OVX836 Heptameric Nucleoprotein Vaccine Generates Lung Tissue-Resident Memory CD8+ T-Cells for Cross-Protection Against Influenza.

Tissue-resident memory (TRM) CD8+ T-cells play a crucial role in the protection against influenza infection but remain difficult to elicit using recombinant protein vaccines. OVX836 is a recombinant protein vaccine, obtained by the fusion of the DNA sequence of the influenza A nucleoprotein (NP) to the DNA sequence of the OVX313 heptamerization domain. We previously demonstrated that OVX836 provides broad-spectrum protection against influenza viruses. Here, we show that OVX836 intramuscular (IM) immunization induces higher numbers of NP-specific IFNγ-producing CD8+ T-cells in the lung, compared to mutant NP (NPm) and wild-type NP (NPwt), which form monomeric and trimeric structures, respectively. OVX836 induces cytotoxic CD8+ T-cells and high frequencies of lung TRM CD8+ T-cells, while inducing solid protection against lethal influenza virus challenges for at least 90 days. Adoptive transfer experiments demonstrated that protection against diverse influenza subtypes is mediated by NP-specific CD8+ T-cells isolated from the lung and spleen following OVX836 vaccination. OVX836 induces a high number of NP-specific lung CD8+ TRM-cells for long-term protection against influenza viruses.

OVX836 a recombinant nucleoprotein vaccine inducing cellular responses and protective efficacy against multiple influenza A subtypes.

Inactivated influenza vaccines (IIVs) lack broad efficacy. Cellular immunity to a conserved internal antigen, the nucleoprotein (NP), has been correlated to protection against pandemic and seasonal influenza and thus could have the potential to broaden vaccine efficacy. We developed OVX836, a recombinant protein vaccine based on an oligomerized NP, which shows increased uptake by dendritic cells and immunogenicity compared with NP. Intramuscular immunization in mice with OVX836 induced strong NP-specific CD4+ and CD8+ T-cell systemic responses and established CD8+ tissue memory T cells in the lung parenchyma. Strikingly, OVX836 protected mice against viral challenge with three different influenza A subtypes, isolated several decades apart and induced a reduction in viral load. When co-administered with IIV, OVX836 was even more effective in reducing lung viral load.

Sialylated Fetuin-A as a candidate predictive biomarker for successful grass pollen allergen immunotherapy.

BACKGROUND: Eligibility to immunotherapy is based on the determination of IgE reactivity to a specific allergen by means of skin prick or in vitro testing. Biomarkers predicting the likelihood of clinical improvement during immunotherapy would significantly improve patient selection. METHODS: Proteins were differentially assessed by using 2-dimensional differential gel electrophoresis and label-free mass spectrometry in pretreatment sera obtained from clinical responders and nonresponders within a cohort of 82 patients with grass pollen allergy receiving sublingual immunotherapy or placebo. Functional studies of Fetuin-A (FetA) were conducted by using gene silencing in a mouse asthma model, human dendritic cell in vitro stimulation assays, and surface plasmon resonance. RESULTS: Analysis by using quantitative proteomics of pretreatment sera from patients with grass pollen allergy reveals that high levels of O-glycosylated sialylated FetA isoforms are found in patients exhibiting a strong decrease in rhinoconjunctivitis symptoms after sublingual immunotherapy. Although FetA is involved in numerous inflammatory conditions, its potential role in allergy is unknown. In vivo silencing of the FETUA gene in BALB/c mice results in a dramatic upregulation of airway hyperresponsiveness, lung resistance, and TH2 responses after allergic sensitization to ovalbumin. Both sialylated and nonsialytated FetA bind to LPS, but only the former synergizes with LPS and grass pollen or mite allergens to enhance the Toll-like receptor 4-mediated proallergic properties of human dendritic cells. CONCLUSIONS: As a reflection of the patient's inflammatory status, pretreatment levels of sialylated FetA in the blood are indicative of the likelihood of clinical responses during grass pollen immunotherapy.

Changes in markers associated with dendritic cells driving the differentiation of either TH2 cells or regulatory T cells correlate with clinical benefit during allergen immunotherapy.

