Proteins accessible to immune surveillance show significant T-cell epitope depletion:implications for vaccine design

T cell activation is the final step in a complex pathway through which pathogen-derived peptide fragments can elicit an immune response. For it to occur, peptides must form stable complexes with Major Histocompatibility Complex (MHC) molecules and be presented on the cell surface. Computational predictors of MHC binding are often used within in silico vaccine design pathways. We have previously shown that, paradoxically, most bacterial proteins known experimentally to elicit an immune response in disease models are depleted in peptides predicted to bind to human MHC alleles. The results presented here, derived using software proven through benchmarking to be the most accurate currently available, show that vaccine antigens contain fewer predicted MHC-binding peptides than control bacterial proteins from almost all subcellular locations with the exception of cell wall and some cytoplasmic proteins. This effect is too large to be explained from the undoubted lack of precision of the software or from the amino acid composition of the antigens. Instead, we propose that pathogens have evolved under the influence of the host immune system so that surface proteins are depleted in potential MHC-binding peptides, and suggest that identification of a protein likely to contain a single immuno-dominant epitope is likely to be a productive strategy for vaccine design.

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.

Developing solid particulate vaccine adjuvants - surface bound antigen favouring a humoural response, whereas entrapped antigen shows a tendency for cell mediated immunity

This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.

Th1 immune responses can be modulated by varying dimethyldioctadecylammonium and distearoyl-sn-glycero-3-phosphocholine content in liposomal adjuvants

Objectives - Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) combined with trehalose 6,6'-dibehenate (TDB) elicit strong cell-mediated and antibody immune responses; DDA facilitates antigen adsorption and presentation while TDB potentiates the immune response. To further investigate the role of DDA, DDA was replaced with the neutral lipid of distearoyl-sn-glycero-3-phosphocholine (DSPC) over a series of concentrations and these systems investigated as adjuvants for the delivery of Ag85B–ESAT-6-Rv2660c, a multistage tuberculosis vaccine. Methods - Liposomal were prepared at a 5?:?1 DDA–TDB weight ratio and DDA content incrementally replaced with DSPC. The physicochemical characteristics were assessed (vesicle size, zeta potential and antigen loading), and the ability of these systems to act as adjuvants was considered. Key findings - As DDA was replaced with DSPC within the liposomal formulation, the cationic nature of the vesicles decreases as does electrostatically binding of the anionic H56 antigen (Hybrid56; Ag85B-ESAT6-Rv2660c); however, only when DDA was completed replaced with DSPC did vesicle size increase significantly. T-helper 1 (Th1)-type cell-mediated immune responses reduced. This reduction in responses was attributed to the replacement of DDA with DSPC rather than the reduction in DDA dose concentration within the formulation. Conclusion - These results suggest Th1 responses can be controlled by tailoring the DDA/DSPC ratio within the liposomal adjuvant system.

Th1 immune responses can be modulated by varying dimethyldioctadecylammonium and distearoyl-sn-glycero-3-phosphocholine content in liposomal adjuvants

Objectives Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) combined with trehalose 6,6′-dibehenate (TDB) elicit strong cell-mediated and antibody immune responses; DDA facilitates antigen adsorption and presentation while TDB potentiates the immune response. To further investigate the role of DDA, DDA was replaced with the neutral lipid of distearoyl-sn- glycero-3-phosphocholine (DSPC) over a series of concentrations and these systems investigated as adjuvants for the delivery of Ag85B-ESAT-6-Rv2660c, a multistage tuberculosis vaccine. Methods Liposomal were prepared at a 5: 1 DDA-TDB weight ratio and DDA content incrementally replaced with DSPC. The physicochemical characteristics were assessed (vesicle size, zeta potential and antigen loading), and the ability of these systems to act as adjuvants was considered. Key findings As DDA was replaced with DSPC within the liposomal formulation, the cationic nature of the vesicles decreases as does electrostatically binding of the anionic H56 antigen (Hybrid56; Ag85B-ESAT6-Rv2660c); however, only when DDA was completed replaced with DSPC did vesicle size increase significantly. T-helper 1 (Th1)-type cell-mediated immune responses reduced. This reduction in responses was attributed to the replacement of DDA with DSPC rather than the reduction in DDA dose concentration within the formulation. Conclusion These results suggest Th1 responses can be controlled by tailoring the DDA/DSPC ratio within the liposomal adjuvant system. © 2013 Royal Pharmaceutical Society.

