Strain-dependent induction of allergic sensitization caused by peanut allergen DNA immunization in mice

To investigate the potential application of allergen gene immunization in the modulation of food allergy, C3H/HeSn (C3H) mice received i.m. injections of pAra h2 plasmid DNA encoding one of the major peanut allergens, Ara h2. Three weeks following pDNA immunization, serum Ara h2-specific IgG2a, IgG1, but not IgE, were increased significantly in a dose-dependent manner. IgG1 was 30-fold higher in multiply compared with singly immunized mice. Ara h2 or peanut protein injection of immunized mice induced anaphylactic reactions, which were more severe in multiply immunized mice. Heat-inactivated immune serum induced passive cutaneous anaphylaxis, suggesting that anaphylaxis in C3H mice was mediated by IgG1. IgG1 responses were also induced by intradermal injection of pAra h2, and by i.m. injection of pOMC, the plasmid DNA encoding the major egg allergen protein, ovomucoid. To elucidate whether the pDNA immunization-induced anaphylaxis was a strain-dependent phenomenon, AKR/J and BALB/c mice also received multiple i.m. pAra h2 immunizations. Injection of peanut protein into these strains at weeks 3 or 5 following immunization did not induce reactions. Although IgG2a was increased significantly from week 2 in AKR/J mice and from week 4 in BALB/c mice and remained elevated for at least 6 wk, no IgG1 or IgE was detected. These results indicate that the type of immune responses to pDNA immunization in mice is strain dependent. Consequently, models for studying human allergen gene immunization require careful selection of suitable strains. In addition, this suggests that similar interindividual variation is likely in humans.     

Contribution of classic and alternative effector pathways in peanut-induced anaphylactic responses

Food allergy affects approximately 5% of children and is the leading cause of hospitalization for anaphylactic reactions in westernized countries. However, the pathways of anaphylaxis in food allergy are still relatively unknown. We investigated the effector pathways of allergic and anaphylactic responses of different strains of mice in a clinical relevant model of peanut allergy. C3H/HeOuJ, C57BL/6 and BALB/c mice were sensitized by intragastric peanut extract and challenged by intragastric or intraperitoneal injection of peanut. Peanut-specific T cell responses, IgE, IgG1 and IgG2a and mucosal mast cell degranulation were induced to different extent in C3H/HeOuJ, C57BL/6 and BALB/c mice. Interestingly, anaphylactic symptoms after systemic challenge were highest in C3H/HeOuJ followed by C57BL/6 but were absent in BALB/c mice. Mechanistic studies showed that the food allergic systemic anaphylaxis was dependent on platelets, FcRγ and mast cells, and partially dependent on platelet activating factor and monocytes/macrophages, depending on mouse strain. These data demonstrate that in three mouse strains, components of the classic and alternative anaphylactic cascade are differently expressed, leading to differential outcomes in parameters of allergic disease and food induced systemic anaphylaxis.     

Role of Maternal Dietary Peanut Exposure in Development of Food Allergy and Oral Tolerance

Background

The impact of maternal ingestion of peanut during pregnancy and lactation on an offspring’s risk for peanut allergy is under debate.

Objective

To investigate the influence of maternal dietary peanut exposure and breast milk on an offspring’s allergy risk.

Methods

Preconceptionally peanut-exposed C3H/HeJ females were either fed or not fed peanut during pregnancy and lactation. The offsprings’ responses to peanut sensitization or oral tolerance induction by feeding antigen prior to immunization were assessed. We also assessed the impact of immune murine milk on tolerance induction pre- or post-weaning. For antigen uptake studies, mice were gavaged with fluorescent peanut in the presence or absence of immune murine milk; Peyer’s patches were harvested for immunostaining.

Results

Preconceptional peanut exposure resulted in the production of varying levels of maternal antibodies in serum (and breast milk), which were transferred to the offspring. Despite this, maternal peanut exposure either preconceptionally or during pregnancy and lactation, when compared to no maternal exposure, had no impact on peanut allergy. When offspring were fed peanut directly, dose-dependent tolerance induction, unaltered by maternal feeding of peanut, was seen. Although peanut uptake into the gut-associated lymphoid tissues was enhanced by immune milk as compared to naïve milk, tolerance induction was not affected by the co-administration of immune milk either pre- or post-weaning.

Conclusion

Maternal peanut exposure during pregnancy and lactation has no impact on the development of peanut allergy in the offspring. Tolerance to peanut can be induced early, even pre-weaning, by giving moderate amounts of peanut directly to the infant, and this is neither enhanced nor impaired by concurrent exposure to immune milk.     