BACKGROUND: Regulatory dendritic cell (DC) markers, such as C1Q, are upregulated in PBMCs of patients with grass pollen allergy exhibiting clinical benefit during allergen immunotherapy (AIT). OBJECTIVES: We sought to define markers differentially expressed in human monocyte-derived DCs differentiated toward a proallergic (DCs driving the differentiation of TH2 cells [DC2s]) phenotype and investigate whether changes in such markers in the blood correlate with AIT efficacy. METHODS: Transcriptomes and proteomes of monocyte-derived DCs polarized toward DCs driving the differentiation of TH1 cells (DC1s), DC2s, or DCs driving the differentiation of regulatory T cells (DCreg cells) profiles were compared by using genome-wide cDNA microarrays and label-free quantitative proteomics, respectively. Markers differentially regulated in DC2s and DCreg cells were assessed by means of quantitative PCR in PBMCs from 80 patients with grass pollen allergy before and after 2 or 4 months of sublingual AIT in parallel with rhinoconjunctivitis symptom scores. RESULTS: We identified 20 and 26 new genes/proteins overexpressed in DC2s and DCreg cells, respectively. At an individual patient level, DC2-associated markers, such as CD141, GATA3, OX40 ligand, and receptor-interacting serine/threonine-protein kinase 4 (RIPK4), were downregulated after a 4-month sublingual AIT course concomitantly with an upregulation of DCreg cell-associated markers, including complement C1q subcomponent subunit A (C1QA), FcγRIIIA, ferritin light chain (FTL), and solute carrier organic anion transporter family member 2B1 (SLCO2B1), in the blood of clinical responders as opposed to nonresponders. Changes in such markers were better correlated with clinical benefit than alterations of allergen-specific CD4(+) T-cell or IgG responses. CONCLUSIONS: A combination of 5 markers predominantly expressed by blood DCs (ie, C1Q and CD141) or shared with lymphoid cells (ie, FcγRIIIA, GATA3, and RIPK4) reflecting changes in the balance of regulatory/proallergic responses in peripheral blood can be used as early as after 2 months to monitor the early onset of AIT efficacy.

A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines.

Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of γ-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells.

Identification of Novel Short Ragweed Pollen Allergens Using Combined Transcriptomic and Immunoproteomic Approaches.

BACKGROUND: Allergy to short ragweed (Ambrosia artemisiifolia) pollen is a serious and expanding health problem in North America and Europe. Whereas only 10 short ragweed pollen allergens are officially recorded, patterns of IgE reactivity observed in ragweed allergic patients suggest that other allergens contribute to allergenicity. The objective of the present study was to identify novel allergens following extensive characterization of the transcriptome and proteome of short ragweed pollen. METHODS: Following a Proteomics-Informed-by-Transcriptomics approach, a comprehensive transcriptomic data set was built up from RNA-seq analysis of short ragweed pollen. Mass spectrometry-based proteomic analyses and IgE reactivity profiling after high resolution 2D-gel electrophoresis were then combined to identify novel allergens. RESULTS: Short ragweed pollen transcripts were assembled after deep RNA sequencing and used to inform proteomic analyses, thus leading to the identification of 573 proteins in the short ragweed pollen. Patterns of IgE reactivity of individual sera from 22 allergic patients were assessed using an aqueous short ragweed pollen extract resolved over 2D-gels. Combined with information derived from the annotated pollen proteome, those analyses revealed the presence of multiple unreported IgE reactive proteins, including new Amb a 1 and Amb a 3 isoallergens as well as 7 novel candidate allergens reacting with IgEs from 20-70% of patients. The latter encompass members of the carbonic anhydrase, enolase, galactose oxidase, GDP dissociation inhibitor, pathogenesis related-17, polygalacturonase and UDP-glucose pyrophosphorylase families. CONCLUSIONS: We extended the list of allergens identified in short ragweed pollen. These findings have implications for both diagnosis and allergen immunotherapy purposes.

Identification of the cysteine protease Amb a 11 as a novel major allergen from short ragweed.

