BACKGROUND:Allergen immunotherapy is effective in allergic individuals however efforts are being made to improve its safety, convenience, and efficacy. It has recently been demonstrated that allergen-linked immunostimulatory DNA (ISS) is effective in stimulating an allergen-specific Th1 response with decreased allergenicity. The objective of this study is to investigate whether ISS linked to purified ragweed allergen Amb-a-1 (AIC) can inhibit local allergen-specific Th2 and induce allergen-specific Th1 responses in explanted nasal mucosa of ragweed-sensitive subjects. In addition, we set out to determine whether AIC is more effective compared to stimulation with unlinked Amb a 1 and ISS. METHODS:Tissue from ragweed-sensitive patients (n = 12) was cultured with whole ragweed allergen (RW), Amb-a-1, AIC, Amb-a-1 and ISS (unlinked), or tetanus toxoid (TT) for 24 hours. IL-4, -5, -13, TNF-alpha and IFN-gamma mRNA-positive cells were visualized by in situ hybridization and T cells, B cells and neutrophils were enumerated using immunocytochemistry. RESULTS:RW or Amb-a-1 increased the number of IL-4, IL-5, and IL-13 mRNA+ cells in the tissue compared to medium alone. AIC had similar cytokine mRNA reactivity as control tissue. AIC and TT increased IFNgamma-mRNA expression. Unlinked Amb-a-1 and ISS showed similar effects to AIC, however this response was weaker. The number of TNF mRNA+ cells, T cells, B cells and neutrophils remained unchanged. CONCLUSIONS:AIC is effective in stimulating a local allergen-specific Th1- and abolishing Th2-cytokine mRNA reactivity in the nose and may be considered as a strong candidate for an improved approach to immunotherapy in ragweed-sensitive individuals.

Background:: Many individuals do not respond to a three-dose series of hepatitis B vaccine (HBV) and most do not achieve a protective antibody response until after dose 2 or 3.Methods:: Healthy, seronegative 18-28 year old adults were randomly assigned in equal numbers to receive two doses of the experimental vaccine (HBV-ISS without alum) (0, 8 weeks) and placebo (24 weeks) or Engerix-B-(R) (0, 8, 24 weeks). Adverse events were collected during the first week and at 4 weeks after each injection. Antibodies were measured 4 weeks after dose 1; before, 1 and 4 weeks after dose 2, and before, 1 and 4 weeks after dose 3 and at 1 year.Results:: Ninety-nine participants were enrolled (65% female; mean age 22.6 years). 79% of HBV-ISS and 12% of Engerix-B-(R) recipients had a protective antibody response 4 weeks post dose 1 (geometric mean concentration [GMC] 23.0 and 1.87 mIU/mL, respectively). By 1 week post dose 2, 100% of HBV-ISS and 18% Engerix-B-(R) recipients had protective levels (GMC 1603 versus 2.40 mIU/mL). Rates of adverse events were low and similar in both groups; headache and fatigue were the most common systemic adverse events in up to 1/3 of both groups. Mild injection-site tenderness was more common after HBV-ISS than Engerix-B-(R) after both doses (74-77% compared to 34-58%; p <= 0.029).Conclusions:: Protective levels are achieved more quickly and after fewer doses of HBV-ISS than Engerix-B-(R). HBV-ISS is well tolerated but associated with more mild injection-site tenderness than Engerix-B-(R). (c) 2005 Elsevier Ltd. All rights reserved.