Developing solid particulate vaccine adjuvants:surface bound antigen favouring a humoural response, whereas entrapped antigen shows a tendency for cell mediated immunity

This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.

Molecular studies in gorgonian alloimmunity : search for gene homologs of the immunoglobulin gene superfamily in Swiftia exserta

Humoral and cells surface molecules of the mammalian immune system, grouped into the Immunoglobulin Gene Superfamily, share protein structure and gene sequence homologies with molecules found among diverse phylogenetic groups. In histocompatibility studies, the gorgonian coral Swiftia exserta has recently demonstrated specific alloimmunity with memory (Salter-Cid and Bigger, 1991. Biological Bulletin Vol 181). In an attempt to shed light on the origins of this gene family and the evolution of the vertebrate immune response, genomic DNA from Swiftia exserta was isolated, purified, and analyzed by Southern blot hybridization with mouse gene probes corresponding to two molecules of the Immunoglobulin Gene Superfamily, the Thy-1 antigen, and the alpha-3 domain of the MHC Class I histocompatibility marker. Hybridizations were conducted under low to non-stringent conditions to allow binding of mismatched homologs that may exist between the mouse gene probes and the Swiftia DNA. Removal of non-specific binding (sequences less than 70% homologous) occurred in washing steps. Results show that with the probes selected, the method chosen, and the conditions applied, no evidence of sequences of 70% or greater homology to the mouse Thy-1 or MHC Class I alpha-3 genes exist in Swiftia exserta genome.

Data compilation on the biological response to ocean acidification: environmental and experimental context of data sets and related literature

The exponential growth of studies on the biological response to ocean acidification over the last few decades has generated a large amount of data. To facilitate data comparison, a data compilation hosted at the data publisher PANGAEA was initiated in 2008 and is updated on a regular basis (doi:10.1594/PANGAEA.149999). By January 2015, a total of 581 data sets (over 4 000 000 data points) from 539 papers had been archived. Here we present the developments of this data compilation five years since its first description by Nisumaa et al. (2010). Most of study sites from which data archived are still in the Northern Hemisphere and the number of archived data from studies from the Southern Hemisphere and polar oceans are still relatively low. Data from 60 studies that investigated the response of a mix of organisms or natural communities were all added after 2010, indicating a welcomed shift from the study of individual organisms to communities and ecosystems. The initial imbalance of considerably more data archived on calcification and primary production than on other processes has improved. There is also a clear tendency towards more data archived from multifactorial studies after 2010. For easier and more effective access to ocean acidification data, the ocean acidification community is strongly encouraged to contribute to the data archiving effort, and help develop standard vocabularies describing the variables and define best practices for archiving ocean acidification data.

An investigation of the molecular interactions between statins and bacterial pathogens, and their combined impact on the human immune system