BACKGROUND: Allergy to pollen from short ragweed (Ambrosia artemisiifolia) is a serious and expanding health problem in the United States and in Europe. OBJECTIVE: We sought to investigate the presence of undescribed allergens in ragweed pollen. METHODS: Ragweed pollen proteins were submitted to high-resolution gel electrophoresis and tested for IgE reactivity by using sera from 92 American or European donors with ragweed allergy. Pollen transcriptome sequencing, mass spectrometry (MS), and recombinant DNA technologies were applied to characterize new IgE-binding proteins. RESULTS: High-resolution IgE immunoblotting experiments revealed that 50 (54%) of 92 patients with ragweed allergy were sensitized to a 37-kDa allergen distinct from Amb a 1. The full-length cDNA sequence for this molecule was obtained by means of PCR cloning after MS sequencing of the protein combined with ragweed pollen RNA sequencing. The purified allergen, termed Amb a 11, was fully characterized by MS and confirmed to react with IgEs from 66% of patients. This molecule is a 262-amino-acid thiol protease of the papain family expressed as a combination of isoforms and glycoforms after proteolytic removal of N- and C-terminal propeptides from a proform. Three-dimensional modeling revealed a high structural homology with known cysteine proteases, including the mite Der p 1 allergen. The protease activity of Amb a 11, as well as its capacity to activate basophils from patients with ragweed allergy, were confirmed. The production of a nonglycosylated recombinant form of Amb a 11 in Escherichia coli established that glycosylation is not required for IgE binding. CONCLUSION: We identified the cysteine protease Amb a 11 as a new major allergen from ragweed pollen. Given the similar physicochemical properties shared by the 2 major allergens, we hypothesize that part of the allergenic activity previously ascribed to Amb a 1 is rather borne by Amb a 11.

Proteomic identification of target proteins in normal but nonfertilizing sperm.

OBJECTIVE: To identify the male molecular causes of failures of IVF (with a deficient binding of spermatozoa to the zona pellucida, without any obvious oocyte anomaly), which are undetected by classical sperm analysis. DESIGN: Case-control prospective study. SETTING: University hospital. PATIENT(S): Proteomic profiles of spermatozoa in patients with a complete failure of fertilization and no spermatozoa bound to the zona pellucida were compared with those of controls (men with normal fertilization and cleavage rates after classical IVF for tubal indication). INTERVENTION(S): All samples were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) after being divided into three fractions according to their isoelectric point. MAIN OUTCOME MEASURE(S): Differentially expressed proteins between infertile men and controls were identified by mass spectrometry. RESULT(S): Seventeen proteins differentially expressed between cases and controls were found. Twelve of these proteins were identified by mass spectrometry, and two may influence gametes interaction: laminin receptor LR67 and L-xylulose reductase (P34H). CONCLUSION(S): This study shows that 2D-DIGE might be useful in finding potential targets for diagnosis and prognosis of idiopathic infertility in IVF.

A regulatory dendritic cell signature correlates with the clinical efficacy of allergen-specific sublingual immunotherapy.

BACKGROUND: Given their pivotal role in the polarization of T-cell responses, molecular changes at the level of dendritic cells (DCs) could represent an early signature indicative of the subsequent orientation of adaptive immune responses during immunotherapy. OBJECTIVE: We sought to investigate whether markers of effector and regulatory DCs are affected during allergen immunotherapy in relationship with clinical benefit. METHODS: Differential gel electrophoresis and label-free mass spectrometry approaches were used to compare whole proteomes from human monocyte-derived DCs differentiated toward either regulatory or effector functions. The expression of those markers was assessed by using quantitative PCR in PBMCs from 79 patients with grass pollen allergy enrolled in a double-blind, placebo-controlled clinical study evaluating the efficacy of sublingual tablets in an allergen exposure chamber over a 4-month period. RESULTS: We identified several markers associated with DC1 and/or DC17 effector DCs, including CD71, FSCN1, IRF4, NMES1, MX1, TRAF1. A substantial phenotypic heterogeneity was observed among various types of tolerogenic DCs, with ANXA1, Complement component 1 (C1Q), CATC, GILZ, F13A, FKBP5, Stabilin-1 (STAB1), and TPP1 molecules established as shared or restricted regulatory DC markers. The expression of 2 of those DCs markers, C1Q and STAB1, was increased in PBMCs from clinical responders in contrast to that seen in nonresponders or placebo-treated patients. CONCLUSION: C1Q and STAB1 represent candidate biomarkers of early efficacy of allergen immunotherapy as the hallmark of a regulatory innate immune response predictive of clinical tolerance.

Expression and characterization of natural-like recombinant Der p 2 for sublingual immunotherapy.

