In silico identified CCR4 antagonists target regulatory T cells and exert adjuvant activity in vaccination

Adjuvants are substances that enhance immune responses and thus improve the efficacy of vaccination. Few adjuvants are available for use in humans, and the one that is most commonly used (alum) often induces suboptimal immunity for protection against many pathogens. There is thus an obvious need to develop new and improved adjuvants. We have therefore taken an approach to adjuvant discovery that uses in silico modeling and structure-based drug-design. As proof-of-principle we chose to target the interaction of the chemokines CCL22 and CCL17 with their receptor CCR4. CCR4 was posited as an adjuvant target based on its expression on CD4(+)CD25(+) regulatory T cells (Tregs), which negatively regulate immune responses induced by dendritic cells (DC), whereas CCL17 and CCL22 are chemotactic agents produced by DC, which are crucial in promoting contact between DC and CCR4(+) T cells. Molecules identified by virtual screening and molecular docking as CCR4 antagonists were able to block CCL22- and CCL17-mediated recruitment of human Tregs and Th2 cells. Furthermore, CCR4 antagonists enhanced DC-mediated human CD4(+) T cell proliferation in an in vitro immune response model and amplified cellular and humoral immune responses in vivo in experimental models when injected in combination with either Modified Vaccinia Ankara expressing Ag85A from Mycobacterium tuberculosis (MVA85A) or recombinant hepatitis B virus surface antigen (rHBsAg) vaccines. The significant adjuvant activity observed provides good evidence supporting our hypothesis that CCR4 is a viable target for rational adjuvant design.

Nanotechnology for the delivery of vaccines

Liposomes offer an ideal platform for the delivery of subunit vaccines, due to their versatility and flexibility, which allows for antigen as well as immunostimulatory lipids and TLR agonists to become associated with these bilayered vesicles. Liposomes have the ability to protect vaccine antigen, as well as enhance delivery to antigen presenting cells, whilst the importance of cationic surface charge for delivery of TB subunit vaccines and formation of an ‘antigen depot’ may play a key role in boosting cell-mediated immunity and Th1 immune responses. The rational design of vaccine adjuvants requires the thorough investigation into the physicochemical characteristics that dictate the function of a liposomal adjuvant. Within this thesis, physicochemical characteristics were investigated in order to show any effects on the biodistribution profiles and the ensuing immune responses of these formulations. Initially the role of liposome charge within the formulation was investigated and subsequently their efficacy as vaccine adjuvants in combination with their biodistribution was measured to allow the role of formulation in vaccine function to be considered. These results showed that cationic surface charge, in combination with high loading of H56 vaccine antigen through electrostatic binding, was crucial in the promotion of the ‘depot-effect’ at the injection site which increases the initiation of Th1 cell-mediated immune responses that are required to offer protection against tuberculosis. To further investigate this, different methods of liposome production were also investigated where antigen incorporation within the vesicles as well as surface adsorption were adopted. Using the dehydration-rehydration (DRV) method (where liposomes are freeze-dried in the presence of antigen to promote antigen encapsulation) and the double emulsion (DE) method, a range of liposomes entrapping antigen were formulated. Variation in the liposome preparation method can lead to antigen entrapment within the delivery system which has been shown to be greater for DRV-formulated liposomes compared to their DE-counterparts. This resulted in no significant effect on the vaccine biodistribution profile, as well as not significantly altering the efficacy of cationic liposomal adjuvants. To further enhance the efficacy of these systems, the addition of TLR agonists either at the vesicle surface as well as within the delivery system has been displayed through variation in the preparation method. Anionic liposomal adjuvants have been formulated, which displayed rapid drainage from the injection site to the draining lymph nodes and displayed a reduction in measured Th1 immune responses. However, variation in the preparation method can alter the immune response profile for anionic liposomal adjuvants with a bias in immune response to Th2 responses being noted. Through the use of high shear mixing and stepwise incorporation, the efficient loading of TLR agonist within liposomes has been shown. However, interestingly the conjugation between lipid and non-electrostatically bound TLR agonist, followed by insertion into the bilayer of DDA/TDB resulted in localised agonist retention at the injection site and further stimulation of the Th1 immune response at the SOI, spleen and draining lymphatics as well as enhanced antibody titres.

