Recombinant Profilin Research Articles

The Recombinant Profilin from Free-Living Amoebae Induced Allergic Immune Responses via TLR2.

Repeated exposure to recombinant profilin from Acanthamoeba (rAc-PF) induces allergic airway responses in vitro and in vivo. Based on the role of toll-like receptors (TLRs) in allergic airway diseases, TLRs play a central role in innate immune responses and the adaptive immune system and regulate responses against antigens through antigen-specific receptors. In this study, we attempted to determine the molecular mechanisms underlying rAc-PF-induced allergic inflammatory responses. We determined the correlation between rAc-PF and TLRs and analyzed changes in allergic immune responses after blocking multiple TLR signaling under rAc-PF treatment conditions in vitro. We also compared allergic inflammatory responses in TLR2 knockout (KO) and wild-type (WT) mice. To investigate the effect of TLR2 on antigen prototyping and T cell activation in the inflammatory response induced by rAc-PF, we assessed maturation of BMDCs and polarization of naïve T cells by rAc-PF stimulation. Additionally, we compared changes in inflammation-related gene expression by rAc-PF treatment in primary lung epithelial cells isolated from TLR2 KO and WT mice. The rAc-PF treatment was increased the expression level of TLR2 and 9 in vitro. But, there were not significantly differ the others TLRs expression by rAc-PF treated group. And then, the mRNA expression levels of inflammation-related genes were reduced in the TLR2 or TLR9 antagonist-treated groups compared to those in the rAc-PF alone, were no difference the treated with the other TLRs (TLR4, 6, and 7/8) antagonist. The difference was higher in the TLR2 antagonist group. Additionally, the levels of airway inflammatory disease indicators were lower in the TLR2 KO group than in the WT group after rAc-PF treatment. Furthermore, the expression of bone marrow-derived dendritic cell (BMDC) surface molecular markers following rAc-PF stimulation was lower in TLR2 KO mice than in WT mice, and TLR2 KO in BMDCs resulted in a remarkable decline in Th2/17-related cytokine production and Th2/17 subset differentiation. In addition, the expression levels of rAc-PF-induced inflammatory genes were reduced inTLR2 KO primary lung cells compared to those in normal primary lung cells. These results suggest that the rAc-PF-induced airway inflammatory response is regulated by TLR2 signaling.

Synergistic effect of GRA7 and profilin proteins in vaccination against chronic Toxoplasma gondii infection

Toxoplasmosis is a zoonotic disease with worldwide prevalence in humans and warm-blooded animal populations. In livestock Toxoplasma gondii is the causal agent of significant economic losses since it can cause abortions in goats and sheep. It is estimated that one third of the world population is infected. Although there are effective therapies for acute infection, these are sometimes poorly tolerated, teratogenic, and have a long administration time. Considering the deficiencies that exist related to the prevention and treatment of toxoplasmosis, the development of a safe and effective vaccine would be extremely valuable in fighting against this infection. In the present work, we characterize for the first time the adjuvant and immunogenic potential of a recombinant profilin protein (rTgPF), in a vaccine formulation alone or in combination with the well-known GRA7 antigen candidate in a murine toxoplasmosis model. Since TgPF acts as a ligand for TLR11 and 12 inducing innate immune responses that promote type 1 adaptive responses, we first study the capacity of the mix rGRA7 + rTgPF to initiate an immune response by evaluating dendritic cell activation. Both rTgPF and rGRA7 induces activation of mouse BMDCs more efficiently than the single proteins, evidenced by increased expression of CD80 and CD86 co-stimulatory proteins and secretion of IL-6, IL-10 and IL-12 cytokines after in vitro stimulation. The sum of the effects of rGRA7 and rTgPF on BMDCs maturation led us to assay them in a vaccination protocol. BALB/c mice vaccinated with this mix elicited a Th1-biased immunity via the induction of lymphocyte proliferation, activation of CD4+T cells and increased IFN-γ production that resulted in enhanced protection against chronic Toxoplama gondii infection. Profilin per se induce only cellular immunity but augments the effect of rGRA7 immune responses when used together, thus allowing us to postulate rTgPF as a potential adjuvant in a protein vaccine.