BACKGROUND: Recombinant allergens with a native conformation represent an alternative to natural extracts for immunotherapy and diagnostic purposes. METHODS: We produced the Der p 2 mite allergen in Pichia pastoris and Escherichia coli. After purification by cation exchange chromatography, recombinant molecules were compared to their natural counterpart based upon structural (disulfide bonds, secondary structure, thermal stability) and immunological properties (antibody reactivity, basophil and T cell activation, tolerance induction in a murine sublingual immunotherapy model). RESULTS: The Der p 2.0101 isoform was confirmed to be prevalent in Dermatophagoides pteronyssinus extracts. It was then produced as a secreted molecule in P. pastoris or refolded from E. coli inclusion bodies. The yeast-expressed rDer p 2 molecule exhibits a natural-like disulfide bridge distribution and secondary structure, whereas the E. coli-derived rDer p 2 presents some heterogeneity in cysteine bonds and a lower stability following thermal stress. The two recombinant as well as natural Der p 2 molecules exhibit comparable IgE recognition and activate basophil and CD4+ T cells. Sublingual immunotherapy of nDer p 2- sensitized mice using either one of the rDer p 2 molecules efficiently decreases airway hyperresponsiveness as well as Th2 responses. CONCLUSIONS: Natural and recombinant Der p 2 molecules produced in P. pastoris and E. coli exhibit comparable immunological properties despite distinct structural features. Natural-like cysteine pairing is a critical parameter to identify stable, well-folded and homogenous proteins appropriate for immunotherapy and diagnostic purposes.

Proteomic analysis of BRCA1-depleted cell line reveals a putative role for replication protein A2 up-regulation in BRCA1 breast tumor development.

PURPOSE: Germline mutations in BRCA1 result in a strong predisposition to breast cancer, with frequent loss of heterozygosity of the remaining wild-type allele. The development of BRCA1 tumors is likely to depend on additional genetic alterations and gene expression changes which follow growth and DNA repair defects associated with BRCA1 deficiency. The identification of these modifications offers an opportunity to find surrogate markers of BRCA1 tumors. Here, we sought to identify differentially expressed proteins related to BRCA1 depletion. EXPERIMENTAL DESIGN: We used isogenic HeLa cells either stably knocked-down or not for BRCA1 (BRCA1(KD) ) and compared protein profiles of these cells by DIGE. RESULTS: We detected increased levels of Replication protein A2 (RPA2) in BRCA1(KD) cells as compared to control cells. RPA2 is an essential protein required for DNA replication and repair. We further demonstrated that depletion of RPA2 subunit delays growth of BRCA1(KD) respect to isogenic control cells. Strikingly, elevated levels of RPA2 were more frequently observed in BRCA1 tumors when triple-negative tumors from BRCA1 mutation carriers (n=13) and non-carriers (n=36) were stained in situ for RPA2. CONCLUSIONS AND CLINICAL RELEVANCE: RPA2 up-regulation may thus be involved in the growth and/or survival of BRCA1 tumor cells and useful in immunohistochemical discrimination of triple-negative BRCA1 tumors.

New molecular markers in resistant B-CLL.

B-chronic lymphocytic leukemia (B-CLL) is characterized by a highly variable clinical course which has long remained a stumbling block for clinicians. This variability appears to arise from complex molecular alterations identified in malignant cells from patient subsets. Recent studies have focused in particular on identifying new molecular markers to help predict the most effective and adapted treatments. In addition to the mutation status of immunoglobulin variable heavy-chain region (IgVH) genes, which is a well-established predictive factor in B-CLL, these new markers include defects of cell factors involved in the maintenance of genome stability, such as telomere function, DNA repair, ATM and p53. Other predictive factors, such as tyrosine kinase Zap-70 and soluble factors found in patient sera, may be associated with B-cell receptor signal transduction. Interestingly, an alteration of these factors fits closely, though not strikingly, with the absence of somatic mutations in IgVH genes, suggesting that the latter may be due either to epigenetic events leading to an unstable genome or to an inherited defect in the immune response of malignant B-cells. Recent lessons from Zap-70 expression/phosphorylation suggest that some of these markers may reflect the defective pathways in B-CLL cells rather than being markers of cell malignancy per se. Furthermore, specific subsets of markers are found in patient cells resistant to treatment. Current studies on gene expression profiling and proteomic analyses should soon lead to a better understanding of how these pathways are affected, especially in multi-drug resistant B-CLL.

Mapping of alkaline proteins in bovine skeletal muscle.

In agricultural sciences, proteomics has become the new hope for analyzing the meat quality traits that are closely related to the skeletal muscle traits. 2-DE muscle maps of many species have been recently reported and used to find molecular markers of meat quality traits. However, one limitation of 2-DE based analyses is due to the limited alkaline protein separation. Considering this problem, there is a need to use recent advances that have markedly improved the 2-DE based analysis of alkaline proteins. Hence, the present study provides additional information concerning the alkaline proteome of bovine skeletal muscle by using an appropriate protocol to characterize proteins over the entire range of pH 7-11. A total of 32 distinct gene products corresponding to 60 protein spots were identified by PMF and grouped in seven categories according to their main function. This 2-D map will contribute to muscle proteome studies since a significant portion of proteins is in the alkaline pH range.

