Publisher Correction: Adjuvanting a viral vectored vaccine against pre-erythrocytic malaria.

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Adjuvanting a viral vectored vaccine against pre-erythrocytic malaria.

The majority of routinely given vaccines require two or three immunisations for full protective efficacy. Single dose vaccination has long been considered a key solution to improving the global immunisation coverage. Recent infectious disease outbreaks have further highlighted the need for vaccines that can achieve full efficacy after a single administration. Viral vectors are a potent immunisation platform, benefiting from intrinsic immuno-stimulatory features while retaining excellent safety profile through the use of non-replicating viruses. We investigated the scope for enhancing the protective efficacy of a single dose adenovirus-vectored malaria vaccine in a mouse model of malaria by co-administering it with vaccine adjuvants. Out of 11 adjuvants, only two, Abisco®-100 and CoVaccineHTTM, enhanced vaccine efficacy and sterile protection following malaria challenge. The CoVaccineHTTM adjuvanted vaccine induced significantly higher proportion of antigen specific central memory CD8+ cells, and both adjuvants resulted in increased proportion of CD8+ T cells expressing the CD107a degranulation marker in the absence of IFNγ, TNFα and IL2 production. Our results show that the efficacy of vaccines designed to induce protective T cell responses can be positively modulated with chemical adjuvants and open the possibility of achieving full protection with a single dose immunisation.

Dengue virus activates cGAS through the release of mitochondrial DNA.

Cyclic GMP-AMP synthetase (cGAS) is a DNA-specific cytosolic sensor, which detects and initiates host defense responses against microbial DNA. It is thus curious that a recent study identified cGAS as playing important roles in inhibiting positive-sense single-stranded RNA (+ssRNA) viral infection, especially since RNA is not known to activate cGAS. Using a dengue virus serotype 2 (DENV-2) vaccine strain (PDK53), we show that infection creates an endogenous source of cytosolic DNA in infected cells through the release of mitochondrial DNA (mtDNA) to drive the production of cGAMP by cGAS. Innate immune responses triggered by cGAMP contribute to limiting the spread of DENV to adjacent uninfected cells through contact dependent gap junctions. Our result thus supports the notion that RNA virus indirectly activates a DNA-specific innate immune signaling pathway and highlights the breadth of the cGAS-induced antiviral response.

Molecular determinants of plaque size as an indicator of dengue virus attenuation.

The development of live viral vaccines relies on empirically derived phenotypic criteria, especially small plaque sizes, to indicate attenuation. However, while some candidate vaccines successfully translated into licensed applications, others have failed safety trials, placing vaccine development on a hit-or-miss trajectory. We examined the determinants of small plaque phenotype in two dengue virus (DENV) vaccine candidates, DENV-3 PGMK30FRhL3, which produced acute febrile illness in vaccine recipients, and DENV-2 PDK53, which has a good clinical safety profile. The reasons behind the failure of PGMK30FRhL3 during phase 1 clinical trial, despite meeting the empirically derived criteria of attenuation, have never been systematically investigated. Using in vitro, in vivo and functional genomics approaches, we examined infections by the vaccine and wild-type DENVs, in order to ascertain the different determinants of plaque size. We show that PGMK30FRhL3 produces small plaques on BHK-21 cells due to its slow in vitro growth rate. In contrast, PDK53 replicates rapidly, but is unable to evade antiviral responses that constrain its spread hence also giving rise to small plaques. Therefore, at least two different molecular mechanisms govern the plaque phenotype; determining which mechanism operates to constrain plaque size may be more informative on the safety of live-attenuated vaccines.

Vaccine delivery by penetratin: mechanism of antigen presentation by dendritic cells.

Cell-penetrating peptides (CPP) or membrane-translocating peptides such as penetratin from Antennapedia homeodomain or TAT from human immunodeficiency virus are useful vectors for the delivery of protein antigens or their cytotoxic (Tc) or helper (Th) T cell epitopes to antigen-presenting cells. Mice immunized with CPP containing immunogens elicit antigen-specific Tc and/or Th responses and could be protected from tumor challenges. In the present paper, we investigate the mechanism of class I and class II antigen presentation of ovalbumin covalently linked to penetratin (AntpOVA) by bone marrow-derived dendritic cells with the use of biochemical inhibitors of various pathways of antigen processing and presentation. Results from our study suggested that uptake of AntpOVA is via a combination of energy-independent (membrane fusion) and energy-dependent pathways (endocytosis). Once internalized by either mechanism, multiple tap-dependent or independent antigen presentation pathways are accessed while not completely dependent on proteasomal processing but involving proteolytic trimming in the ER and Golgi compartments. Our study provides an understanding on the mechanism of antigen presentation mediated by CPP and leads to greater insights into future development of vaccine formulations.