Novel murine mAbs define specific and cross-reactive epitopes on the latex profilin panallergen Hev b 8

The production of specific antibodies able to recognize allergens from different sources or block interactions between allergens and antibodies mediating allergic reactions is crucial for developing successful tools for diagnostics and therapeutics. Panallergens are highly conserved proteins present in widely different species, implicated in relevant cross-reactions. The panallergen latex profilin (Hev b 8) has been associated with the latex-food-pollen syndrome. We generated five monoclonal IgGs and one IgE from murine hybridomas against recombinant Hev b 8 and evaluated their interaction with this allergen using ELISA and biolayer interferometry (BLI). Affinity purified mAbs exhibited high binding affinities towards rHev b 8, with KD1 values ranging from 10−10 M to 10−11 M. Some of these antibodies also recognized the recombinant profilins from maize and tomato (Zea m 12 and Sola l 1), and the ash tree pollen (Fra e 2). Competition ELISA demonstrated that some mAb pairs could bind simultaneously to rHev b 8. Using BLI, we detected competitive, non-competitive, and partial-competition interactions between pairs of mAbs with rHev b 8, suggesting the existence of at least two non-overlapping epitopes on the surface of this allergen. Three-dimensional models of the Fv of 1B4 and 2D10 IgGs and docking simulations of these Fvs with rHev b 8 revealed these epitopes. Furthermore, these two mAbs inhibited the interaction of polyclonal IgE and IgG4 antibodies from profilin-allergic patients with rHev b 8, indicating that the mAbs and the antibodies present in sera from allergic patients bind to overlapping epitopes on the allergen. These mAbs can be useful tools for immune-localization studies, immunoassay development, or standardization of allergenic products.

Inclusion of PD-L1 into a recombinant profilin antigen enhances immunity against Babesia microti in a murine model

Pathogens and cancer cells employ the programmed cell death-Ligand 1 (PD-L1)/ programmed cell death-1 (PD-1) signaling pathway to inhibit the immune response. Hence, blockade of PD-L1/PD-1 recognition through monoclonal antibodies enhances the immune response. Antibodies that block PD-L1 and PD-1 binding have been used for the prevention and therapy of human pathogenic diseases, but have not yet been evaluated for the treatment of infectious diseases of livestock. In the present study, a recombinant vaccine named PROF-PDL1E, was designed comprising the Babesia microti-derived vaccine candidate profilin and the host PD-L1 protein, and its effect on immunization against murine B. microti infection was evaluated. PD-L1-specific antibodies generated after vaccination blocked PD-L1 and PD-1 binding as shown by in vitro assays. PROF-PDL1E reduced the burden of B. microti in a mouse model and decreased PD-1 expression in T cells. Furthermore, no tissue damage could be observed after PROF-PDL1E vaccination as verified by hematoxylin and eosin tissue staining of essential organs. In conclusion, vaccines targeting immune checkpoints seem to be a promising strategy for anti-Babesia vaccine development.

Production and Use of Recombinant Profilins Amb a 8, Art v 4, Bet v 2, and Phl p 12 for Allergenic Sensitization Studies.

Four recombinant (r) allergens (rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101) were successfully produced and used for sensitization studies. The allergens belong to the profilin family which is one of the most numerous allergen families. These four proteins represent allergens originating from pollen of weeds (rAmb a 8.0101 and rArt v 4.0101), tree (rBet v 2.0101) and grass (rPhl p 12.0101). The recombinant allergens were characterized using various biochemical and biophysical methods and tested for their ability to bind patient-derived antibodies. One hundred patients aged 2 to 50 years sensitized to pollen and plant-derived food allergens (IgE > 0.35 kU/L) were included. Sensitization to individual allergen sources and components of birch and timothy pollens was evaluated using multiparameter immunoblots. The presence of IgE to pollen-derived recombinant profilins rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101 in serum was evaluated using ELISA method. The presence of IgE against pollen profilins was detected in 20 out of 100 studied patients. High correlation was seen between IgE ELISA results with individual pollen profilins. In summary, it was shown that the recombinant versions of the four allergenic profilins can be used for sensitization studies and for component-resolved allergy diagnostics.