Background: Immunostimulatory DNA sequences (ISS) are potent immunomodulators that can drive T(H)1 responses to antigens or allergens. This effect can be dramatically enhanced by direct linkage of ISS to the protein.Objective: Evaluate the effects of the number of ISS bound to the major ragweed allergen Amb a 1 on inummogenicity and allergenicity. Methods: Immunogenicity in mice and allergenicity using PBMC or sera from subjects with ragweed allergy were assayed.Results: Both antibody induction in vivo and antibody recognition in vitro were highly sensitive to the number of ISSs linked. IgE recognition of Amb a 1 in competitive ELISA or histamine release assays was inhibited by ISS linkage and showed an inverse relationship to the number of ISSs bound. Type and magnitude of antibody induction in mice was also highly dependent on the number of ISS bound. At the highest ISS to protein ratios, antibody induction was very low. Moderate ISS to protein ratios induced high antibody responses in which IgG(2a) generally predominated. Low ISS to protein ratios produced the highest overall antibody responses in which IgG(1) predominated. In contrast, varied ISS to protein ratios did not affect T-cell responses. In both in vivo mouse studies and in vitro human PBMC studies, all ISS to protein ratios evaluated induced similar responses represented by high levels of IFN-gamma and low levels of T(H)2 cytokines.Conclusion: Controlling the number of ISS bound to a protein allows manipulation of antibody recognition and induction while retaining the potent T(H)1 properties of an ISS-linked protein.Clinical implications: Immunostimulatory DNA sequence-linked Amb a 1 conjugate represents a safe, novel therapeutic approach for treating ragweed allergy.

BACKGROUND:In animal models administration of immunostimulatory DNA sequences preferentially elicits T(H)1-dominated (type 1-dominated) immunity and can inhibit developing or ongoing T(H)2 (type 2) responses.OBJECTIVE:Our objective was to investigate this phenomenon in humans.METHODS:In a randomized, third party-blinded, placebo-controlled, proof-of-concept study conducted entirely in the winter in 19 adults with ragweed allergy, we administered 6 subcutaneous injections of purified Amb a 1 linked to the 22-base-long immunostimulatory phosphorothioate oligodeoxyribonucleotide 1018 (Amb a 1-immunostimulatory DNA sequence conjugate [AIC]). Before the course of AIC or placebo injections and 2 and 16 weeks afterward, we measured recall responses to ragweed, streptokinase, and PHA in short-term primary culture of fresh PBMCs after restimulation with antigen. We quantified regulatory cytokine and chemokine responses characteristic of T(H)2 immunity (IL-5, IL-13, CCL17 [TARC], and CCL22 [MDC]), and T(H)1 immunity (IFN-gamma, CXCL9 [Mig], and CXCL10 [IP-10]), as well as IL-10, a cytokine sometimes linked to regulatory T-cell populations.RESULTS:We demonstrated for the first time that human systemic in vivo ragweed-specific T(H)2 responses were selectively redirected toward T(H)1 responses, with significant increases in IFN-gamma, CXCL9, and CXCL10 and significant decreases in IL-5, CCL17, and CCL22 found at 2 and 16 weeks after the sixth injection. Cytokine and chemokine responses to the unrelated bacterial antigen streptokinase and the global capacity to mount immune responses on polyclonal activation with PHA did not change. No clinically significant systemic or local allergic reactions were associated with AIC or placebo injections.CONCLUSIONS:AIC, injected in concentrations that were approximately 40-fold lower than those used in most murine studies published to date, led to a prolonged shift from T(H)2 immunity toward T(H)1 immunity and appeared to be safe. This novel approach has the potential for immune redirection in human immediate hypersensitivity diseases.