Particulate delivery systems for vaccines:what can we expect?

In our attempts to thwart the unwanted attentions of microbes by prophylactic and therapeutic vaccination, the knowledge of interactions at the molecular level may prove to be an invaluable asset. This article examines how particulate delivery systems such as liposomes and polymer microspheres can be applied in the light of recent advances in immunological understanding. Some of the biological interactions of these delivery systems are discussed with relevance for antigen trafficking and molecular pathways of immunogenicity and emphasis on the possible interaction of liposomal components. In particular, traditional concepts such as antigen protection, delivery to antigen presenting cells and depot formation remain important aspects, whilst the inclusion of selected co-adjuvants and enhanced delivery of these moieties in conjunction with antigen now has a firm rationale. © 2006 The Authors.

Recent developments in particulate-based vaccines

Vaccines remain a key tool in the defence against major diseases. However, in the development of vaccines a trade off between safety and efficacy is required with newer vaccines, based on sub-unit proteins and peptides, displaying improved safety profiles yet suffering from low efficacy. Adjuvants can be employed to improve their potency, but currently there are only a limited number of adjuvant systems licensed for clinical use. Of the new adjuvants being investigated, particulate systems offer several advantages including: passive targeting to the antigen-presenting cells within the immune system, protection against adjuvant degradation, and ability for sustained antigen release. There has been a range of particulate vaccine delivery systems outlined in recent patents including polymer-based microspheres (which are generally more focused on the use of synthetic polymers, in particular the polyesters) and surfactant-based vesicles. Within these formulations, several patented systems are exploiting the use of cationic lipids which, despite their limitations in gene therapy, clearly offer strong potential as adjuvants. Within this review, the current range of particulate system technologies being investigated as potential adjuvants are discussed with regard to both their respective advantages and the potential hurdles which must be overcome for such systems to be converted into successful pharmaceutical products.

Radiolabelling of antigen and liposomes for vaccine biodistribution studies

A relatively simple and effective method to follow the movement of pharmaceutical preparations such as vaccines in biodistribution studies is to radiolabel the components. Whilst single radiolabelling is common practice, in vaccine systems containing adjuvants the ability to follow both the adjuvant and the antigen is favourable. To this end, we have devised a dual-radiolabelling method whereby the adjuvant (liposomes) is labelled with 3H and the antigen (a subunit protein) with 125I. This model is stable and reproducible; we have shown release of the radiolabels to be negligible over periods of up to 1 week in foetal calf serum at 37° C. In this paper we describe the techniques which enable the radiolabelling of various components, assessing stability and processing of samples which all for their application in biodistribution studies. Furthermore we provide examples derived from our studies using this model in tuberculosis vaccine biodistribution studies. © 2010 by the authors.

A case-study investigating the physicochemical characteristics that dictate the function of a liposomal adjuvant

A range of particulate delivery systems have been considered as vaccine adjuvants. Of these systems, liposomes offer a range of advantages including versatility and flexibility in design format and their ability to incorporate a range of immunomodulators and antigens. Here we briefly outline research, from within our laboratories, which focused on the systematic evaluation of cationic liposomes as vaccines adjuvants. Our aim was to identify physicochemical characteristics that correlate with vaccine efficacy, with particular consideration of the interlink between depot-forming action and immune responses. A variety of parameters were investigated and over a range of studies we have confirmed that cationic liposomes, based on dimethyldioctadecylammonium bromide and trehalose 6,6'-dibehenate formed a depot at the injection site, which stimulates recruitment of antigen presenting cells to the injection site and promotes strong humoral and cell-mediated immune responses. Physicochemical factors which promote a strong vaccine depot include the combination of a high cationic charge and electrostatic binding of the antigen to the liposome system and the use of lipids with high transition temperatures, which form rigid bilayer vesicles. Reduction in vesicle size of cationic vesicles did not promote enhanced drainage from the injection site. However, reducing the cationic nature through substitution of the cationic lipid for a neutral lipid, or by masking of the charge using PEGylation, resulted in a reduced depot formation and reduced Th1-type immune responses, while Th2-type responses were less influenced. These studies confirm that the physicochemical characteristics of particulate-based adjuvants play a key role in the modulation of immune responses.