Evaluation of cleaning methods for residual orange extract on different cookware materials using ELISA with profilin allergen indicator

AbstractUnintentional contamination of cookware with allergen residues causes unexpected allergic reactions to individuals with food allergies. Our aim was to find an efficient cleaning method for residual orange extract as a food allergen on cookware fabricated from four types of materials—polypropylene (PP), wood, stainless steel, and glass—after food preparation. To evaluate the cleaning methods, we developed a novel enzyme‐linked immunosorbent assay system using a rabbit polyclonal antibody against a recombinant orange profilin allergen. Using this assay system with a limit of quantification of 2,500 µg/mL, we measured the residual orange extract on the cookware surfaces using swabbing tests. Rinsing with 1 L of water showed a >95% removal efficiency for stainless steel and glass cookware, whereas half the PP and wood cookware required scrubbing with a detergent‐containing sponge for complete cleanliness. Our findings may help reduce the burden for cooks and food manufacturers in controlling fruit allergens.Practical applicationsThe cleaning process is essential in preventing cross‐contamination of food allergens through the equipment surfaces of kitchens and food processing lines. There are no established standard cleaning processes for fruit allergens. The extracts often remain on cookware surfaces after food preparation because of their viscosity. Since even small amounts of allergens can elicit symptoms of food allergies, removing food allergens by cleaning the equipment surfaces is important to avoid unexpected food‐related allergic reactions and produce safe foods for those with food allergies. Here, we investigated efficient cleaning methods using laboratory scale tests for controlling orange allergens. We used orange extract as a representative fruit‐generated adhesive extract on different cookware materials—polypropylene, wood, stainless steel, and glass. Since these materials are commonly used in food processing equipment, our findings could help develop an efficient and economical allergy control method for both cooks and manufacturers of processed fruit without excessive cleaning.

Detection of the tomato allergen Sola l 1 and evaluation of its reactivity after heat and papain treatment

ABSTRACTTo detect the tomato allergen profilin (Sola l 1) from fresh tomato and tomato-containing processed foods, we generated a rabbit polyclonal anti-Sola l 1 peptide antibody. An indirect enzyme-linked immunosorbent assay (iELISA) based on the antibody was established for semiquantification of Sola l 1, and standard curves were generated using Sola l 1 homologue recombinant birch profilin (rBet v 2) as a calibrator. The mean concentration of tomato extracts was 25.6 ± 9.1 µg/g, and the concentrations of tomato-containing processed foods ranged from 13.3 to 24.8 µg/g. The reactivity of Sola l 1 after heat and papain treatment of tomato extracts was evaluated using competitive iELISA, and the results showed that Sola l 1 reactivity was stable with or without treatment. Our findings suggested that tomato-containing processed foods and fresh tomatoes both contained Sola l 1 and could have the potential to induce allergic symptoms in patients with tomato allergies.

In ovo vaccination using Eimeria profilin and Clostridium perfringens NetB proteins in Montanide IMS adjuvant increases protective immunity against experimentally-induced necrotic enteritis.

ObjectiveThe effects of vaccinating 18-day-old chicken embryos with the combination of recombinant Eimeria profilin plus Clostridium perfringens (C. perfringens) NetB proteins mixed in the Montanide IMS adjuvant on the chicken immune response to necrotic enteritis (NE) were investigated using an Eimeria maxima (E. maxima)/C. perfringens co-infection NE disease model that we previously developed.MethodsEighteen-day-old broiler embryos were injected with 100 μL of phosphate-buffered saline, profilin, profilin plus necrotic enteritis B-like (NetB), profilin plus NetB/Montanide adjuvant (IMS 106), and profilin plus Net-B/Montanide adjuvant (IMS 101). After post-hatch birds were challenged with our NE experimental disease model, body weights, intestinal lesions, serum antibody levels to NetB, and proinflammatory cytokine and chemokine mRNA levels in intestinal intraepithelial lymphocytes were measured.ResultsChickens in ovo vaccinated with recombinant profilin plus NetB proteins/IMS106 and recombinant profilin plus NetB proteins/IMS101 showed significantly increased body weight gains and reduced gut damages compared with the profilin-only group, respectively. Greater antibody response to NetB toxin were observed in the profilin plus NetB/IMS 106, and profilin plus NetB/IMS 101 groups compared with the other three vaccine/adjuvant groups. Finally, diminished levels of transcripts encoding for proinflammatory cytokines such as lipopolysaccharide-induced tumor necrosis factor-α factor, tumor necrosis factor superfamily 15, and interleukin-8 were observed in the intestinal lymphocytes of chickens in ovo injected with profilin plus NetB toxin in combination with IMS 106, and profilin plus NetB toxin in combination with IMS 101 compared with profilin protein alone bird.ConclusionThese results suggest that the Montanide IMS adjuvants potentiate host immunity to experimentally-induced avian NE when administered in ovo in conjunction with the profilin and NetB proteins, and may reduce disease pathology by attenuating the expression of proinflammatory cytokines and chemokines implicated in disease pathogenesis.