DNA vaccines for allergy treatment

In the past 10 years, a great number of studies have demonstrated that injection of plasmid DNA coding for certain genes results in the induction of humoral and cellular immune responses against the respective gene product. This vaccination approach covers a broad range of possible applications, including the induction of protective immunity against viral, bacterial, and parasitic infections, and it opens new perspectives for treatment of cancer. Surprisingly, DNA immunization also turned out as a promising novel type of immunotherapy against allergy. In this paper, we describe the construction of DNA vaccines for application in allergy models. Beyond, we offer a palette of recently developed modulations to optimize DNA vaccines for allergy treatment by increasing their immunogenicity and minimizing their anaphylactic potential.     

Protective Immunity Induced by Oral Immunization with a Rotavirus DNA Vaccine Encapsulated in Microparticles

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.     

CTLA-4 signaling regulates the intensity of hypersensitivity responses to food antigens, but is not decisive in the induction of sensitization

Although food allergy has emerged as a major health problem, the mechanisms that are decisive in the development of sensitization to dietary Ag remain largely unknown. CTLA-4 signaling negatively regulates immune activation, and may play a crucial role in preventing induction and/or progression of sensitization to food Ag. To elucidate the role of CTLA-4 signaling in responses to food allergens, a murine model of peanut allergy was used. During oral exposure to peanut protein extract (PPE) together with the mucosal adjuvant cholera toxin (CT), which induces peanut allergy, CTLA-4 ligation was prevented using a CTLA-4 mAb. Additionally, the effect of inhibition of the CTLA-4 pathway on oral exposure to PPE in the absence of CT, which leads to unresponsiveness to peanut Ag, was explored. During sensitization, anti-CTLA-4 treatment considerably enhanced IgE responses to PPE and the peanut allergens, Ara h 1, Ara h 3, and Ara h 6, resulting in elevated mast cell degranulation upon an oral challenge. Remarkably, antagonizing CTLA-4 during exposure to PPE in the absence of CT resulted in significant induction of Th2 cytokines and an elevation in total serum IgE levels, but failed to induce allergen-specific IgE responses and mast cell degranulation upon a PPE challenge. These results indicate that CTLA-4 signaling is not the crucial factor in preventing sensitization to food allergens, but plays a pivotal role in regulating the intensity of a food allergic sensitization response. Furthermore, these data indicate that a profoundly Th2-biased cytokine environment is insufficient to induce allergic responses against dietary Ag.     

Elevated Antigen-Driven IL-9 Responses Are Prominent in Peanut Allergic Humans

Food allergies, and peanut allergy in particular, are leading causes of anaphylactic fatalities worldwide. The immune mechanisms that underlie food allergy remain ill-defined and controversial, in part because studies in humans typically focus on analysis of a limited number of prototypical Th1/Th2 cytokines. Here we determine the kinetics and prevalence of a broad panel of peanut-driven cytokine and chemokine responses in humans with current peanut allergy vs those with stable, naturally occurring clinical tolerance to peanut. Our primary focus is identification of novel indicators of immune dysregulation. Antigen-specific cytokine mRNA and protein responses were elicited in primary culture via peanut or irrelevant antigen (Leishmania extract, milk antigens) mediated stimulation of fresh peripheral blood cells from 40 individuals. Peanut extract exposure in vitro induced a broad panel of responses associated with Th2/Th9-like, Th1-like and Th17-like immunity. Peanut-dependent Type 2 cytokine responses were frequently found in both peanut allergic individuals and those who exhibit clinical tolerance to peanut ingestion. Among Th2/Th9-associated cytokines, IL-9 responses discriminated between allergic and clinically tolerant populations better than did commonly used IL-4, IL-5 or IL-13 responses. Comparison with responses evoked by unrelated control antigen-mediated stimulation showed that these differences are antigen-dependent and allergen-specific. Conversely, the intensity of IL-12, IL-17, IL-23 and IFN-γ production was indistinguishable in peanut allergic and peanut tolerant populations. In summary, the ability to generate and maintain cytokine responses to peanut is not inherently distinct between allergic and peanut tolerant humans. Quantitative differences in the intensity of cytokine production better reflects clinical phenotype, with optimally useful indicators being IL-9, IL-5, IL-13 and IL-4. Equivalent, and minimal, Ag-dependent pro-inflammatory cytokine levels in both healthy and peanut allergic volunteers argues against a key role for such cytokines in maintenance of clinical tolerance to food antigens in humans.     