Correlating liposomal adjuvant characteristics to in-vivo cell-mediated immunity using a novel Mycobacterium tuberculosis fusion protein:a multivariate analysis study

Objective In this study, we have used a chemometrics-based method to correlate key liposomal adjuvant attributes with in-vivo immune responses based on multivariate analysis. Methods The liposomal adjuvant composed of the cationic lipid dimethyldioctadecylammonium bromide (DDA) and trehalose 6,6-dibehenate (TDB) was modified with 1,2-distearoyl-sn-glycero-3-phosphocholine at a range of mol% ratios, and the main liposomal characteristics (liposome size and zeta potential) was measured along with their immunological performance as an adjuvant for the novel, postexposure fusion tuberculosis vaccine, Ag85B-ESAT-6-Rv2660c (H56 vaccine). Partial least square regression analysis was applied to correlate and cluster liposomal adjuvants particle characteristics with in-vivo derived immunological performances (IgG, IgG1, IgG2b, spleen proliferation, IL-2, IL-5, IL-6, IL-10, IFN-γ). Key findings While a range of factors varied in the formulations, decreasing the 1,2-distearoyl-sn-glycero-3-phosphocholine content (and subsequent zeta potential) together built the strongest variables in the model. Enhanced DDA and TDB content (and subsequent zeta potential) stimulated a response skewed towards a cell mediated immunity, with the model identifying correlations with IFN-γ, IL-2 and IL-6. Conclusion This study demonstrates the application of chemometrics-based correlations and clustering, which can inform liposomal adjuvant design.

Improving T cell-induced response to subunit vaccines:opportunities for a proteomic systems approach

Prophylactic vaccines are an effective strategy to prevent development of many infectious diseases. With new and re-emerging infections posing increasing risks to food stocks and the health of the population in general, there is a need to improve the rationale of vaccine development. One key challenge lies in development of an effective T cell-induced response to subunit vaccines at specific sites and in different populations. Objectives: In this review, we consider how a proteomic systems-based approach can be used to identify putative novel vaccine targets, may be adopted to characterise subunit vaccines and adjuvants fully. Key findings: Despite the extensive potential for proteomics to aid our understanding of subunit vaccine nature, little work has been reported on identifying MHC 1-binding peptides for subunit vaccines generating T cell responses in the literature to date. Summary: In combination with predictive and structural biology approaches to mapping antigen presentation, proteomics offers a powerful and as yet un-tapped addition to the armoury of vaccine discovery to predict T-cell subset responses and improve vaccine design strategies.

Vaccines

Vaccines continue to offer the key line of protection against a range of infectious diseases; however, the range of vaccines currently available is limited. One key consideration in the development of a vaccine is risk-versus-benefit, and in an environment of perceived low risk, the benefit of vaccination may not be recognised. To address this, there has been a move towards the use of subunit-based vaccines, which offer low side-effect profiles but are generally weakly immunogenic. This can be compensated for by the development of effective adjuvants. Nanotechnology offers key attributes in this field through the ability of nanoparticulates to incorporate and protect antigens from rapid degradation, combined with their potential to effectively deliver the antigens to appropriate cells within the immune system. These characteristics can be exploited in the development of new adjuvants. This chapter will outline the applications of nanosystems in vaccine formulations and consider the mechanisms of action behind a range of formulations.