Splenic lymphocyte immune response induced by intranasal immunization with recombinant Toxoplasma gondii profilin in mice

To observe the splenocytes immune response elicited by different concentrations of recombinant Toxoplasma gondii profiling (rTgPRF) through the nasal route, and determine the optimal dose. Fifty female BALB/c mice were randomly divided into 5 groups. The immunized groups were intranasally administered with 10, 20, 30 μg or 40 μg of rTgPRF that was separately dissolved in 20 μl of phosphate-buffered saline (PBS) on days 0, 14, and 21 respectively, while the control mice were given PBS solution instead. Two weeks after the last immunization, all mice were killed. Under asceptic conditions, the spleens from the immunized mice were dissected, and then the splenocyte proliferative responses in vitro were tested by CCK-8 kit. The levels of IFN-γ, IL-2, IL-4 and IL-10 of splenocyte culture supernatant were detected by ELISA. Compared to the control group, the splenocytes from the 30 μg and 40 μg groups exhibited a significantly higher proliferative response to rTgPRF (P < 0.05), and SI from the 30 μg rTgPRF group was higher than that from the 40 μg group (P < 0.05). The levels of IFN-γ in all the immunized groups (P < 0.05) and IL-2 in the 20, 30 μg and 40 μg groups were significantly stronger than those in the control (P < 0.05), and the 30 μg group presented the highest concentrations of IFN-γ (P < 0.01) and IL-2 (P < 0.01). There were no statistical differences among the groups in the levels of IL-4 and IL-10. The intranasal immunization with rTgPRF can induce the splenocyteproliferation and Th1-type mediated immunity. The best immunized dose is confirmed as 30 μg.

Fusion of foreign T-cell epitopes and addition of TLR agonists enhance immunity against Neospora caninum profilin in cattle.

We demonstrated recently that immunization with recombinant Neospora caninum profilin (rNcPRO) induces limited protection and a regulatory T-cell response in mice. The aim of this study was to evaluate the immune response elicited by rNcPRO in cattle and assess a strategy to enhance its immunogenicity, combining the addition of T-cell epitopes and immune modulators. We developed a chimeric recombinant profilin fused to functional T-cell epitopes present in the N-terminal sequence of vesicular stomatitis virus (VSV) glycoprotein G (rNcPRO/G). Groups of three cattle were immunized with two doses (2weeks apart) of rNcPRO or rNcPRO/G formulated with alum hydroxide or a nanoparticulated soya-based adjuvant enriched with Toll-like receptor (TLR) 2 and TLR9 agonists, aimed to tackle the MyD88 pathway (AVECplus). rNcPRO induced only a primary immune response (IgM mediated), while antibodies in rNcPRO/G-vaccinated animals switched to IgG1 after the booster. The vaccine formulated with rNcPRO/G and AVECplus improved the production of systemic IFN-γ and induced long-term recall B-cell responses. Overall, our study provides data supporting the use of T-cell epitopes from VSV glycoprotein G and TLR agonists to enhance and modulate immunity to peptide antigens in bovines, particularly when using small proteins from parasites for which immune responses are usually feeble.

Neuronal profilins in health and disease: Relevance for spine plasticity and Fragile X syndrome

Learning and memory, to a large extent, depend on functional changes at synapses. Actin dynamics orchestrate the formation of synapses, as well as their stabilization, and the ability to undergo plastic changes. Hence, profilins are of key interest as they bind to G-actin and enhance actin polymerization. However, profilins also compete with actin nucleators, thereby restricting filament formation. Here, we provide evidence that the two brain isoforms, profilin1 (PFN1) and PFN2a, regulate spine actin dynamics in an opposing fashion, and that whereas both profilins are needed during synaptogenesis, only PFN2a is crucial for adult spine plasticity. This finding suggests that PFN1 is the juvenile isoform important during development, whereas PFN2a is mandatory for spine stability and plasticity in mature neurons. In line with this finding, only PFN1 levels are altered in the mouse model of the developmental neurological disorder Fragile X syndrome. This finding is of high relevance because Fragile X syndrome is the most common monogenetic cause for autism spectrum disorder. Indeed, the expression of recombinant profilins rescued the impairment in spinogenesis, a hallmark in Fragile X syndrome, thereby linking the regulation of actin dynamics to synapse development and possible dysfunction.