Mannan adjuvants intranasally administered inactivated influenza virus in mice rendering low doses inductive of strong serum IgG and IgA in the lung.

BACKGROUND: H1N1 influenza viruses mutate rapidly, rendering vaccines developed in any given year relatively ineffective in subsequent years. Thus it is necessary to generate new vaccines every year, but this is time-consuming and resource-intensive. Should a highly virulent influenza strain capable of human-to-human transmission emerge, these factors will severely limit the number of people that can be effectively immunised against that strain in time to prevent a pandemic. An adjuvant and mode of administration capable of rendering ordinarily unprotective vaccine doses protective would thus be highly advantageous. METHODS: The carbohydrate mannan was conjugated to whole inactivated H1N1 influenza virus at a range of ratios, and mixed with it at a range of ratios, and various doses of the resulting preparations were administered to mice via the intranasal (IN) route. Serum immunity was assessed via antigen-specific IgG ELISA and the haemagglutination-inhibition (HI) assay, and mucosal immunity was assessed via IgA ELISA of bronchio-alveolar lavages. RESULTS: IN-administered inactivated H1N1 mixed with mannan induced higher serum IgG and respiratory-tract IgA than inactivated H1N1 conjugated to mannan, and HIN1 alone. Adjuvantation was mannan-dose-dependent, with 100 µg of mannan adjuvanting 1 µg of H1N1 more effectively than 10 or 50 µg of mannan. Serum samples from mice immunised with 1 µg H1N1 adjuvanted with 10 µg mannan did not inhibit agglutination of red blood cells (RBCs) at a dilution factor of 10 in the HI assay, but samples resulting from adjuvantation with 50 and 100 µg mannan inhibited agglutination at dilution factors of ≥ 40. Both serum IgG1 and IgG2a were induced by IN mannan-adjuvanted H1N1 vaccination, suggesting the induction of humoral and cellular immunity. CONCLUSIONS: Mixing 100 µg of mannan with 1 µg of inactivated H1N1 adjuvanted the vaccine in mice, such that IN immunisation induced higher serum IgG and respiratory tract IgA than immunisation with virus alone. The serum from mice thus immunised inhibited H1N1-mediated RBC agglutination strongly in vitro. If mannan similarly adjuvants low doses of influenza vaccine in humans, it could potentially be used for vaccine 'dose-sparing' in the event that a vaccine shortage arises from an epidemic involving a highly virulent human-to-human transmissable influenza strain.

The chemotherapeutic agent DMXAA as a unique IRF3-dependent type-2 vaccine adjuvant.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a potent type I interferon (IFN) inducer, was evaluated as a chemotherapeutic agent in mouse cancer models and proved to be well tolerated in human cancer clinical trials. Despite its multiple biological functions, DMXAA has not been fully characterized for the potential application as a vaccine adjuvant. In this report, we show that DMXAA does act as an adjuvant due to its unique property as a soluble innate immune activator. Using OVA as a model antigen, DMXAA was demonstrated to improve on the antigen specific immune responses and induce a preferential Th2 (Type-2) response. The adjuvant effect was directly dependent on the IRF3-mediated production of type-I-interferon, but not IL-33. DMXAA could also enhance the immunogenicity of influenza split vaccine which led to significant increase in protective responses against live influenza virus challenge in mice compared to split vaccine alone. We propose that DMXAA can be used as an adjuvant that targets a specific innate immune signaling pathway via IRF3 for potential applications including vaccines against influenza which requires a high safety profile.

Small cationic DDA:TDB liposomes as protein vaccine adjuvants obviate the need for TLR agonists in inducing cellular and humoral responses.