Statins are a class of drug that inhibits cholesterol biosynthesis, and are used to treat patients with high serum cholesterol levels. They exert this function by competitively binding to the enzyme 3-hydroxy-3-methylglutaryl-CoenzymeA reductase (HMGR), which catalyses the formation of mevalonate, a rate-limiting step in cholesterol biosynthesis. In addition, statins have what are called “pleiotropic effects”, which include the reduction of inflammation, immunomodulation, and antimicrobial effects. Statins can also improve survival of patients with sepsis and pneumonia. Cystic fibrosis (CF) is the most common recessive inherited disease in the Caucasian population, which is characterised by factors including, but not limited to, excessive lung inflammation and increased susceptibility to infection. Therefore, the overall objective of this study was to examine the effects of statins on CFassociated bacterial pathogens and the host response. In this work, the prevalence of HMGR was examined in respiratory pathogens, and several CF-associated pathogens were found to possess homologues of this enzyme. HMGR homology was analysed in Staphylococcus aureus, Burkholderia cenocepacia and Streptococcus pneumoniae, and the HMGR of B. cenocepacia was found to have significant conservation to that of Pseudomonas mevalonii, which is the most widely-characterised bacterial HMGR. However, in silico analysis revealed that, unlike S. aureus and S. pneumoniae, B. cenocepacia did not possess homologues of other mevalonate pathway proteins, and that the HMGR of B. cenocepacia appeared to be involved in an alternative metabolic pathway. The effect of simvastatin was subsequently tested on the growth and virulence of S. aureus, B. cenocepacia and S. pneumoniae. Simvastatin inhibited the growth of all 3 species in a dose-dependent manner. In addition, statin treatment also attenuated biofilm formation of all 3 species, and reduced in vitro motility of S. aureus. Interestingly, simvastatin also increased the potency of the aminoglycoside antibiotic gentamicin against B. cenocepacia. The impact of statins was subsequently tested on the predominant CF-associated pathogen Pseudomonas aeruginosa, which does not possess a HMGR homologue. Mevastatin, lovastatin and simvastatin did not influence the growth of this species. However, sub-inhibitory statin concentrations reduced the swarming motility and biofilm formation of P. aeruginosa. The influence of statins was also examined on Type 3 toxin secretion, quorum sensing and chemotaxis, and no statin effect was observed on any of these phenotypes. Statins did not appear to have a characteristic effect on the P. aeruginosa transcriptome. However, a mutant library screen revealed that the effect of statins on P. aeruginosa biofilm was mediated through the PvrR regulator and the Cup fimbrial biosynthesis genes. Furthermore, proteomic analysis demonstrated that 6 proteins were reproducibly induced by simvastatin in the P. aeruginosa swarming cells. The effect of statins on the regulation of the host-P. aeruginosa immune response was also investigated. Statin treatment increased expression of the pro-inflammatory cytokines IL-8 and CCL20 in lung epithelial cells, but did not attenuate P. aeruginosa-mediated inflammatory gene induction. In fact, simvastatin and P. aeruginosa caused a synergistic effect on CCL20 expression. The expression of the transcriptional regulators KLF2 and KLF6 was also increased by statins and P. aeruginosa, with the induction of KLF6 by simvastatin proving to be a novel effect. Interestingly, both statins and P. aeruginosa were capable of inducing alternative splicing of KLF6. P. aeruginosa was found to induce KLF6 alternative splicing by way of the type 3 secreted toxin ExoS. In addition, a mechanistic role was elucidated for KLF6 in the lung, as it was determined that statin-mediated induction of this protein was responsible for the induction of the host response genes CCL20 and iNOS. Moreover, statin treatment caused a slight increase in infection-related cytotoxicity, and increased bacterial adhesion to cells. Taken together, these data demonstrate that statins can reduce the virulence of CFassociated bacterial pathogens and alter host response effectors. Furthermore, novel statin effectors were identified in both bacterial and host cells.

Why Serological Responses during Cystitis are Limited.

The high frequency of urinary tract infections (UTIs), some of which appear to be endogenous relapses rather than reinfections by new isolates, point to defects in the host's memory immune response. It has been known for many decades that, whereas kidney infections evoked an antibody response to the infecting bacteria, infections limited to the bladder failed to do so. We have identified the existence of a broadly immunosuppressive transcriptional program associated with the bladder, but not the kidneys, during infection of the urinary tract that is dependent on bladder mast cells. This involves the localized secretion of IL-10 and results in the suppression of humoral immune responses in the bladder. Mast cell-mediated immune suppression could suggest a role for these cells in critically balancing the needs to clear infections with the imperative to prevent harmful immune reactions in the host.