Immunochemical and molecular characterization of allergenic profilin (Koc s 2) from Kochia scoparia pollen

Kochia scoparia pollen has been demonstrated as an important cause of pollinosis in tropical and sub-tropical regions of the world. The aim of this study was to characterize the IgE-binding protein of the Kochia pollen extract and production of recombinant form of allergenic profilin of this weed. To predict its allergenic cross-reactivity with profilins of common allergenic plants, nucleotide sequence homology of Kochia profilin was evaluated. Specific ELISA and immunoblotting assay were applied to determine the IgE-binding reactivity of 28 sera collected from patients who were sensitized to Kochia pollen. In cloning procedure, the Kochia profilin-coding sequence was inserted into PTZ57R/T vector and expressed using pET-21b(+) vector. IgE-binding competence of purified recombinant Kochia profilin (rKoc s 2) was analyzed by in vitro assays. There were several protein bands in Kochia pollen extract with molecular weights approximately ranging from 14.5 to 85 kDa. Nucleotide sequencing revealed an open reading frame of 399 bp encoding for 133 amino acid residues which belonged to the profilin family; 15 patients (15/28, 53.1 %) had significant specific IgE levels for the rKoc s 2. Immunodetection and inhibition assays indicated that the purified rKoc s 2 might be the same as that in the crude extract. Koc s 2, the first allergen from the Kochia pollen was identified as a member of the family of profilins. High degree of homology was found among amino acid sequences of Kochia profilin and several profilin molecules from unrelated plant families.

Evaluation of Montanide™ ISA 71 VG adjuvant against coccidiosis (VET2P.1030)

Abstract Chickens were immunized subcutaneously with an Eimeria recombinant profilin protein plus MontanideTM ISA 70 VG (ISA70) or MontanideTM ISA 71 VG (ISA 71) water-in-oil adjuvants, or with profilin alone. While immunization with profilin/ISA 70 vs. profilin alone altered the levels of more total transcripts compared with profilin/ISA 71 vs. profilin alone (509 vs. 296), the latter was associated with a greater number of unique biological functions, and a larger number of genes within these functions, compared with the former. Canonical pathway analysis identified 10 pathways related with genes encoding the altered transcripts in animals immunized with profilin/ISA 71 vs. profilin alone, compared with only 2 pathways in profilin/ISA 70 vs. profilin alone. Vaccination with profilin/ISA 71 was associated with greater body weight gain following E. acervulina infection, and decreased parasite fecal shedding after E. maxima infection, compared with profilin alone. Anti-profilin antibody levels were higher in sera of E. maxima- and E. tenella-infected chickens vaccinated with profilin/ISA 71 compared with profilin alone. The levels of transcripts encoding interferon-γ, interleukin (IL)-2, IL-10, and IL-17A were increased in intestinal lymphocytes from E. acervulina-, E. maxima-, and/or E. tenella-infected chickens vaccinated with profilin/ISA 71 compared with profilin alone.

Evaluation of Montanide™ ISA 71 VG Adjuvant during Profilin Vaccination against Experimental Coccidiosis

Chickens were immunized subcutaneously with an Eimeria recombinant profilin protein plus Montanide™ ISA 70 VG (ISA 70) or Montanide™ ISA 71 VG (ISA 71) water-in-oil adjuvants, or with profilin alone, and comparative RNA microarray hybridizations were performed to ascertain global transcriptome changes induced by profilin/ISA 70 vs. profilin alone and by profilin/ISA 71 vs. profilin alone. While immunization with profilin/ISA 70 vs. profilin alone altered the levels of more total transcripts compared with profilin/ISA 71 vs. profilin alone (509 vs. 296), the latter was associated with a greater number of unique biological functions, and a larger number of genes within these functions, compared with the former. Further, canonical pathway analysis identified 10 pathways that were associated with genes encoding the altered transcripts in animals immunized with profilin/ISA 71 vs. profilin alone, compared with only 2 pathways in profilin/ISA 70 vs. profilin alone. Therefore, ISA 71 was selected as a candidate adjuvant in conjunction with profilin vaccination for in vivo disease protection studies. Vaccination with profilin/ISA 71 was associated with greater body weight gain following E. acervulina infection, and decreased parasite fecal shedding after E. maxima infection, compared with profilin alone. Anti-profilin antibody levels were higher in sera of E. maxima- and E. tenella-infected chickens vaccinated with profilin/ISA 71 compared with profilin alone. Finally, the levels of transcripts encoding interferon-γ, interleukin (IL)-2, IL-10, and IL-17A were increased in intestinal lymphocytes from E. acervulina-, E. maxima-, and/or E. tenella-infected chickens vaccinated with profilin/ISA 71 compared with profilin alone. None of these effects were seen in chickens injected with ISA 71 alone indicating that the adjuvant was not conferring non-specific immune stimulation. These results suggest that profilin plus ISA 71 augments protective immunity against selective Eimeria species in chickens.

