Development of peptide vaccines in dengue

Dengue is one of the most important arboviral infection worldwide, infecting up to 390 million people and causing 25,000 deaths annually. Although a licensed dengue vaccine is available, it is not efficacious against dengue serotypes that infect people living in South East Asia, where dengue is an endemic disease. Hence, there is an urgent need to develop an efficient dengue vaccine for this region. Data from different clinical trials indicate that a successful dengue vaccine must elicit both neutralizing antibodies and cell mediated immunity. This can be achieved by designing a multi-epitope peptide vaccine comprising B, CD8+ and CD4+ T cell epitopes. As recognition of T cell epitopes are restricted by human leukocyte antigens (HLA), T cell epitopes which are able to recognize several major HLAs will be preferentially included in the vaccine design. While peptide vaccines are safe, biocompatible and cost-effective, it is poorly immunogenic. Strategies to improve its immunogenicity by the use of long peptides, adjuvants and nanoparticle delivery mechanisms are discusse

Design of Peptide-Based Nanovaccines Targeting Leading Antigens From Gynecological Cancers to Induce HLA-A2.1 Restricted CD8+ T Cell Responses

Gynecological cancers are a leading cause of mortality in women. CD8+ T cell immunity largely correlates with enhanced survival, whereas inflammation is associated with poor prognosis. Previous studies have shown polystyrene nanoparticles (PSNPs) are biocompatible, do not induce inflammation and when used as vaccine carriers for model peptides induce CD8+ T cell responses. Herein we test the immunogenicity of 24 different peptides, from three leading vaccine target proteins in gynecological cancers: the E7 protein of human papilloma virus (HPV); Wilms Tumor antigen 1 (WT1) and survivin (SV), in PSNP conjugate vaccines. Of relevance to vaccine development was the finding that a minimal CD8+ T cell peptide epitope from HPV was not able to induce HLA-A2.1 specific CD8+ T cell responses in transgenic humanized mice using conventional adjuvants such as CpG, but was nevertheless able to generate strong immunity when delivered as part of a specific longer peptide conjugated to PSNPs vaccines. Conversely, in most cases, when the minimal CD8+ T cell epitopes were able to induce immune responses (with WT1 or SV super agonists) in CpG, they also induced responses when conjugated to PSNPs. In this case, extending the sequence around the CD8+ T cell epitope, using the natural protein context, or engineering linker sequences proposed to enhance antigen processing, had minimal effects in enhancing or changing the cross-reactivity pattern induced by the super agonists. Nanoparticle approaches, such as PSNPs, therefore may offer an alternative vaccination strategy when conventional adjuvants are unable to elicit the desired CD8+ T cell specificity. The findings herein also offer sequence specific insights into peptide vaccine design for nanoparticle-based vaccine carriers

Dendritic Cells and Myeloid Derived Suppressor Cells Fully Responsive to Stimulation via Toll-Like Receptor 4 Are Rapidly Induced from Bone-Marrow Cells by Granulocyte-Macrophage Colony-Stimulating Factor

Dendritic cells (DCs) are commonly generated from bone marrow (BM) progenitor cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or in combination with interleukin 4 (IL-4). These cells are often harvested post day 5, when they acquire maturation markers and can stimulate T cells. Apart from DCs, myeloid derived suppressor cells (MDSCs) are also found within these cultures. However, little is known about the functional characteristics of DCs and MDSCs before day 5. Herein, using a murine model, it is shown that early DCs and MDSCs, even in cultures with GM-CSF alone, upregulate fully maturation and activation surface molecules in response to the toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) stimulation. Despite initially displaying lower marker expression levels, these cells efficiently induced T cell stimulation and cytokine production. Interestingly, Gr-1int MDSCs increased their T cell co-stimulatory activity upon TLR4 stimulation. Additionally, early DCs and MDSCs exhibited differential endocytic capacity for viral sized nanoparticles and bacterial sized microparticles. DCs internalized both particle sizes, whilst MDSCs only internalized the larger microparticles, with reduced endocytic activity over time in the culture. These findings have unveiled an important role for the rapid initiation of productive immunity by GM-CSF, with promising implications for future vaccine and DC immunotherapy developments

A Synthetic Nanoparticle Based Vaccine Approach Targeting MSP4/5 Is Immunogenic and Induces Moderate Protection Against Murine Blood-Stage Malaria