Transcriptomic Analysis of the Host Response and Innate Resilience to Enterotoxigenic Escherichia coli Infection in Humans.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a globally prevalent cause of diarrhea. Though usually self-limited, it can be severe and debilitating. Little is known about the host transcriptional response to infection. We report the first gene expression analysis of the human host response to experimental challenge with ETEC. METHODS: We challenged 30 healthy adults with an unattenuated ETEC strain, and collected serial blood samples shortly after inoculation and daily for 8 days. We performed gene expression analysis on whole peripheral blood RNA samples from subjects in whom severe symptoms developed (n = 6) and a subset of those who remained asymptomatic (n = 6) despite shedding. RESULTS: Compared with baseline, symptomatic subjects demonstrated significantly different expression of 406 genes highlighting increased immune response and decreased protein synthesis. Compared with asymptomatic subjects, symptomatic subjects differentially expressed 254 genes primarily associated with immune response. This comparison also revealed 29 genes differentially expressed between groups at baseline, suggesting innate resilience to infection. Drug repositioning analysis identified several drug classes with potential utility in augmenting immune response or mitigating symptoms. CONCLUSIONS: There are statistically significant and biologically plausible differences in host gene expression induced by ETEC infection. Differential baseline expression of some genes may indicate resilience to infection.

Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer

BACKGROUND: Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance.

METHODS: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided.

RESULTS: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner.

CONCLUSIONS: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies.

The complexity of Drosophila innate immunity

Metazoans rely on efficient mechanisms to oppose infections caused by pathogens. The immediate and first-line defense mechanism(s) in metazoans, referred to as the innate immune system, is initiated upon recognition of microbial intruders by germline encoded receptors and is executed by a set of rapid effector mechanisms. Adaptive immunity is restricted to vertebrate species and it is controlled and assisted by the innate immune system. Interestingly, most of the basic signaling cascades that regulate the primeval innate defense mechanism(s) have been well conserved during evolution, for instance between humans and the fruit fly, Drosophila melanogaster. Being devoid of adaptive signaling and effector systems, Drosophila has become an established model system for studying pristine innate immune cascades and reactions. In general, an immune response is evoked when microorganisms pass the fruit fly’s physical barriers (e.g. cuticle, epithelial lining of gut and trachea), and it is mainly executed in the hemolymph, the equivalent of the mammalian blood. Innate immunity in the fruit fly consists of a phenoloxidase (PO) response, a cellular response (hemocytes), an antiviral response, and the NF-κB dependent production of antimicrobial peptides referred to as the humoral response. The JAK/STAT and Jun kinase signaling cascades are also implicated in the defence against pathogens.

Augmentation de l'immunogénicité de la nucléoprotéine de l'influenza par multimérisation in vitro

Les travaux effectués au cours de ce mémoire ont permis de développer une alternative aux vaccins présentement utilisés contre le virus de l’influenza. Nous avons utilisé la nucléoprotéine (NP) de l’influenza comme base vaccinale puisque cette protéine est conservée chez les souches d’influenza A et qu’elle possède un potentiel de protection croisée. Nous avons montré que la multimérisation de la NP grâce à un gabarit d’ARN permet d’augmenter son immunogenicité. Cette multimérisation en pseudo-nucléoparticule virale (NLP) a augmenté la réponse humorale et cellulaire spécifique à NP et l’ajout d’un adjuvant (PAL) a permis d’amplifier davantage la réponse humorale contre NP. Une dose du vaccin candidat NLP-PAL n’a pas réussi à protéger des souris contre une infection létale avec une souche homotypique d’influenza. Cependant, des résultats avec un régime de deux immunisations montrent des résultats encourageants qui permettent d’espérer une protection envers une infection virale.