB database. Experimental observations indicated that rare and semi-common 5-mers generated stronger cellular responses in comparison with common-occurring sequences. We hypothesized that the biological process responsible for this enhanced immunogenicity could be used to positively modulate immune responses with potential application for vaccine development. Initially, twelve rare 5-mers, 9-mers, and 13-mers were incorporated in frame at the end of an H5N1 hemagglutinin (HA) antigen and expressed from a DNA vaccine. The presence of some 5-mer peptides induced improved immune responses. Adding one 5-mer peptide exogenously also offered improved clinical outcome and/or survival against a lethal H5N1 or H1N1 influenza virus challenge in BALB/c mice and ferrets, respectively. Interestingly, enhanced anti-HBsAg antibody production by up to 25-fold in combination with a commercial Hepatitis B vaccine (Engerix-B, GSK) was also observed in BALB/c mice. Mechanistically, NK cell activation and dependency was observed with enhancing peptides ex vivo and in NK-depleted mice. Overall, the data suggest that rare or non-existent oligopeptides can be developed as immunomodulators and supports the further evaluation of some 5-mer peptides as potential vaccine adjuvants.

IL-7 induces rapid internalization of CD127 by clathrin mediated endocytosis, leading to subsequent degradation by the proteasome. (174.1)

Abstract IL-7, a potent immunomodulatory cytokine, is currently being investigated as a potential therapy and vaccine adjuvant in the treatment of HIV infection. In order to maximize these therapeutic strategies, we must first understand how the IL-7 receptor is regulated. We show here that in pimary human CD8 T cells, IL-7 decreases surface CD127 expression on CD8 T-cells in a dose- and time-dependent manner with initial suppression as early as 20 minutes and maximal suppression at 12 hours. This decrease at the cell surface is followed by loss of total CD127 protein within the cell as demonstrated by western blot. IL-7 induces internalization of surface CD127 through endocytic vesicles. The endocytosis inhibitors Filipin, and Dynasore both prevent IL-7 induced down regulation of CD127 at the cell membrane. Consistent with this we have shown by confocal microscopy that in resting CD8 T-cells CD127 is distributed evenly throughout the cell. After treatment with IL-7, CD127 forms multiple intracellular punctae with increased co-localization with clathrin by 5 minutes followed by co-localization with the early endosomal marker EEA1 after 30 minutes. By 2 hours CD127 staining associates with the late endosome marker RAB7 and the proteasomal 20S subunit, while at the same time showing decreased colocalization with the lysosomal marker LAMP1. Interestingly, the proteasome inhibitors lactacystin and MG132 both blocked IL-7’s ability to down regulate CD127.

Non-inflammatory and additive-free adjuvant for cutaneous vaccination (113.29)

Abstract Skin has been considered an attractive site for vaccine delivery for more than 3000 years, but it is still not commonly used today, because of technical difficulties and a lack of inflammation-free adjuvant. Considerable progress made in generating convenient intradermal (i.d.) injection devices in the past decade raises an urgent need of developing potent vaccine adjuvants that introduce little skin inflammation. Many current vaccine adjuvants cause severe skin inflammation that jeopardizes the integrity of the skin and thus cannot be accepted for cutaneous vaccination. We develop a laser-based vaccine adjuvant (LVA) capable of boosting immune responses without incurring inflammation. LVA was induced by brief (2 min) illumination of a small area of the skin with a safe laser prior to i.d. administration of vaccines at the site of laser illumination. The pre-illumination augmented the hemagglutination inhibition (HAI) titers against seasonal or 2009-pandemic influenza vaccine by 10~20-folds when compared to i.m. immunization. And, while i.d. immunization blocked viral production in the lung by 2-fold over i.m. vaccination, i.d. immunization after laser illumination increased the blockade to 186-fold. In comparison with all current vaccine adjuvants that are either chemical compounds or biological agents, LVA is a risk- and additive-free adjuvant and has distinct advantages over traditional vaccine adjuvants for cutaneous vaccination.

Abstract 1536: A novel immunocytokine generates a CD8+ T cell-mediated anti-tumor response in vivo