Malaria remains a significant health problem in many tropical and sub-tropical regions. The development of vaccines against the clinically active blood-stage of infection needs to consider variability and polymorphism in target antigens, and an adjuvant system able to induce broad spectrum immunity comprising both antibodies and helper T cells. Moreover, recent studies have shown some conventional pro-inflammatory adjuvants can also promote expansion of immunosuppressive regulatory T cells (Treg) and myeloid derived suppressor cells (MDSC), both of which could negatively impact malaria disease progression. Herein, we explore the ability of a model nanoparticle delivery system (polystyrene nanoparticles; PSNPs), previously proven to not induce conventional inflammation, Treg or MDSC, to induce immunity to MSP4/5 from Plasmodium yoelii, a member of the MSP4 and MSP5 family of proteins which are highly conserved across diverse malaria species including P. falciparum. The results show PSNPs-MSP4/5 conjugates are highly immunogenic, inducing immune responses comprising both T helper 1 (Th1) and Th2 cellular immunity, and a spectrum of antibody subclasses including IgG1, IgG2a, and IgG2b. Benchmarked against Alum and Complete Freund's Adjuvant (CFA), the immune responses that were induced were of comparable or higher magnitude, for both T cell frequencies by ELISpot and antibody responses in terms of ELISA end titer. Importantly, immunization with PSNPs-MSP4/5 induced partial protection against malaria blood-stage infection (50–80%) shown to be mechanistically dependent on interferon gamma (IFN-γ) production. These results expand the scope of adjuvants considered for malaria blood-stage vaccine development to those that do not use conventional adjuvant pathways and emphasizes the critical role of cellular immunity and specifically IFN-γ producing cells in providing moderate protection against blood-stage malaria comparable to Freunds adjuvant

Engineered hydrogen-bonded glycopolymer capsules and their interactions with antigen presenting cells

Hollow glycopolymer microcapsules were fabricated by hydrogen-bonded layer-by-layer (LbL) assembly, and their interactions with a set of antigen presenting cells (APCs), including dendritic cells (DCs), macrophages (MACs), and myeloid derived suppressor cells (MDSCs), were investigated. The glycopolymers were obtained by cascade postpolymerization modifications of poly(oligo(2-ethyl-2-oxazoline methacrylate)-stat-glycidyl methacrylate) involving the modification of the glycidyl groups with propargylamine and the subsequent attachment of mannose azide by copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC). Multilayer assembly of the hydrogen-bonding pair (glycopolymer/poly(methacrylic acid) (PMA)) onto planar and particulate supports (SiO2 particles, d = 1.16 μm) yielded stable glycopolymer films upon cross-linking by CuAAC. The silica (SiO2) particle templates were removed yielding hollow monodisperse capsules, as demonstrated by fluorescence and scanning electron microscopy. Cellular uptake studies using flow cytometry revealed the preferential uptake of the capsules by DCs when compared to MACs or MDSCs. Mannosylated capsules showed a cytokine independent cis-upregulation of CD80 specifically on DCs and a trans-downregulation of PDL-1 on MDSCs. Thus, the glycopolymer capsules may have potential as vaccine carriers, as they are able to upregulate costimulatory molecules for immune cell stimulation on DCs and at the same time downregulate immune inhibitory receptors on suppressor APC such as MDSCs

The Key Role of TNF-TNFR2 Interactions in the Modulation of Allergic Inflammation: A Review

Tumor necrosis factor-alpha (TNF) is a pleiotropic cytokine, which is thought to play a major role in the pathogenesis of inflammatory diseases, including allergy. TNF is produced at the early stage of allergen sensitization, and then continues to promote the inflammation cascade in the effector phase of allergic reactions. Consequently, anti-TNF treatment has been proposed as a potential therapeutic option. However, recent studies reveal anti-intuitive effects of TNF in the activation and proliferative expansion of immunosuppressive Tregs, tolerogenic DCs and MDSCs. This immunosuppressive effect of TNF is mediated by TNFR2, which is preferentially expressed by immunosuppressive cells. These findings redefine the role of TNF in allergic reaction, and suggest that targeting TNF-TNFR2 interaction itself may represent a novel strategy in the treatment of allergy

Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films

Daily assessment of the percentage of erythrocytes that are infected ('percent-parasitaemia') across a time-course is a necessary step in many experimental studies of malaria, but represents a time-consuming and unpopular task among researchers. The most common method is extensive microscopic examination of Giemsa-stained thin blood-films. This study explored a method for the assessment of percent-parasitaemia that does not require extended periods of microscopy and results in a descriptive and permanent record of parasitaemia data that is highly amenable to subsequent 'data-mining'. Digital photography was utilized in conjunction with a basic purpose-written computer programme to test the viability of the concept. Partial automation of the determination of percent parasitaemia was then explored, resulting in the successful customization of commercially available broad-spectrum image analysis software towards this aim. Lastly, automated discrimination between infected and uninfected RBCs based on analysis of digital parameters of individual cell images was explored in an effort to completely automate the calculation of an accurate percent-parasitaemia