Most subunit vaccines require adjuvants in order to induce protective immune responses to the targeted pathogen. However, many of the potent immunogenic adjuvants display unacceptable local or systemic reactogenicity. Liposomes are spherical vesicles consisting of single (unilamellar) or multiple (multilamellar) phospholipid bi-layers. The lipid membranes are interleaved with an aqueous buffer, which can be utilised to deliver hydrophilic vaccine components, such as protein antigens or ligands for immune receptors. Liposomes, in particular cationic DDA:TDB vesicles, have been shown in animal models to induce strong humoral responses to the associated antigen without increased reactogenicity, and are currently being tested in Phase I human clinical trials. We explored several modifications of DDA:TDB liposomes--including size, antigen association and addition of TLR agonists--to assess their immunogenic capacity as vaccine adjuvants, using Ovalbumin (OVA) protein as a model protein vaccine. Following triple homologous immunisation, small unilamellar vesicles (SUVs) with no TLR agonists showed a significantly higher capacity for inducing spleen CD8 IFNγ responses against OVA in comparison with the larger multilamellar vesicles (MLVs). Antigen-specific antibody reponses were also higher with SUVs. Addition of the TLR3 and TLR9 agonists significantly increased the adjuvanting capacity of MLVs and OVA-encapsulating dehydration-rehydration vesicles (DRVs), but not of SUVs. Our findings lend further support to the use of liposomes as protein vaccine adjuvants. Importantly, the ability of DDA:TDB SUVs to induce potent CD8 T cell responses without the need for adding immunostimulators would avoid the potential safety risks associated with the clinical use of TLR agonists in vaccines adjuvanted with liposomes.

Mixed vector immunization with recombinant adenovirus and MVA can improve vaccine efficacy while decreasing antivector immunity.

Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (Ad+MVA) enhanced CD8(+) T cell-dependant protection of mice against challenge with Plasmodium berghei. Moreover, the degree of protection could be enhanced after homologous boosting with the same Ad+MVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8(+) T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the Ad+MVA mixture led to consistent boosting of the transgene-specific CD8(+) response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens.

Reactive oxygen species level defines two functionally distinctive stages of inflammatory dendritic cell development from mouse bone marrow.

Reactive oxygen species (ROS) have been implicated in various physiological activities. However, their role in dendritic cell (DC) activation and generation has not been investigated. Using the bone marrow-derived GM-CSF-induced ex vivo DC model, we characterize how induction of ROS correlates with inflammatory DC functionality and expansion. We describe that the functionality of GM-CSF-induced DCs is distinct in two developmental stages. Whereas division of DC-committed hematopoietic progenitor cells (HPCs) neared completion by day 6, the level of ROS soared after day 4. Day 3 ROS(lo) DCs were highly responsive to TLR stimuli such as LPS and zymosan by rapid upregulation of CD80, CD86, and MHC class II, in contrast to the low response of day 6 ROS(hi) DCs. ROS(hi) DCs could not initiate and sustain a significant level of NF-kappaB phosphorylation in response to LPS and zymosan, although demonstrating hyperactivation of p38 MAPK by LPS, in a fashion disparate to ROS(lo) DCs. ROS(lo) DCs stimulated a higher level of allogeneic and OVA-specific T cell proliferative responses, although ROS(hi) DCs were much more proficient in processing OVA. In response to pathogenic stimuli, ROS(hi) DCs also demonstrated rapid cellular adhesion and H(2)O(2) release, indicating their role in immediate microbial targeting. Moreover, HPC expansion and DC generation were dependent on the surge of ROS in an NADPH oxidase-independent manner. These findings point to the potential role of cellular ROS in mediating functionality and development of DCs from HPCs during inflammation.

Cell-penetrating peptides: application in vaccine delivery.

Recent years have seen a surge in interest in cell-penetrating peptides (CPP) as an efficient means for delivering therapeutic targets into cellular compartments. The cell membrane is impermeable to hydrophilic substances yet linking to CPP can facilitate delivery into cells. Thus the unique translocatory property of CPP ensures they remain an attractive carrier, with the capacity to deliver cargoes in an efficient manner having applications in drug delivery, gene transfer and DNA vaccination. Fundamental for an effective vaccine is the delivery of antigen epitopes to antigen-presenting cells, ensuing processing and presentation and induction of an immune response. Vaccination with proteins or synthetic peptides incorporating CTL epitopes have proven limited due to the failure for exogenous antigens to be presented efficiently to T cells. Linking of antigens to CPP overcomes such obstacles by facilitating cellular uptake, processing and presentation of exogenous antigen for the induction of potent immune responses. This review will encompass the various strategies for the delivery of whole proteins, T cell epitopes and preclinical studies utilizing CPP for cancer vaccines.

Intracellular detection and immune signaling pathways of DNA vaccines.

Parallel to attenuated and subunit vaccines, DNA vaccines require adjuvant signals in addition to antigen presentation for the induction of adaptive immune responses. As opposed to common beliefs, increasing evidence is showing that Toll-like receptor 9 activation by CpG motifs present in DNA vaccines are not vital for the induction of immune responses in vivo. Investigations on the signaling pathways of the adjuvant effect mediated by DNA vaccines have revealed other important mediators. DNA-dependent activator of interferon regulatory factors (DAI) and absent in melanoma (AIM)2 were recently identified as cytosolic DNA sensors that respond with the release of type I interferon and proinflammatory cytokines. Both are distinct molecules with different signaling pathways. AIM2 acts through inflammasomes to activate caspase-1, whilst DAI activates the transcription factors, NF-kappaB and interferon regulatory factor 3. Most significantly, the noncanonical IkappaB kinase, TANK-binding kinase-1, was identified as the essential signaling component in DNA vaccines that is responsible for the generation of immune responses. This review provides an update on the cellular detection and the subsequent signaling pathways mediated by DNA vaccines.