Engineered recombinant peanut protein and heat-killed Listeria monocytogenes coadministration protects against peanut-induced anaphylaxis in a murine model

Peanut allergy (PNA) is the major cause of fatal and near-fatal anaphylactic reactions to foods. Traditional immunotherapy using peanut (PN) protein is not an option for PNA therapy because of the high incidence of adverse reactions. We investigated the effects of s.c. injections of engineered (modified) recombinant PN proteins and heat-killed Listeria monocytogenes (HKLM) as an adjuvant on anaphylactic reactions in a mouse model of PN allergy. PN-allergic C3H/HeJ mice were treated s.c. with a mixture of the three major PN allergens and HKLM (modified (m)Ara h 1-3 plus HKLM). The effects on anaphylactic reactions following PN challenge and the association with Ab levels and cytokine profiles were determined. Although all mice in the sham-treated groups exhibited anaphylactic symptoms with a median symptom score of 3, only 31% of mice in the mAra h 1-3 plus HKLM group developed mild anaphylaxis, with a low median symptom score of 0.5. Alterations in core body temperature, bronchial constriction, plasma histamine, and PN-specific IgE levels were all significantly reduced. This protective effect was markedly more potent than in the mAra h 1-3 protein alone-treated group. HKLM alone did not have any protective effect. Reduced IL-5 and IL-13, and increased IFN-gamma levels were observed only in splenocytes cultures from mAra h 1-3 plus HKLM-treated mice. These results show that immunotherapy with modified PN proteins and HKLM is effective for treating PN allergy in this model, and may be a potential approach for treating PNA.     

Genetic immunization by jet injection of targeted pDNA-coated nanoparticles

Genetic immunization strategies have largely focused on the use of "naked" plasmid DNA or the gene gun. However, there remains a clear need to further improve the efficiency and/or cost of potential DNA vaccines. The theoretical basis of our research is to rationally design genetic immunization methodologies for nanoparticle-based delivery systems of plasmid DNA, perhaps in combination with already commercially available needle-free devices, such as the Biojector 2000. These methodologies may both reduce the dose of pDNA required and enhance the breadth and depth of protective immune responses (i.e., humoral and cellular). The purpose of this article is to provide detailed experimental methods to (1) engineer and characterize pDNA-coated cationic nanoparticles (<100nm) directly from oil-in-water microemulsion precursors and (2) enhance both the breadth and depth of immune responses after immunization of mice with pDNA-coated nanoparticles by different routes of administration, including intradermal, using a needle-free jet injection device.     

HIV-1 p24 DNA和P24蛋白联合免疫小鼠的效果

DNA疫苗能够诱导机体产生特异的细胞免疫和体液免疫反应,在肿瘤和感染性疾病的疫苗开发中显示出巨大的潜能。以HIV-1核心蛋白P24为抗原基因,构建pVAX1-p24 DNA,经Western blotting和动物活体成像检测证明,pVAX1 DNA携带的外源基因可以在293T 细胞和小鼠肌肉组织有效表达。采用不同的免疫策略免疫BALB/c小鼠 (DNA/DNA,DNA/Protein),实验结果表明：pVAX1-p24单独免疫BALB/c小鼠,可诱导明显的体液免疫及细胞免疫反应;pVAX1-p24与P24蛋白联合免疫诱导的体液免疫反应高于pVAX1-p24单独免疫,所获得的抗体滴度是单独免疫的7.3~8.0倍,但细胞免疫反应则不及单独免疫组。研究结果表明采取不同的免疫策略可以诱导产生不同的免疫反应,根据具体情况调整免疫策略将获得更好的免疫效果。这些研究为艾滋病疫苗的研发提供了实验依据。     

Protective efficiency of dendrosomes as novel nano-sized adjuvants for DNA vaccination against birch pollen allergy

We evaluated the use of a novel gene porter (Den123--a nontoxic self-assembled dendritic spheroidal nanoparticle made of biodegradable monomers), aiming to enhance and improve the desired immune response in protection from allergy. Footpad DNA immunization in Balb/c mice was done three times using the Bet v 1a gene with or without Den123 with 2-week intervals followed by sensitization with rBetv1 (5 microg) in alum twice in a weekly interval. Different doses of pCMV-Betv1 were used (10 microg and 100 microg). The protective role of different formulations was evaluated by measuring the IgG1, IgG2a and IgE antibody production, cytokine release of isolated splenocytes and beta-hexosaminidase release from the RBL cells. Higher and increasing ratios of IgG2a/IgG1 were seen in mice which received plasmids in combination with Den123. Den123 and DNA vaccine synergistically enhanced the Interferon gamma released from splenocytes. In the presence of Den123, IgE inhibition was independent of the dose and type of the injected DNA. All DNA-pre-immunized mice demonstrated low basophil degranulation. It is therefore concluded that administration of the DNA entrapped in Den123 nanoparticles results in sustained release of plasmids, Th1/Th2 balanced immune response with promising IgE inhibition. Also higher amounts of DNA contributed to stronger Th1 response.     