Background: Amb a 1-immunostimulatory phosphorothioate oligonucleotide conjugate (AIC) is a novel immunotherapeutic compound consisting of purified Amb a 1 from short ragweed proteins covalently linked to an immunostimulatory phosphorothioate oligodeoxyribonucleotide. In sensitized animals AIC can stimulate an Amb a 1-specific T(H)1 response and decrease pulmonary reactivity to ragweed challenge. Clinical trials have documented reduced allergic response to AIC in comparison with licensed ragweed extract.Objectives: We sought to determine the in vivo effect,of shortcourse immunotherapy with AIC on eosinophilia and cytokine mRNA expression in the nasal mucosa of ragweed-sensitive patients.Methods: Ragweed-sensitive patients with allergic rhinitis were treated with 6 escalating doses of AIC (0.06-12 mug, n 28) or placebo (n = 29) at weekly intervals immediately before the 2001 ragweed season. Symptom scores and medication use were recorded for the 2001 and 2002 ragweed seasons for all patients. A subset of patients (12 receiving AIC and 7 receiving placebo) consented to have nasal biopsy specimens taken before immunization and before and after the first ragweed season. The preseason and postseason biopsy specimens were taken 24 hours after ragweed allergen challenge and compared with the initial unchallenged biopsy specimen to assess cytokine and inflammatory cell responses by using immunocytochemistry and in situ hybridization.Results: AIC was safe and well tolerated by all patients. There was no difference between the AIC and placebo groups in the number of allergen-induced major basic protein-, IL-4-, IL-5-, or IFN-gamma-positive cells in the mucosa in the first weeksafter AIC immunization. On rechallenge and rebiopsy after the end of the 2001 ragweed season, however, AIC-treated patients had a significantly reduced increase in eosinophils and IL-4 mRNA-positive cells and an increased number of IFN-gamma mRNA-positive cells compared with placebo-treated patients. No difference between treatment groups was observed in symptom scores or medication use during the first ragweed season. During the second ragweed season, however, there was a significant decrease in chest symptoms and a trend toward reduced nasal symptoms in the AIC-treated group.Conclusion: Short-course immunotherapy with AIC can modify the response of nasal mucosa to allergen challenge by increasing T(H)1 cytokine production and decreasing T(H)2 cytokine production and eosinophilia. This modification was not immediate but was observed 4 to 5 months after completion of immunotherapy and seasonal ragweed-pollen exposure. The T-cell subset shift after immunization and seasonal exposure was followed by evidence of clinical efficacy in the second ragweed season without additional AIC immunizations.

Certain oligodeoxynuclotides with CpG motifs provide enhanced immune response to co-delivered antigens. We performed a phase I, observer-blinded, randomized study in healthy anti-hepatitis B surface antigen (anti-HBsAg) antibody negative adults to explore safety and immunogenicity of co-injection of recombinant HBsAg combined with an immunostimulatory DNA sequence (ISS) 1018 ISS. Four ISS dosage groups (N=12 per group) were used: 300, 650, 1000 or 3000μg. For each group, two controls received 20μg HBsAg alone, two controls received ISS alone, and eight subjects received ISS+20μg HBsAg. Subjects received two doses 8 weeks apart. Injection site reactions (tenderness and pain on limb movement) were more frequent at higher ISS+HBsAg doses but were mainly mild and of short duration. Higher anti-HBsAg antibody levels were associated with higher ISS doses. Four weeks after the first dose, a seroprotective titer (≥10mIU/ml) was noted for 0, 25, 75, and 87.5% of subjects by increasing ISS dose group (P<0.05) for those who received ISS+HBsAg; 1 month after the second dose this increased to 62.5, 100, 100, and 100%, respectively. Geometric mean anti-HBsAg antibody levels by increasing ISS+HBsAg dose were 1.22, 5.78, 24.75, and 206.5mIU/ml after the first dose and 65.37, 877.6, 1545, and 3045mIU/ml after the second dose. We conclude that 1018 ISS+HBsAg was well tolerated and immunogenic in this phase I study in healthy adults and may offer the potential for enhancement of hepatitis B virus (HBV) immunization and protection after one or two doses or in individuals who fail to respond to the standard vaccine regimen.