Molecular basis of improved immunogenicity in DNA vaccination mediated by a mannan based carrier.

Receptor mediated gene delivery is an attractive non-viral method for targeting genetic material to specific cell types. We have previously utilized oxidized (OMPLL) and reduced mannan poly-L-lysine (RMPLL) to target DNA vaccines to antigen presenting cells and demonstrated that it could induce far stronger immune responses in mice compared to naked DNA immunization. In this study, we describe the immune enhancing attributes of mannan-PLL mediated DNA vaccination at the molecular level. Several attributes observed in similar gene delivery conjugates, such as entry via the endocytic pathway, low toxicity, protection from nucleases and compaction of particle size, were also evident here. In addition, OMPLL and RMPLL conjugates had profound effects on the antigen presentation functions of dendritic cells and macrophages, through the stimulation of cytokine production and maturation of dendritic cells. Interestingly, we demonstrate that OMPLL-DNA and RMPLL-DNA are able to mediate dendritic cell activation via toll-like receptor 2 as opposed to mannan alone which mediates via toll-like receptor 4. Overall, this report leads to greater understanding of how oxidized and reduced mannan mediated gene delivery could augment immune responses to DNA vaccination and provide insights into ways of further improving its immunogenicity.

Strategies used for MUC1 immunotherapy: preclinical studies.

The development of effective immunotherapeutic approaches against cancer has been a major focus of research in the last 10-15 years. Despite the impressive progress in the last 10 years, which has incorporated purified proteins, DNA and targeting to dendritic cells and/or the use of Toll-like receptor ligands, there are still many hurdles to overcome in order to elicit effective immune responses that could totally eradicate cancer cells. MUC1 has attracted interest as a target for immunotherapy of malignancies, including solid cancers, such as breast, pancreas and ovary, and blood cancers, including multiple myeloma. Numerous methods have been shown to elicit humoral, cellular and tumor protective responses in preclinical settings. Many of these have entered into human clinical trials, which aim to evaluate the immunogenicity of MUC1 and its suitability for use in immunotherapy/vaccine for many malignancies.

Strategies used for MUC1 immunotherapy: human clinical studies.

MUC1 is a high-molecular-weight glycoprotein that is overexpressed in adenocarcinomas and hematological cancers. Numerous preclinical studies in mice have demonstrated that MUC1 is immunogenic, and that cellular and humoral immune responses could be induced depending on the MUC1 vaccine formulation. MUC1-based vaccines have quickly entered into human clinical trials, and immune responses and some clinical responses have been reported. Here, we give an up-to-date review of some of the MUC1-based vaccines that have entered clinical trials and their results in cancer patients.

Protein/peptide and DNA vaccine delivery by targeting C-type lectin receptors.

C-type lectin receptors (CLRs) are a class of pathogen-recognition receptors that are actively investigated in the field of vaccine delivery. Many of their properties have functions linked to the immune system. These receptors are expressed abundantly on antigen-presenting cells and are considered to be the sentinels of immune surveillance owing to their endocytic nature and the ability to recognize a diverse range of pathogens through recognition of pathogen-associated molecular patterns. CLRs are also involved in the processes of antigen presentation mediated through the induction of dendritic cell maturation and cytokine production. These properties engender CLRs to be ideal for vaccine targeting. Conversely, CLRs also function to recognize glycosylated self-antigens to induce homeostatic control and tolerance. In this review, we will describe the various preclinical/clinical vaccination strategies to target antigens and plasmid DNA to this diverse class of receptors.

Oxidized and reduced mannan mediated MUC1 DNA immunization induce effective anti-tumor responses.

DNA immunization is an attractive form of vaccination, which has shown promising results only in small animal models. There is a need to develop efficient gene delivery systems. We previously demonstrated that oxidized (OM) and reduced mannan (RM) complexed to ovalbumin DNA via poly-l-lysine (PLL), were able to generate potent immune responses in mice. Herein, we further investigated the suitability of OMPLL and RMPLL as carriers for mucin 1 (MUC1) DNA vaccination for cancer immunotherapy. Studies presented here showed that immune responses in C57BL/6 mice induced by OMPLL-MUC1 DNA and RMPLL-MUC1 DNA immunization were more immunogenic compared to MUC1 DNA alone. Moreover, tumor protection was evident at a dose as low as 0.5 microg. In addition, strong T cell responses were induced in HLA-A2 transgenic and human MUC1 transgenic mice. These results demonstrate the potential of OM and RM as efficient non-viral gene delivery carriers for DNA vaccines for use in cancer immunotherapy.

Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo.

Antigen mannosylation has been shown to be an effective approach to potentiate antigen immunogenicity, due to the enhanced antigen uptake and presentation by APC. To overcome disadvantages associated with conventional methods used to mannosylate antigens, we have developed a novel mannose-based antigen delivery system that utilizes a polyamidoamine (PAMAM) dendrimer. It is demonstrated that mannosylated dendrimer ovalbumin (MDO) is a potent immune inducer. With a strong binding avidity to DC, MDO potently induced OVA-specific T cell response in vitro. It was found that the immunogenicity of MDO was due not only to enhanced antigen presentation, but also to induction of DC maturation. Mice immunized with MDO generated strong OVA-specific CD4(+)/CD8(+) T cell and antibody responses. MDO also targeted lymph node DC to cross-present OVA, leading to OTI CD8(+) T cell proliferation. Moreover, upon challenge with B16-OVA tumor cells, tumors in mice pre-immunized with MDO either did not grow or displayed a much more delayed onset, and had slower kinetics of growth than those of OVA-immunized mice. This mannose-based antigen delivery system was applied here for the first time to the immunization study. With several advantages and exceptional adjuvanticity, we propose mannosylated dendrimer as a potential vaccine carrier.

Structure and design of polycationic carriers for gene delivery.

The development of safe and effective gene delivery methods is a major challenge to enable gene therapy or DNA vaccines to become a reality. Currently there are two major approaches for delivery of genetic material, viral and non-viral. The majority of on-going clinical trials in gene therapy or DNA vaccines use retroviruses and adenoviruses for delivering genetic materials. Viral delivery systems are far more effective than non-viral delivery however there are concerns regarding toxicity, immunogenicity and possible integration of viral genetic material into the human genome. Given the negative charge of the phosphate backbone of DNA, polycationic molecules have been the major focus as carriers of DNA. There are several physiological barriers to overcome for effective systemic delivery of DNA. The ideal vector must be stable in the systemic circulation, escape the reticuloendothelial system, able to extravasate tissues, enter the target cell, escape lysosomal degradation and transport DNA to the nucleus to be transcribed. With increasing understanding of the physicochemical properties essential to overcome the various barriers, it is possible to apply rational design to the cationic carriers. A number of poly-amino acids, cationic block co-polymers, dendrimers and cyclodextrins have been rationally designed to optimize gene delivery. This review will discuss approaches that have been used to design various synthetic polycations with enhanced DNA condensing ability, serum stability and endosomolytic capability for efficient gene transfer in vitro and in vivo.

Mannan derivatives induce phenotypic and functional maturation of mouse dendritic cells.

Mannan, a polysaccharide isolated from yeast binds to C-type lectins of the mannose receptor family, expressed by antigen-presenting cells (APCs) including dendritic cells (DCs) and macrophages. As these receptors mediate endocytosis, they have been targeted with ligands to deliver antigens into APCs to initiate immune responses. Immunization with tumour antigen MUC1 conjugated to oxidized mannan (OM) or reduced mannan (RM) induced differential immune responses in mice, and only mice immunized with OM-MUC1 elicited strong MUC1-specific cytotoxic T lymphocyte responses and protected mice from a MUC1 tumour challenge. In this study, the adjuvant effect of mannan and its derivatives including OM and RM, in comparison to lipopolysaccharide, on DCs were investigated. Mannan, OM and RM were capable of stimulating mouse bone marrow-derived DC in vitro, eliciting enhanced allogeneic T-cell proliferation and enhancing OTI/OTII peptide-specific T-cell responses. Injection of mice with mannan, OM and RM induced a mature phenotype of lymph node and splenic DCs. Analysis by reverse transcription-polymerase chain reaction indicated that Manna, OM and RM also stimulated up-regulation of inflammatory cytokines including interleukin-1beta and tumour necrosis factor-alpha, and differential T helper 1 (Th1)/Th2 cytokines. Subsequent experiments demonstrated that activation of DCs was Toll-like receptor-4-dependent. The data presented here, together with evidence reported previously on OM and RM in induction of immune responses in vivo, suggest that OM and RM exert a dual capacity to target antigen to APCs as well as mature DCs.