Profilin 1 as a Target for Cathepsin X Activity in Tumor Cells

Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.

Effects of Caryota mitis profilin-loaded PLGA nanoparticles in a murine model of allergic asthma

BackgroundPollen allergy is the most common allergic disease. However, tropical pollens, such as those of Palmae, have seldom been investigated compared with the specific immunotherapy studies done on hyperallergenic birch, olive, and ragweed pollens. Although poly(lactic-co-glycolic acid) (PLGA) has been extensively applied as a biodegradable polymer in medical devices, it has rarely been utilized as a vaccine adjuvant to prevent and treat allergic disease. In this study, we investigated the immunotherapeutic effects of recombinant Caryota mitis profilin (rCmP)-loaded PLGA nanoparticles and the underlying mechanisms involved.MethodsA mouse model of allergenic asthma was established for specific immunotherapy using rCmP-loaded PLGA nanoparticles as the adjuvant. The model was evaluated by determining airway hyperresponsiveness and levels of serum-specific antibodies (IgE, IgG, and IgG2a) and cytokines, and observing histologic sections of lung tissue.ResultsThe rCmP-loaded PLGA nanoparticles effectively inhibited generation of specific IgE and secretion of the Th2 cytokine interleukin-4, facilitated generation of specific IgG2a and secretion of the Th1 cytokine interferon-gamma, converted the Th2 response to Th1, and evidently alleviated allergic symptoms.ConclusionPLGA functions more appropriately as a specific immunotherapy adjuvant for allergen vaccines than does conventional Al(OH)3 due to its superior efficacy, longer potency, and markedly fewer side effects. The rCmP-loaded PLGA nanoparticles developed herein offer a promising avenue for specific immunotherapy in allergic asthma.

Effects of Novel Vaccine/Adjuvant Complexes on the Protective Immunity Against Eimeria acervulina and Transcriptome Profiles

SUMMARY. This study investigated the ability of two novel adjuvant formulations, QCDC (Quil A/cholesterol/DDA/ Carbopol) and QCDCR (QCDC/Bay R1005), in combination with a recombinant profilin vaccine, to modulate host protective immunity and to alter gene expression during experimental avian coccidiosis. Vaccination with profilin plus QCDCR significantly reduced the severity of intestinal lesions and increased mitogen-induced lymphocyte proliferation in infected chickens compared with immunization with profilin alone or profilin plus QCDC. Immunization with profilin plus QCDC or profilin plus QCDCR increased body weight gain but had no effect on fecal oocyst shedding of chickens infected with Eimeria acervulina compared with birds vaccinated with profilin alone. The results of global gene expression analysis revealed that, compared with PBS controls, (a) chickens vaccinated with profilin alone had 71 up-regulated and 56 down-regulated mRNA transcripts, (b) chickens immunized with profilin plus QCDC had 198 up-regulated and 247 down-regulated mRNAs, and (c) birds immunized with profilin plus QCDCR had 210 up-regulated and 267 down-regulated mRNAs. Compared with birds vaccinated with profilin alone, (a) chickens given profilin plus QCDC had 60 up-regulated and 104 down-regulated transcripts and (b) chickens immunized with profilin plus QCDCR had 103 up-regulated and 130 down-regulated mRNAs. Finally, chickens vaccinated with profilin plus QCDCR had 193 up-regulated and 204 down-regulated transcripts compared with birds given profilin plus QCDC. Biological function and network analysis revealed that the majority of altered transcripts were encoded by immune-related genes.

