IFN-γ and IL-12 plasmid DNAs as vaccine adjuvant in a murine model of grass allergy

doi:10.1067/mai.2001.117261

Journal of Allergy and Clinical Immunology

Volume 108, Issue 3, September 2001, Pages 402-408

https://doi.org/10.1067/mai.2001.117261 Get rights and content

Abstract

Background: Plasmids encoding cytokines such as IFN-γ and IL-12 are potential genetic adjuvants that might increase the effectiveness of allergen vaccines. Objective: The role of plasmids expressing the cytokines IFN-γ (pIFN-γ) and/or IL-12 (pIL-12) as adjuvants in modulating allergic immune responses, inflammation, and asthma was investigated in a murine model of Kentucky blue grass (KBG) allergy. Methods: Groups of naive B6D2F1 mice were vaccinated subcutaneously with KBG allergens and administered intramuscularly with pIFN-γ, pIL-12, pIFN-γ plus pIL-12, or a vector control. Mice were then sensitized with KBG allergens in alum (intraperitoneally) and later challenged intranasally. Mice were examined for modulation of specific immunity, prevention of the development of airway hyperresponsiveness, and inflammation. Results: Mice vaccinated with cytokine plasmid adjuvants had relatively lower levels of total serum IgE and higher levels of grass allergen–specific IgG2a in comparison with control mice. The lowest IgE and highest IgG2a levels were found in mice vaccinated with the combination of pIFN-γ and pIL-12 as an adjuvant. The vaccination of mice with both pIFN-γ and pIL-12 as an adjuvant induced the highest level of T_H1 cytokines, IFN-γ, and IL-2 in comparison with mice given either of the plasmids alone. The most profound decrease in airway hyperresponsiveness and pulmonary inflammation was observed in mice receiving both pIFN-γ and pIL-12 as an adjuvant. Conclusion: These results demonstrate that pIFN-γ and pIL-12 together provide an effective adjuvant to parenteral grass allergen vaccines and show that this adjuvant can significantly enhance the effectiveness of allergen immunotherapy in human beings. (J Allergy Clin Immunol 2001;108:402-8.)

Section snippets

Animals

Female B6D2F1 mice, 6 to 8 weeks old, from Jackson Laboratory (Bar Harbor, Me) were maintained in pathogen-free conditions at the animal center at the James A. Haley Veterans Hospital. All procedures were reviewed and approved by the committee on animal research at the James A. Haley VA Medical Center and the University of South Florida College of Medicine.

Vaccination protocol

Four groups of naive mice (n = 12) were intramuscularly vaccinated 3 times at intervals of 2 days, each in its right quadriceps muscle, with

Plasmid constructs and expression of IL-12 and IFN-γ

The 2 subunits of murine IL-12, p35 and p40, were cloned into the same pcDNA3.1 vector. The cloning strategy was such that each subunit was under the transcriptional control of an individual CMV immediate-early promoter and also had its own BGH poly A sequences derived from the vector pcDNA3.1. Mice given pIFN-γ or pIL-12 exhibited expression of the IFN-γ or IL-12 p40 subunit, respectively, in their muscle, as observed by immunohistochemical staining of the muscle tissues (Fig 1, A , a and c ).

Discussion

Previously, it was shown in a mouse model that effective parenteral vaccination of mice with grass allergens required subcutaneous injection of a high dose (250 μg to 1 mg per mouse) of allergens.¹⁴ By corollary, an even higher dose of allergen mixture would be required to induce an effective immune deviation from an allergic response. The results of this study demonstrate that in a mouse, a substantially lower dosage (50 μg) of allergens, when administered along with a pDNA cytokine(s)