Identification and characterization of a hypoallergenic ortholog of Ara h 2.01

Peanut (Arachis hypogaea L.), can elicit type I allergy becoming the most common cause of fatal food-induced anaphylactic reactions. Strict avoidance is the only effective means of dealing with this allergy. Ara h 2, a peanut seed storage protein, has been identified as the most potent peanut allergen and is recognized by approximately 90% of peanut hypersensitive individuals in the US. Because peanut has limited genetic variation, wild relatives are a good source of genetic diversity. After screening 30 Arachis duranensis accessions by EcoTILLing, we characterized five different missense mutations in ara d 2.01. None of these polymorphisms induced major conformational modifications. Nevertheless, a polymorphism in the immunodominant epitope #7 (S73T) showed a 56–99% reduction in IgE-binding activity and did not affect T cell epitopes, which must be retained for effective immunotherapy. The identification of natural hypoallergenic isoforms positively contributes to future immunological and therapeutic studies and peanut cultivar development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Oral administration with papillomavirus pseudovirus encoding IL-2 fully restores mucosal and systemic immune responses to vaccinations in aged mice

Infectious diseases are one of the major threats for the elderly because their immune system is often compromised, and vaccinations to prevent these infections are not effective. A major defect in their immune system seems to be the inability of T cells to produce IL-2. We used papillomavirus (PV) pseudoviruses (PSVs) as a model vaccine and a gene delivery vector to address how to enhance immune responses to vaccinations. We found that oral immunization with PV PSV induced minimal mucosal and systemic Abs and CTLs specific for the PSVs in aged mice compared with young adult mice. In addition, fewer specific Th cells were generated in the aged mice. When aged mice were immunized with PV PSVs encoding human IL-2, specific Th cells were generated, producing murine IL-2, IL-4, and IFN-gamma. Further, specific Abs and CTLs were induced, resulting in protection against mucosal viral challenge. Thus, this study provided a basis for clinical trials using PV PSVs encoding IL-2 for vaccination of the elderly.     

An enteric helminth infection protects against an allergic response to dietary antigen

Although helminths induce a polarized Th2 response they have been shown, in clinical studies, to confer protection against allergies. To elucidate the basis for this paradox, we have examined the influence of an enteric helminth infection on a model of food allergy. Upon Ag challenge, mice fed peanut (PN) extract plus the mucosal adjuvant cholera toxin (CT) produced PN-specific IgE that correlated with systemic anaphylactic symptoms and elevated plasma histamine. PN-specific IgE was not induced in helminth-infected mice fed PN without CT. Moreover, when PN plus CT was fed to helminth-infected mice, both PN-specific IgE and anaphylactic symptoms were greatly diminished. The down-regulation of PN-specific IgE was associated with a marked reduction in the secretion of IL-13 by PN-specific T cells. When helminth-infected PN plus CT-sensitized mice were treated with neutralizing Abs to IL-10, the PN-specific IgE response and anaphylactic symptoms were similar to, or greater than, those seen in mice that receive PN and CT alone. Taken together, these results suggest that helminth-dependent protection against allergic disease involves immunoregulatory mechanisms that block production of allergen-specific IgE.     

Human immunodeficiency virus type 1 gag-specific mucosal immunity after oral immunization with papillomavirus pseudoviruses encoding gag

Mucosal surfaces are the primary portals for human immunodeficiency virus (HIV) transmission. Because systemic immunization, in general, does not induce effective mucosal immune responses, a mucosal HIV vaccine is urgently needed. For this study, we developed papillomavirus pseudoviruses that express HIV-1 Gag. The pseudoviruses are synthetic, nonreplicating viruses, yet they can produce antigens for a long time in the immune system. Here we show that oral immunization of mice by the use of papillomavirus pseudoviruses encoding Gag generated mucosal and systemic Gag-specific cytotoxic T lymphocytes that effectively lysed Gag-expressing target cells. Furthermore, the pseudoviruses generated Gag-specific gamma interferon-producing T cells and serum immunoglobulin G (IgG) and mucosal IgA. In contrast, oral immunization with plasmid DNA encoding HIV-1 Gag did not induce specific immune responses. Importantly, oral immunization with the pseudoviruses induced Gag-specific memory cytotoxic T lymphocytes and protected mice against a rectal mucosal challenge with a recombinant vaccinia virus expressing HIV-1 Gag. Thus, papillomavirus pseudoviruses encoding Gag are a promising mucosal vaccine against AIDS.     