RATIONALE: Seasonal trends in asthma-related hospitalizations are widely recognized, however, little is known about trends in asthma-related intensive care unit (ICU) admissions or intubations.This analysis was performed to examine monthly rates of ICU admissions and/or intubations as a percent of total asthma-related admissions.METHODS: A subset of 215 hospitals from a large database of approximately 250-300 hospitals representing over 3 million annual inpatient visits was used to identify patients admitted to the hospital with a primary diagnosis of asthma (493.xx)during calendar year 2000.RESULTS: A total of 29,430 hospital admissions were identified with a primary diagnosis of asthma.Nearly 11% (n=3,149) were admitted to the ICU and/or intubated, with the majority among patients over 35 years of age (58%).The greatest proportion of total asthma-related hospital admissions occurred September through January (9.6-10.8%),with declining rates in February through May (7.6-8.8%), and the lowest in June through August (4.8-6.3%).ICU admissions and/or intubations remained relatively constant as a percent of total asthma-related hospitalizations, ranging from 9.7-13.5%.Although peak hospitalizations occurred in the winter months, the proportion oflCU admissions and/or intubations were the lowest in December through February (9.7%) and highest during July (13.5%).CONCLUSIONS: While overall asthma-related hospital admissions dip during the summer months, ICU admissions and/or intubations remain constant as a percent of total admissions and increase during the summer.These findings suggest a need for year round vigilance, without summer holidays from medication, in the treatment of patients with persistent asthma.

AIM: The aim of this review was to evaluate the safety and efficacy of bee venom immunotherapy in children at the Women's and Children's Hospital, Adelaide, Australia.METHODS: This was a retrospective review of all children who were enrolled in the immunotherapy program between 1994 and 2000.Only children who had bee sting anaphylaxis with significant respiratory and/or cardiovascular involvement were enrolled.Two protocols were used to achieve a maintenance dose of 100ug of bee venom monthly.Children were either given weekly injections (commencing dose-0.1ug)or were hospitalised for a 5 day rush protocol (commencing dose-0.001ug).Immunotherapy was continued for 36 months.Clinical notes were reviewed and the parents of all children were contacted.RESULTS: 71 children underwent immunotherapy with a median age of 7.5 years (range 4.5-15.9).76% were males, 6% had persistent asthma at the time of the sting, and 15% were admitted to an intensive care unit following the presenting sting.A total of 1,188 injections were administered to achieve the maintenance dose of 100ug.21 children experienced 81 general reactions however adrenaline was only required for 7 reactions (1 per 170 injections).During the maintenance program 18 children experienced 36 reactions but adrenaline was given to only 2 children.55 field stings were experienced by children enrolled in the program with 5 reactions requiting adrenaline.Assuming a 50% recurrence rate of anaphylaxis immunotherapy significantly reduced the expected rate of recurrence (RR 0.62, CI 0.45 to 0.85) as well as the rate of hospitalisation (RR 0.17, CI 0.09-0.32).88% of parents reported that immunotherapy was worthwhile, 89% would recommend immunotherapy to others and 71% reported being less anxious following immunotherapy.Parents were more likely to report this if their child had been stung following immunotherapy.CONCLUSION: Bee venom immunotherapy is safe and effective in children.

ABSTRACT Synthetic oligonucleotides containing CpG motifs in specific sequence contexts have been shown to induce potent immune responses. We have evaluated mucosal administration of two immunostimulatory sequence (ISS)-containing phosphorothioate-stabilized oligonucleotides for antiherpetic efficacy in animal models. The ISS oligonucleotides, suspended in phosphate-buffered saline, were tested in mouse and guinea pig vaginal models of herpes simplex virus type 2 (HSV-2) infection. For comparison, groups of untreated, non-ISS oligonucleotide-treated, and acyclovir-treated animals also were monitored. The results indicated that vaginal epithelial application of ISS (up to 6 h after viral inoculation) with mice lethally challenged with HSV-2 delayed disease onset and reduced the number of animals that developed signs of disease (P = 0.003). ISS application significantly increased survival rates over those of controls (P = 0.0014). The ISS also impacted an established infection in the guinea pig model of HSV-2 disease. A single administration of ISS (21 days after viral inoculation) significantly reduced the frequency and severity of HSV-2 lesions compared to results with non-ISS oligonucleotide-treated and untreated guinea pigs (P < 0.01). HSV-2 is shed from the vaginal cavity of the guinea pig in the absence of lesions, similar to the case with humans. As an additional indication of ISS efficacy, the magnitude of viral shedding also was significantly reduced in ISS-treated animals (P < 0.001). These effects appeared to be immunologically mediated, since ISS had no direct effect on HSV-2 replication in vitro using standard plaque assays. These data suggest that ISS may be useful in the treatment and control of genital herpes in humans.