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Recombinant plasmids containing CpG with porcine host defense peptides (PR-39/pBD-1) modulates the innate and adaptive intestinal immune responses (including maternal-derived) in piglets
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To address this deficiency, scientists used plasmids directly as vaccine adjuvants [26]. Mukesh Kumar et al. (2001) constructed a recombinant plasmid containing the IFN-γ and IL-12 genes, which proved to be effective as a vaccine adjuvant to improve human allergen immunity [27]. A similar finding was reported by Krieg AM et al. that the presence of a CpG motif provided the necessary immunoadjuvant signal for DNA vaccination [28].
CpG oligodeoxynucleotides (CpG-ODN) is an immunoenhancer, which is composed of unmethylated cytosine and guanine. Host Defense Peptides (HDPs) are small molecule polypeptides with various immunological activities that have been shown to induce a stronger innate immune response in piglets with synthetic CpG-ODN. Therefore, combination of CpG-ODN and HDPs was expected to be a novel immunoadjuvant with high efficiency, low toxicity and great potential. However, cost of synthetic HDPs or CpG-ODN is too high to be advantageous for animal farming. In this study, in order to improve the immune function of vaccine and reduce cost, a series of recombinant plasmids (containing HDPs gene (PR-39/pBD-1) and different numbers of CpG motifs) were constructed. In vitro, porcine lymphocytes were stimulated by recombinant plasmids to verify the immunostimulatory function of recombinant plasmids. In vivo, recombinant plasmids were used to immunize piglets with Enterotoxigenic Escherichia coli (ETEC) vaccine to analyze effects of recombinant plasmids on the mucosal immune responses. In addition, dosage screening and capability of maternal antibody responses were also investigated. Our results showed that recombinant plasmids had strong adjuvant effects especially the plasmid pVAX49-PR-39 and pVAX49-pBD-1. Moreover, there was no diarrhea in piglets using pVAX49-PR-39 or pVAX49-pBD-1 as adjuvants. These findings suggested that recombinant plasmids (containing PR-39/pBD-1 and CpG) as adjuvants of vaccines could enhance immune stimulation better than HDPs or CpG alone. It has a good protective effect on maintaining health of newborn piglets. Among them, both plasmids pVAX49-PR-39 and pVAX49-pBD-1 could be used as effective vaccine adjuvants for piglets.
Effects of a DNA vaccine in an animal model of Alternaria alternata sensitivity
2009, Revista Iberoamericana de Micologia
Sensitivity to the fungus Alternaria is associated with asthma persistence and severity. Current therapeutic options for treating Alternaria-induced airway inflammation are limited. In this study, Brown Norway rats are used to study the effectiveness of a DNA-based vaccine delivered to the airway in attenuating the response to a major Alternaria allergen, rAlt a 2. Compared to untreated sensitized animals, or animals receiving an “out-of-frame” DNA-based vaccine, animals treated with “in-frame” DNA vaccine showed an attenuation in specific IgE antibody titers to rAlt a 2, an increase in IgG^2b (a Th1 response), a reduction in spontaneous IL-13 release by peribronchial lymph node cell suspensions, and an attenuation in the decrease in total lung capacity 72 h post-allergen challenge. Further, histopathologic examination of the lung tissues revealed reduced pulmonary inflammation post-allergen challenge in the DNA-vaccine-treated compared to sensitized, untreated animals. We conclude that a DNA-based vaccine delivered to the airway significantly influences the immunologic, pulmonary physiologic, and histological alterations induced by challenge with a major Alternaria allergen, rAlt a 2, in sensitized animals.
La sensibilidad al hongo Alternaria se asocia con la persistencia y gravedad del asma. Las opciones terapéuticas actuales para el tratamiento de la inflamación de las vías aéreas inducida por Alternaria son limitadas. En este estudio se han utilizado ratas Brown Norway para estudiar la eficacia de una vacuna de ADN administrada por vía aérea para atenuar la respuesta al alérgeno mayor de Alternaria rAlt a 2. Comparados con los animales sensibilizados sin tratar, o con los animales que recibieron una vacuna de ADN con un marco de lectura incorrecto, los animales tratados con una vacuna ADN con un marco de lectura correcto mostraron una atenuación en los títulos de anticuerpos específicos IgE frente a rAlt a 2, un aumento en IgG_2b (una respuesta Th1), una reducción en la liberación espontánea de IL-13 por suspensiones celulares de nódulos linfáticos peribronquiales y una atenuación en la diminución en la capacidad pulmonar total 72 h después de la administración del alérgeno. El examen histopatológico de los tejidos pulmonares reveló una inflamación pulmonar reducida tras la administración del alérgeno en los animales tratados con ADN comparados con los animales sensibilizados sin tratamiento. Se concluye que una vacuna de ADN administrada por vía aérea ejerce una influencia significativa sobre las alteraciones inmunológicas, fisiológicas pulmonares e histológicas inducidas por el alérgeno mayor de Alternaria rAlt a 2 en animales sensibilizados.
Vaccination to treat noninfectious diseases: Surveying the opportunities
2006, Immunopotentiators in Modern Vaccines
Vaccination for the treatment and prevention of infectious diseases has been the most successful medical intervention from a public health perspective. The application of vaccination for treating noninfectious diseases is becoming more common and there are many recent examples of applying vaccine technology to a diverse range of noninfectious diseases. This chapter categorizes the immunological requirements of different classes of noninfectious diseases to show the underlying immunological mechanisms that have to be considered in the design of appropriate vaccines. Broadly, this encompasses treatments requiring the strong induction of immune responses, both humoral and cell mediated, or the down modulation of an existing pathological immune response, either through immune deviation or suppression. The chapter highlights the desired adjuvant qualities required for each approach and takes a look at some of the potential dangers that may result from using vaccine formulations that push the immune system too far in one particular direction.
Vaccination to treat noninfectious diseases: Surveying the opportunities
2005, Immunopotentiators in Modern Vaccines
Vaccination for the treatment and prevention of infectious diseases has been the most successful medical intervention from a public health perspective. The application of vaccination for treating noninfectious diseases is becoming more common and there are many recent examples of applying vaccine technology to a diverse range of noninfectious diseases. This chapter categorizes the immunological requirements of different classes of noninfectious diseases to show the underlying immunological mechanisms that have to be considered in the design of appropriate vaccines. Broadly, this encompasses treatments requiring the strong induction of immune responses, both humoral and cell mediated, or the down modulation of an existing pathological immune response, either through immune deviation or suppression. The chapter highlights the desired adjuvant qualities required for each approach and takes a look at some of the potential dangers that may result from using vaccine formulations that push the immune system too far in one particular direction.
Rationale for new treatments aimed at IgE immunomodulation
2004, Annals of Allergy, Asthma and Immunology
To review potential or current therapies that decrease IgE synthesis or effects.
Relevant literature in peer-reviewed journals and abstracts from national meetings.
Key articles were selected by the authors.
Modulation of IgEmediated diseases can occur at several levels. Transcription factors may be altered to differentiate lymphocytes into a T_H1 phenotype, thus decreasing T_H2-driven IgE production. This may be accomplished by inhibiting GATA-3 with peroxisome proliferatoractivated receptor agonists or promoting T-bet expression with CpG motifs. Inhibiting IgE-promoting cytokines may be accomplished by blocking the effects or synthesis of interleukin 4 (IL-4) or IL-13 by suplatast tosilate. Cytokine therapy with anti–IL-4 or anti-IL-13 has the potential to directly influence IgEmediated diseases, but strategies aimed at IL-4 alone have been disappointing. Clinical trials with interferon-γ or IL-12, 2 cytokines important in promoting T_H1 and inhibiting T_H2 responses, have been fraught with adverse effects that make their use limited. The use of plasmids encoding interferon-γ or IL-12 has shown promise in animal models. Inhibition of IgE synthesis has been demonstrated with anti-CD23 antibodies. Early human studies have been very encouraging, and larger studies are under way. The only IgE immunomodulator currently available for use is omalizumab. Omalizumab is effective for allergic asthma in children and adults.
Newer therapies hold great promise for the future treatment of allergic respiratory diseases, but clinical trials are necessary to accurately evaluate risk-benefit ratios of IgE immunomodulators.
Allergen rush immunotherapy increases interleukin (IL)-12 production and IL-12 receptor β2 chain expression in patients with allergic asthma
2004, Cellular Immunology
Citation Excerpt :
IL-12 is an important cytokine for the differentiation of naive T cells to Th1 cells. Administration of IL-12 to mice by inhalation or gene delivery into epithelial cells inhibits Th2 cytokine production, eosinophilic airway inflammation, and airway hyperresponsiveness in the mouse model of asthma [22–26]. In patients with allergic asthma, expression of IL-12 p35 mRNA is decreased in bronchial biopsy specimens [10].
Interleukin (IL)-12 production and IL-12 receptor (IL-12R) β2 chain expression were investigated in patients with allergic asthma successfully treated with rush immunotherapy (RIT) and control patients with mild allergic asthma. Peripheral blood mononuclear cells (PBMCs) were stimulated with Dermatophagoides farinae (Der f), and production of cytokines was measured. Furthermore, the effects of cytokines on IL-12R β2 chain expression on CD4⁺ T cells were investigated. Production by PBMCs of IL-12 and IFN-γ was significantly higher and production of IL-4 was significantly lower after stimulation with Der f allergen in RIT-treated patients than in control patients. Significant increases in the expression of IL-12R β2 chain before and after stimulation of CD4⁺ T cells with IL-12 or IFN-γ were observed in RIT-treated patients compared with that in control patients. Allergen RIT increases IL-12 production and IL-12R β2 chain expression and thus may convert cytokine production from Th2 to Th1 or Th0 type in allergic asthma.