Abstract Interleukin-12 (IL-12) is a potent TH1 cytokine produced by dendritic cells and macrophages to activate NK cells and T cells. Data from preclinical studies in mice indicate that this cytokine may be useful for the treatment of cancer. However, cases of severe toxicity in human trials have impeded its successful clinical development. Targeting delivery of IL-12 to a tumor while reducing systemic IL-12 exposure could make this a safer, more effective cancer therapeutic agent. One strategy is to conjugate IL-12 to a tumor-binding antibody, as antibody conjugates have previously been used to deliver radioisotopes, other cytokines, and small molecule therapeutics to tumors in vivo. NHS-IL12 is a novel immunocytokine consisting of two molecules of the IL-12 heterodimer covalently fused to a fully humanized antibody (NHS76). This antibody targets necrotic portions of tumors because of its high affinity for single- and double-stranded DNA, which are often exposed as tumors outgrow their blood supply. NHS-IL12 may therefore be useful against a wide range of solid tumors, and mouse models can be used to study its mechanisms of action. Initial imaging studies clearly showed superior in vivo localization of IL-12 to mouse tumors following subcutaneous NHS-IL12 injection as compared to administration of recombinant IL-12 (rIL-12) or IL-12 fused to a control antibody (BC1). In the MC38/CEA+ colorectal carcinoma model in CEA.Tg mice, treatment with NHS-IL12 significantly delayed subcutaneous tumor growth more effectively than treatment with rIL-12. NHS-IL12 did not inhibit MC38/CEA+ tumor cell growth in vitro, suggesting that NHS-IL12 acts indirectly on tumors in vivo most probably via an immune-mediated mechanism. Subsequent findings revealed that tumor-bearing mice treated with NHS-IL12 developed a strong CD8+ T cell response directed against the endogenous p15E tumor antigen. In one study, a subset of mice bearing MC38/CEA+ tumors had complete tumor regression following NHS-IL12 treatment. Each of these mice still had a strong p15E-specific CTL response more than two months after tumor rejection, indicating that p15E may be the major tumor rejection antigen for this in vivo tumor model. Subsequent in vivo depletion studies are planned to investigate which immune cell subsets contribute to the anti-tumor efficacy of this agent. Taken together, these data show that targeted delivery of IL-12 to tumors can overcome the immunosuppressive tumor microenvironment and prime a robust CD8+ T cell response. These findings suggest that NHS-IL12 may be a promising cancer therapeutic as a single agent and/or as a potent vaccine adjuvant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1536. doi:1538-7445.AM2012-1536

Abstract 1582: Therapeutic vaccine for pancreatic cancer

Abstract Cadi-05 (Heat killed mycobacterium w) is a potent TLR 2 agonist and induces pure potent Th1 response. In advanced Non small cell lung cancer, Cadi-05 is found to potentiate efficacy of chemotherapy (Paclitaxel + Cisplatin) with improvement in response rate and overall survival in a randomized controlled clinical trial. Cadi-05 is also a potent vaccine adjuvant for prophylactic vaccines providing faster, higher and sustained antibody titers in animals and humans. The therapeutic vaccine for pancreatic cancer, with killed pancreatic cancer cells as an antigen with Cadi-05 as an adjuvant was evaluated for its ability to induce cell mediated immune responses (by ELISPOT for IFN - α, IL-2 and Granzyme B) and effector function (By BrDu incorporation). The vaccine made using Mia PaCa-2 cell line induced cell mediated immune responses against the Mia PaCA-2 as well as against the other cell lines (Panc-1, ASPC-1, SW-1990). The vaccine also generated effector function of similar magnitude against all four cell lines. Effect of vaccine on tumor size and survival following intradermal immunization was also studied. For evaluating efficacy, C57BL/6 BYJ mice were injected Pan-02 cells (1x106) subcutaneously for developing pancreatic cancer model. At day 10 post tumor induction tumor size achieved was∼200-225 mm3. Mice were randomized to receive vaccine or placebo (18 mice each) on day 10. No other therapy was used in the experiment. In Vaccine arm tumor regression was seen in 61% of animals before progression. Tumor regression was not seen in placebo arm. Tumor progression was found to be slower in vaccinated animals with mean tumor size 35% and 59% of placebo arm on day7 (633 mm3 vs. 223 mm3) and day 12 (740 mm3 vs. 437 mm3) respectively resulting in survival benefit of 36% on day 40 following immunisation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1582. doi:1538-7445.AM2012-1582

Structure–Activity Relationships in Human Toll-like Receptor 2-Specific Monoacyl Lipopeptides

Toll-like receptor 2-agonistic lipopeptides typified by S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (PAM(2)CS) compounds are potential vaccine adjuvants. We had previously determined that at least one acyl group of optimal length (C(16)) and an appropriately orientated ester carbonyl group is essential for TLR2-agonistic activity. We now show that these structurally simpler analogues display agonistic activities with human, but not murine, TLR2. SAR studies on the monoacyl derivatives show that the optimal acyl chain length is C(16), and aryl substituents are not tolerated. A variety of alkyl and acyl substituents on the cysteine amine were examined. All N-alkyl derivatives were inactive. In contradistinction, short-chain N-acyl analogues were found to be highly active, with a clear dependence on the chain length. A cysteine N-acetyl analogue was found to be the most potent (EC(50): 1 nM), followed by the N-butyryl analogue. The N-acetyl analogue is human TLR2-specific, with its potency comparable to that of PAM(2)CS.