Toll-like receptor 4 signaling by intestinal microbes influences susceptibility to food allergy

The mechanisms by which signaling by the innate immune system controls susceptibility to allergy are poorly understood. In this report, we show that intragastric administration of a food allergen with a mucosal adjuvant induces allergen-specific IgE, elevated plasma histamine levels, and anaphylactic symptoms in three different strains of mice lacking a functional receptor for bacterial LPS (Toll-like receptor 4 (TLR4)), but not in MHC-matched or congenic controls. Susceptibility to allergy correlates with a Th2-biased cytokine response in both the mucosal (mesenteric lymph node and Peyer's patch) and systemic (spleen) tissues of TLR4-mutant or -deficient mice. TLR4-mutant mice are not inherently impaired in their ability to regulate Th1 cytokine production because they respond to stimulation via TLR9. Coadministration of CpG oligodeoxynucleotides during sensitization of TLR4-mutant mice with allergen plus CT abrogates anaphylactic symptoms and Ag-specific IgE, and results in a Th1-polarized cytokine response. When the composition of the bacterial flora is reduced and altered by antibiotic administration (beginning at 2 wk of age), TLR4 wild-type mice become as susceptible to the induction of allergy as their TLR4-mutant counterparts. Both allergen-specific IgE and Th2 cytokine responses are reduced in antibiotic-treated mice in which the flora has been allowed to repopulate. Taken together, our results suggest that TLR4-dependent signals provided by the intestinal commensal flora inhibit the development of allergic responses to food Ags.     

携带HBV包膜大蛋白DNA疫苗的减毒沙门菌在小鼠诱导免疫应答

探索一种简便、有效的乙型肝炎病毒DNA疫苗免疫方法。将编码绿色荧光蛋白的真核表达质粒pEGFPN1转化到减毒鼠伤寒沙门菌SL7207,灌胃饲服BALB/c小鼠,流式细胞术检测出小鼠脾细胞内表达的绿色荧光蛋白;构建编码HBV包膜大蛋白的DNA疫苗pCIS1S2S,分别以SL7207为载体的口服途径或直接肌肉注射途径免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,结果表明两种免疫途径均能在小鼠体内诱生细胞和体液免疫应答,但口服途径诱导免疫应答的强度明显强于肌肉注射途径。口服携带HBV DNA疫苗的减毒伤寒沙门菌可能代表一种简便、有效的治疗乙型肝炎的新方法。      

Adjuvanted oral vaccines should not induce allergic responses to dietary antigens

Oral vaccines may contain adjuvants which might elicit allergies against dietary proteins. Four antigens were used to measure such an effect--ovalbumin, soya bean protein, lactalbumin and gluten. Neither guinea pigs nor mice showed IgE responses after oral administration of adjuvanted vaccines containing lactalbumin and gluten. No IgE responses were detected in mice with any of these antigens after oral immunization, but, in the guinea pig, nine out of 18 animals reacted to either ovalbumin or soya bean protein and none reacted to lactalbumin and gluten. It is concluded that the risk of allergy induction against normal dietary proteins is low but such tests should be applied to potential adjuvanted oral vaccines to measure any possible contraindication, especially with atopic individuals.     

Evaluation of genetic immunization adjuvants to improve the effectiveness of a human immunodeficiency virus type 2 (HIV-2) envelope DNA vaccine

In an effort to develop a more effective genetic immunization strategy for HIV, we developed an HIV-2 env DNA vaccine and evaluated three adjuvant formulations. The gp140 gene from HIV-2(UC2 )was synthesized using mammalian codons and cloned into a plasmid vector that expresses eukaryotic genes at high levels. We found that after three immunizations in mice, a novel cationic liposome formulation (Vaxfectin) was superior at inducing systemic and mucosal antibody responses compared to a naked DNA, a controlled release device (an Alzet minipump) and polysaccharide microparticles made from chitosan (P = 0.027). Vaxfectin also induced higher levels of systemic antibodies for each isotype and IgG subclass as well as levels of HIV-2-specific mucosal IgA (P = 0.034). When different routes of immunization were used with the Vaxfectin formulation, gp140-specific systemic antibody responses were highest by the intradermal route, mucosal antibody responses were highest by the intramuscular route, while the intranasal route was the least effective. These results suggest that this cationic liposome formulation is an important adjuvant to improve the effectiveness of genetic immunization strategies for AIDS, and that multiple routes of immunization should be employed for optimal efficacy for HIV vaccine candidates.