Fas-FasL expression and interactions in mouse tumor cell lines: Implications for tumor immune escape

The Fas system, comprising the Fas receptor (Fas/Apo-1/CD95) and its ligand, Fas ligand (FasL), is a central mediator of programmed cell death in various physiological and pathological processes. FasL exists as transmembrane and soluble forms and induces apoptosis on crosslinking with Fas receptor. Recent evidence indicated that tumor cells exploit this system for their immunologic escape that includes the loss of Fas and the gain of FasL expression. In the present study, nine mouse tumor cell lines of diverse origin were examined immunocytochemically for the expression of Fas and FasL. Nine of nine cell lines expressed FasL, and five of nine cell lines expressed Fas. FasL expression in these tumor cell lines was demonstrated to be functional by its induction of apoptosis in Fas-sensitive target cells in coculture experiments. These results suggest that FasL may be a prevalent mediator of immune privilege in mouse malignancies, and support the recently proposed "counterattack model" for local elimination of tumor-reactive immune cells by tumor cell-derived FasL.^ Culture supernatant of four cell lines expressing FasL showed cytotoxic effect on Fas-sensitive target cells, indicating the possibility of secreted FasL in the medium. The Fas-expressing cell lines were sensitized to anti-Fas antibody cytotoxicity following treatment with IL-2 and IFN-$\gamma$, suggesting cytokine stimulation as an effective target for future immunotherapeutic strategies. ^

Fungicidas protetores no manejo da ferrugem da soja, processos fisiológicos e produtividade da cultura

Currently, the management recommendations for asian soybean rust (ASR) has been based on the application of protective fungicides mixed with triazoles and stronilurins. Thus, this study aimed at assessing whether the increased productivity provided by the application of protective fungicides is due solely to the fungicidal action of the product or some physiological changes in the plant and which the latter would be. The experiment was conducted from March to July 2015 at the experimental station of Udi Research and Development in Uberlândia-MG, with the cultivar 97Y07 RR. The experimental design chosen for this study was comprised of a randomized block with four replications and 16 treatments: check, fluxapyroxad + pyraclostrobin (116.55 + 58.45 g ha-1), azoxystrobin + benzovindiflupir (90 + 45 g ha-1), trifloxystrobin + prothioconazole (60 + 70 g ha-1), tebuconazole + picoxystrobin (100 + 60 g ha-1), picoxystrobin + cyproconazole (60 + 24 g ha-1), mancozeb (1125 g ha-1), azoxistrobina + tebuconazole + difenoconazole (60 + 75 + 120 g ha-1), azoxystrobin + tebuconazole + difenoconazole + chlorothalonil ( 60 + 120 + 75 + 1440 g ha-1), and mistures fluxapyroxad + pyraclostrobin + mancozeb, azoxystrobin + benzovindiflupir + mancozeb, trifloxystrobin + prothioconazole + mancozeb, tebuconazole + picoxystrobin + mancozeb, picoxystrobin + cyproconazole + mancozeb, azoxystrobin + tebuconazole + difenoconazole + mancozeb, and azoxystrobin + benzovindiflupir + chlorothalonil, from the aforesaid doses. The first application of the treatments occurred in R1, in the absence of symptoms. The number of applications, intervals and the use of adjuvants were performed according to the recommendations by manufacturers. The variables analyzed were: disease severity, concentration of chlorophylls and carotenoids, photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs), internal carbon concentration (Ci), instantaneous efficiency in water use (A/E), intrinsic water use efficiency (A/gs), and carboxylation efficiency (A/C). With these data collected, this study set to date the progress curve of each variable (AUPC). At the end of the crop cycle, the average of pods per plant was quantified, grain per pod, productivity and weight of 1,000 grains. It was concluded that: the addition of mancozeb to fluxapyroxad + pyraclostrobin, azoxystrobin + benzovindiflupir, trifloxystrobin + prothioconazole and tebuconazole + picoxystrobin potentiated the ASR control; adding mancozebe to the mixture azoxystrobin + benzovindiflupir provided better control of the disease compared to the addition of chlorothalonil; mancozeb amounts to AUPC concentration of photosynthetic pigments and when added to axozystrobin + tebuconazole + difenoconazole, increases the AUPC for total chlorophyll concentration, as well as when chlorothalonil was added; mancozeb added to the mix fluxapyroxad + pyraclostrobin raised the AUPC for A/Ci and A/gs, increasing the W1,000G and crop productivity; the addition of protectors similarly reflected on the productivity of culture.