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^☆: Supported by an American Lung Association of Florida Career Development Award to S.S.M. and partly by grants from the VA Merit Review Award and the American Heart Association, Florida, Affiliate Award to S.S.M. Generous support was also given by the Joy McCann Culverhouse endowment to the Division of Allergy and Immunology.

^☆☆: Reprint requests: Shyam S. Mohapatra, PhD, Department of Internal Medicine, Division of Allergy and Immunology—Joy McCann Culverhouse Airway Disease Center, University of South Florida and VA Hospital, 12901 Bruce B Downs Blvd, Tampa, FL 33612.

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Original Articles: Mechanisms of AllergyIFN-γ and IL-12 plasmid DNAs as vaccine adjuvant in a murine model of grass allergy☆,☆☆

Abstract

Section snippets

Animals

Vaccination protocol

Plasmid constructs and expression of IL-12 and IFN-γ

Discussion

Immunol Allergy Clin North Am

Immunol Allergy Clin North Am

J Allergy Clin Immunol

Vaccine

J Allergy Clin Immunol

Immunostimulatory DNA sequences function as Th1 inducing adjuvants

Nat Med

Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs

Proc Natl Acad Sci U S A

CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation

EMBO J

CpG oligodeoxynucleotide vaccination suppresses IgE induction but may fail to downregulate ongoing IgE responses in mice

Int Immunol

Cytokines and costimulatory molecules as genetic adjuvants

Immunol Cell Biol