Adjuvant effect of ginsenoside-based nanoparticles (ginsomes) on the recombinant vaccine against Eimeria tenella in chickens

An experiment was conducted to study the adjuvant effect of ginsomes on the recombinant profilin in coccidian-infected breeding birds. Three-day-old chickens were vaccinated with Eimeria tenella recombinant profilin antigen (10, 50, and 100 μg per chicken) with or without 50 μg ginsomes per chicken. The boost vaccination was carried out 14 days later. Two weeks after the booster, the chickens were challenged with 1.5 × 10(4) homologous sporulated oocysts. The specific antibody response, lymphocyte proliferation, and IL-1 release from lymphocyte were measured at 1-42 days after boost vaccination. Seven days post-challenge, the rate of survival, body weight gains (BWG) were examined then all chickens were sacrificed and lesion scores and oocysts per gram were monitored to evaluate the protective effects of the vaccination after challenge. Compared with the group of vaccinating with profilin only, groups of 50 and 100 μg antigen plus ginsomes significantly enhanced lymphocyte proliferation and IL-1 secretion. The profilin specific antibody level in the four vaccinated groups was significantly higher than in the control group and in groups vaccinated with profilin containing ginsomes than profilin only. In the groups vaccinated with profilin plus ginsomes, the BWG was significantly higher than that of group of profilin only, but there was no significant difference between profilin plus adjuvant ginsomes, diclazuril medicated and uninfected-unmedicated-unvaccinated control groups. The lesion scores in groups immunized with profilin plus ginsomes was significantly lower than that both of groups unimmunized-challenged-unmedicated control and group vaccinated with profilin only. Oocyst excretion in groups vaccinated with 50 or 100 μg profilin plus ginsomes was lower than that of groups vaccinated with profilin only. These results demonstrate that the adjuvant ginsomes can promote subunit vaccine to induce a strong immune response and protective effects.

The clinical role of positive latex IgE in patients with food/pollen adverse reactions

Natural rubber latex allergy (NRL-A) is an international problem of public health. About 50-60% of NRL-A patients may present adverse reactions after ingestion cross-reacting vegetable foods. This condition is called Latex-fruit Syndrome and is matter of research. The aim of our study is distinguishing between clinical/subclinical latex-fruit syndrome and cross-sensitization to latex and food/pollens allergens on the basis of latex recombinant allergens. We studied 19 patients with food hypersensitivity and serological evidences of NRL sensitization. They underwent an accurate allergological evaluation (skin prick test with latex, food and pollen extracts, specific IgE to latex and recombinant allergens, challenge provocation tests). The patients were divided in two groups: group 1) 9 patients allergic to fruits/vegetables and/or pollens, with serological, but not clinical NRL-A; group 2) 10 patients with clinical and serological latex and fruits/vegetables allergy. The same number of patients was positive to pollens in each group. All group 1 patients presented negative provocation challenges and a monosensitization to Hev b8 (and other recombinant profilins), probably linked to a cross-sensitization to pollens and foods. These data suggest that profilin sensitization determines false positivity from NRL-A diagnostic test. Consequently the observed in vitro reactivity to NRL is not clinically relevant in patients with adverse food reactions and it does not determine always a latex-fruit syndrome. Instead, in group 2 we observed positive NRL provocation challenges and sensitization to rHev b 8 , but also to other recombinant latex allergens (rHev b 5, rHev b 6.01, rHev b 6.02). These patients probably presented a latex-fruit syndrome. We supposed that the available panel of recombinant latex allergens could be effective to demonstrate a significant difference between latex-fruit syndrome and cross-sensitization to latex and food/pollens allergens. So further investigations are needed with a larger group of patients.

Introductory lecture: Pollen food allergy syndrome

The term pollen-food allergy syndrome (PFAS) defines a series of clinical symptoms appearing shortly after the ingestion of plant-derived foods in subjects with pollen allergy. The patients with PFAS are primarily allergic to pollen and subsequently react to food allergens as a consequence of the homology between pollen and plant-food proteins. The two highly conserved proteins responsible for the large majority of cases of pollen-food allergy syndrome are the pathogenesis-related proteins group 10 (PR-10), including the major birch pollen allergen Bet v 1 and homologous proteins in different fruits and vegetables, and profilin, a plant pan-allergen present in cell structure of all the vegetable kingdom. Although it has been generally thought that the clinical expression of the pollen food-allergy syndrome is uniquely the so-called “oral allergy syndrome”, recent reports suggest that the ingestion of particular foods may be associated with systemic symptoms as well. Recombinant PR-10 proteins and recombinant profilins from different sources are presently available for diagnostic purposes. The presentation will review the available data about the clinical expression, diagnosis, and therapy of the pollen-food allergy syndrome.