The administration route is decisive for the ability of the vaccine adjuvant CAF09 to induce antigen-specific CD8+ T-cell responses:the immunological consequences of the biodistribution profile

A prerequisite for vaccine-mediated induction of CD8+ T-cell responses is the targeting of dendritic cell (DC) subsets specifically capable of cross-presenting antigen epitopes to CD8+ T cells. Administration of a number of cationic adjuvants via the intraperitoneal (i.p.) route has been shown to result in strong CD8+ T-cell responses, whereas immunization via e.g. the intramuscular (i.m.) or subcutaneous (s.c.) routes often stimulate weak CD8+ T-cell responses. The hypothesis for this is that self-drainage of the adjuvant/antigen to the lymphoid organs, which takes place upon i.p. immunization, is required for the subsequent activation of cross-presenting lymphoid organ-resident CD8α+ DCs. In contrast, s.c. or i.m. immunization usually results in the formation of a depot at the site of injection (SOI), which hinders the self-drainage and targeting of the vaccine to cross-presenting CD8α+ DCs. We investigated this hypothesis by correlating the biodistribution pattern and the adjuvanticity of the strong CD8+ T-cell inducing liposomal cationic adjuvant formulation 09 (CAF09), which is composed of dimethyldioctadecylammonium bromide/monomycoloyl glycerol liposomes with polyinosinic:polycytidylic acid electrostatically adsorbed to the surface. Biodistribution studies with radiolabeled CAF09 and a surface-adsorbed model antigen [ovalbumin (OVA)] showed that a significantly larger fraction of the vaccine dose localized in the draining lymph nodes (dLNs) and the spleen 6 h after i.p. immunization, as compared to after i.m. immunization. Studies with fluorescently labelled OVA + CAF09 demonstrated a preferential association of OVA + CAF09 to DCs/monocytes, as compared to macrophages and B cells, following i.p. immunization. Administration of OVA + CAF09 via the i.p. route did also result in DC activation, whereas no DC activation could be measured within the same period with unadjuvanted OVA and OVA + CAF09 administered via the s.c. or i.m. routes. In the dLNs, the highest level of activated, cross-presenting CD8α+ DCs was detected at 24 h post immunization, whereas an influx of activated, migrating and cross-presenting CD103+ DCs to the dLNs could be measured after 48 h. This suggests that the CD8α+ DCs are activated by self-draining OVA + CAF09 in the lymphoid organs, whereas the CD103+ DCs are stimulated by the OVA + CAF09 at the SOI. These results support the hypothesis that the self-drainage of OVA + CAF09 to the draining LNs is required for the activation of CD8α+ DCs, while the migratory CD103+ DCs may play a role in sustaining the subsequent induction of strong CD8+ T-cell responses.