Background: Recent studies have demonstrated that bacterially derived immunostimulatory sequences (ISSs) of DNA can activate the mammalian innate immune system and promote the development of TH1 cells. Promotion of TH1 immunity by means of immunotherapy in allergic patients has led to the alleviation of symptoms that result from allergen-specific TH2 responses. Objective: Our purpose was to investigate whether the TH1-enhancing properties of ISSs could be used to alter the TH2-dominated immune response of allergic PBMCs in vitro. Methods: Ragweed protein-linked ISS (PLI) was generated from a specific, highly active 22-base ISS and Amb a 1, the immunodominant allergen in ragweed pollen, to combine the TH1-enhancing properties of ISSs with allergen selectivity, and its activity was investigated in PBMC cultures from subjects with ragweed allergy. Results: PLI was markedly successful at reversing the dominant allergen-induced TH2 profile while greatly enhancing IFN-γ production. Delivering ISSs in a linked form proved to be much more effective at modulating the resulting cytokine profile than delivering free ISSs in a mixture with unlinked Amb a 1. PLI also demonstrated cytokine-modulating properties, even when used to stimulate cells that had already been primed for 6 days with Amb a 1. The antigen specificity of the action of PLI was confirmed by the observations that PLI enhances Amb a 1–specific T-cell proliferation. Conclusion: These data indicate that delivery of ISSs within an antigen-specific context exhibits potent cytokine-modulating activity and, combined with its reduced allergenicity, makes this molecule a strong candidate for use in improved immunotherapy applications. (J Allergy Clin Immunol 2001;108:191-7.)

BACKGROUND:Allergen immunotherapy is inconvenient and associated with the risk of anaphylaxis. Efforts to improve the safety of immunotherapy by means of chemical modification of allergens have not been successful because it greatly reduced their antigenicity. Recently, immunostimulatory DNA sequences (ISS or CpG motifs) have been shown to act as strong T(H)1 response-inducing adjuvants.OBJECTIVE:We sought to determine whether conjugation of ISS to the major short ragweed allergen Amb a 1 results in enhanced immunotherapeutic potential in mice and decreased allergenicity in human subjects.METHODS:A 22-mer ISS oligodeoxynucleotide (ISS-ODN) was coupled to Amb a 1 and used for immunization of mice, rabbits, and monkeys.RESULTS:In mice the Amb a 1-ISS conjugate induced a T(H)1 response (IFN-gamma secretion), whereas Amb a 1 induced a T(H)2 response (IL-5 secretion). The T(H)1 response was not observed with an Amb a 1-non-ISS conjugate. Coinjection of Amb a 1 with ISS-ODN was much less effective in inducing a T(H)1 response. In mice primed for a T(H)2 response, injection with Amb a 1-ISS conjugate induced a de novo T(H)1 response and suppressed IgE antibody formation after challenge with Amb a 1. Amb a 1-ISS conjugate induced high-titer anti-Amb a 1 IgG antibodies in rabbits and cynomolgus monkeys, whereas Amb a 1 alone or Amb a 1 coinjected with ISS-ODN did not induce a detectable response. Amb a 1-ISS conjugate was less allergenic than Amb a 1 alone, as shown by a 30-fold lower histamine release from human basophils of patients with ragweed allergy, whereas mixing ISS-ODN with Amb a 1 did not reduce histamine release.CONCLUSION:Amb a 1-ISS conjugate has an enhanced T(H)1-biased immunogenicity and reduced allergenicity. It may offer a more effective and safer approach for allergen immunotherapy than currently available methods.