Data analysis methods for detection of differential protein expression in two-dimensional gel electrophoresis.

The recent development of microarray technology has led statisticians and bioinformaticians to develop new statistical methodologies for comparing different biological samples. The objective is to identify a small number of differentially expressed genes from among thousands. In quantitative proteomics, analysis of protein expression using two-dimensional gel electrophoresis shows some similarities with transcriptomic studies. Thus, the goal of this study was to evaluate different data analysis methodologies widely used in array analysis using different proteomic data sets of hundreds of proteins. Even with few replications, the significance analysis of microarrays method appeared to be more powerful than the Student's t test in truly declaring differentially expressed proteins. This procedure will avoid wasting time due to false positives and losing information with false negatives.

Proteomic analysis of bovine skeletal muscle hypertrophy.

Myostatin plays a major role in muscle growth and development and animals with disruption of this gene display marked increases in muscle mass. Little is known about muscle physiological adaptations in relation to this muscle hypertrophy. To provide a more comprehensive view, we analyzed bovine muscles from control, heterozygote and homozygote young Belgian blue bulls for myostatin deletion, which results in a normal level of inactive myostatin. Heterozygote and homozygote animals were characterized by a higher proportion of fast-twitch glycolytic fibers in Semitendinosus muscle. Differential proteomic analysis of this muscle was performed using two-dimensional gel electrophoresis followed by mass spectrometry. Thirteen proteins, corresponding to 28 protein spots, were significantly altered in response to the myostatin deletion. The observed changes in protein expression are consistent with an increased fast muscle phenotype, suggesting that myostatin negatively controls mainly fast-twitch glycolytic fiber number. Finally, we demonstrated that differential mRNA splicing of fast troponin T is altered by the loss of myostatin function. The structure of mutually exclusive exon 16 appears predominantly expressed in muscles from heterozygote and homozygote animals. This suggests a role for exon 16 of fast troponin T in the physiological adaptation of the fast muscle phenotype.

Mapping of bovine skeletal muscle proteins using two-dimensional gel electrophoresis and mass spectrometry.

The large individual variation in meat quality seen both within and between animals is not fully understood. Consequently, our long-term goal is to identify reliable proteins which control or determine bovine meat quality. Using a proteomic approach, bovine skeletal muscle samples were analyzed by two-dimensional gel electrophoresis (2-DE) using an immobilized pH 4-7 gradient in the first dimension and mass spectrometry. We first tested the reproducibility of the method. These experiments showed slightly greater intersample than intrasample variability. In order to evaluate the type of visualized proteins in 2-DE, we initiated the construction of a protein reference map of bovine Semitendinosus muscle. In total, 129 protein spots corresponding to 75 different gene products were identified. Of these proteins, the largest portion is involved in metabolism (25.5%), cell structure (17%), cell defense (16%) and contractile apparatus (14.5%). One quarter of the identified proteins are represented by two or several protein spots and multiple isoforms of troponin T are present. Peptide mass fingerprint results indicate that these isoforms are partly generated by alternative splicing. The data presented here are an important step for further proteome analyses on bovine muscle. This may lead to progress in understanding the mechanisms controlling postmortem muscle metabolism and meat quality.

Location of myostatin expression during bovine myogenesis in vivo and in vitro.

Mutations in the myostatin gene lead to double-muscling in cattle indicating that it is a negative regulator of the total number of muscle fibres. Myostatin expression was analysed by RT-PCR in three developing bovine muscles. It decreased during differentiation in Semitendinosus and Biceps femoris, and increased in the late differentiating Masseter during gestation. A combination of in situ hybridisation and immuno-histochemical detection of myosin heavy chains (MHC) allowed us to locate the expression in myofibres containing only developmental MHC at different stages and in fast IIA fibres at the end of gestation. In vitro, myostatin was undetectable during proliferation, peaked at the onset of fusion and decreased during terminal differentiation. It was not detected in myotubes by in situ hybridisation. The inhibition of differentiation by BrdU prevented the decrease in expression. Our results show that the peak in myostatin expression coincides with early differentiation indicating a regulatory role in cattle myogenesis.