Rapid quantification and validation of lipid concentrations within liposomes

Quantification of the lipid content in liposomal adjuvants for subunit vaccine formulation is of extreme importance, since this concentration impacts both efficacy and stability. In this paper, we outline a high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method that allows for the rapid and simultaneous quantification of lipid concentrations within liposomal systems prepared by three liposomal manufacturing techniques (lipid film hydration, high shear mixing, and microfluidics). The ELSD system was used to quantify four lipids: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, dimethyldioctadecylammonium (DDA) bromide, and D-(+)-trehalose 6,6′-dibehenate (TDB). The developed method offers rapidity, high sensitivity, direct linearity, and a good consistency on the responses (R2 > 0.993 for the four lipids tested). The corresponding limit of detection (LOD) and limit of quantification (LOQ) were 0.11 and 0.36 mg/mL (DMPC), 0.02 and 0.80 mg/mL (cholesterol), 0.06 and 0.20 mg/mL (DDA), and 0.05 and 0.16 mg/mL (TDB), respectively. HPLC-ELSD was shown to be a rapid and effective method for the quantification of lipids within liposome formulations without the need for lipid extraction processes.

ROLE OF STAT1 AND CXCL10 IN THE TUMOUR IMMUNE MICROENVIRONMENT OF HIGH-GRADE SEROUS OVARIAN CANCER

High-grade serous ovarian cancer (HGSC) is the most prevalent epithelial ovarian cancer characterized by late detection, metastasis and resistance to chemotherapy. Previous studies on the tumour immune microenvironment in HGSC identified STAT1 and CXCL10 as the most differentially expressed genes between treatment naïve chemotherapy resistant and sensitive tumours. Interferon-induced STAT1 is a transcription factor, which induces many genes including tumour suppressor genes and those involved in recruitment of immune cells to the tumour immune microenvironment (TME), including CXCL10. CXCL10 is a chemokine that recruits tumour infiltrating lymphocytes (TILs) and exhibits angiostatic function. The current study was performed to determine the effects of differential STAT1 and CXCL10 expression on HGSC disease progression and TME. STAT1 expression and intratumoural CD8+ T cells were evaluated as prognostic and predictive biomarkers via immunohistochemistry on 734 HGSC tumours accrued from the Terry Fox Research Institute-Canadian Ovarian Experimental Unified Resource. The combined effect of STAT1 expression and CD8+ TIL density was confirmed as prognostic and predictive companion biomarkers in the second independent biomarker validation study. Significant positive correlation between STAT1 expression and intratumoral CD8+ TIL density was observed. The effects of enforced CXCL10 expression on HGSC tumour growth, vasculature and immune tumour microenvironment were studied in the ID8 mouse ovarian cancer cell engraftment in immunocompetent C57BL/6 mice. Significant decrease in tumour progression in mice injected with ID8 CXCL10 overexpressing cells compared to mice injected with ID8 vector control cells was observed. Multiplexed cytokine analysis of ascites showed differential expression of IL-6, VEGF and CXCL9 between the two groups. Endothelial cell marker staining showed differences in tumour vasculature between the two groups. Immune transcriptomic profiling identified distinct expression profiles in genes associated with cytokines, chemokines, interferons, T cell function and apoptosis between the two groups. These findings provide evidence that STAT1 is an independent biomarker and in combination with CD8+ TIL density could be applied as novel immune-based biomarkers in HGSC. These results provide the basis for future studies aimed at understanding mechanisms underlying differential tumour STAT1 and CXCL10 expression and its role in pre-existing tumour immunologic diversity, thus potentially contributing to biomarker guided immune modulatory therapies.

Anti-N-methyl-D-Aspartate Receptor Encephalitis with Positive Serum Antithyroid Antibodies, IgM Antibodies Against Mycoplasma Pneumoniae and Human Herpesvirus 7 PCR in the CSF

We report the case of a boy with an encephalopathy associated with extrapyramidal and psychiatric symptoms and anti-N-methyl-D-aspartate receptor antibodies. He had positive serum antithyroid antibodies, IgM antibodies against Mycoplasma pneumoniae and human herpesvirus 7 polymerase chain reaction in the cerebrospinal fluid. He was successfully treated with rituximab, after steroids, intravenous immunoglobulin and plasma exchange. The pathophysiology of this disorder may be post-infectious and autoimmune.