Cytotoxic T-cells specific for natural IgE peptides downregulate IgE production

Immunoglobulin E (IgE) plays a central role in IgE-mediated immediate type hypersensitivity. Since production of IgE depends on Th2, efforts to block IgE production and control allergic reactions include tolerization of Th2 or deviating development of Th2. We hypothesized that cytotoxic T lymphocytes targeting natural IgE peptides/MHC I complexes can eliminate IgE-producing cells and inhibit centrally IgE production. CTL to self-IgE peptides were elicited in mice immunized with nonameric p109-117, p113-121, and p103-141 (CHepsilon2 domain), which encompass both peptides with an OVA helper peptide (OVAp restricted for H-2d/b) in liposomes and presented by dendritic cells (DC). CTL from BALB/c lysed IgE peptide-pulsed P815 target as well as IgE-producing 26.82 hybridomas (H-2d). Natural tolerance to self-IgE peptides was tested in IgE sufficient (IgE +/+) as well as IgE-deficient (IgE -/-) 129/SvEv mice (H-2b). Comparable magnitude of CTL responses was observed in both strains immunized with p109-117 or p103-141 concomitantly with CD4 T-cell costimulation. CTL from 129/SvEv lysed not only IgE peptide-pulsed EL-4 but also IgE-producing B4 hybridomas (H-2b). This observation strongly suggests a correspondence of epitope of immunogenic peptide to that of physiologically processed IgE peptides presented on IgE-producing cells. Moreover, CTL were generated in 129/SvEv, immunized with the recombinant antigenized antibody in liposomes encompassing p107-123, p109-117, and p113-121 expressed in CDR3 of VH62/human gamma1. Polyclonal IgE production was inhibited by coincubation with MHC I-restricted CTL in vitro. Furthermore, antigen-specific IgE responses were inhibited in mice, immunized with p109-117 and p103-141 while IgG responses were not suppressed. Since IgE peptide sequences of CHepsilon2 are ubiquitous to all murine IgE heavy chain, peptides made as such can serve as a universal IgE vaccine to prevent allergy for a myriad of allergens in rodents. This observation suggests that similar human IgE peptides should be identified and employed to downregulate human IgE production.     

Effect of vaccination against pneumococcal infection and influenza in children with asthma

Assessment of clinical course of asthma and IgG response in children with asthma immunized with pneumococcal polysaccharide vaccine (Pneumo 23) and influenza vaccine (Vaxigrip). 78 children aged 4 - 17 years old were allocated to two groups. Children from the 1st group were immunized against pneumococcal infection and influenza; children from 2nd group were immunized against pneumococcal infection only. Rate of asthma exacerbations in the 1st group of children decreased by 1.7 times compared with the period before vaccination, whereas the same rate in the 2nd group of children decreased by 1.5 times. It was accompanied by the increase of IgG level to antigens of pneumococcalvaccine in blood, which was observed in both groups. Vaccination did not result in increase of IgE levels. Immunization of children with asthma against pneumococcal infection with polysaccharide vaccine or combined immunization against pneumococcal infection and influenza reduced rate of asthma exacerbations and led to formation of immunity to vaccine strains of Streptococcus pneumoniae. Vaccination did not lead to sensitization of children.     

Immunization with cathepsin L proteinases CL1 and CL2 secreted by Fasciola hepatica elicit a preferential type 1 response based on IgG2a antibodies in rats

Cathepsin L proteinases (CL1 and CL2), the major components of Fasciola hepatica excretion/secretion products (E/S) are considered potential antigens of a vaccine against fascioliasis. The humoral response elicited by CL1 and CL2 in rats either immunized with the enzymes or infected with F. hepatica has been analysed, examining specific IgE and IgG subclass dynamics. The experiment was continued for 10 weeks and peripheral blood eosinophilia was also determined. Infected rats presented peaks of eosinophilia at weeks 3 and 7 post-infection, while those immunized with CL1 and CL2 were no different from controls. Total IgE in infected rats increased up to week 5, reaching 30 microg(-1) in some cases, then decreased slowly and rising again towards the end of the experiment. Determination of specific IgE, carried out in sera previously absorbed with Protein G-Sepharose, reached a peak in infected rats between weeks 2 and 5, depending on the individual. In immunized rats both total and specific IgE levels remained around the pre-immunization values. With regard to the IgG subclass responses to E/S products, in infected rats IgG1 predominated over IgG2a, and the reverse was true in rats immunized with CL1 and CL2 and tested against the respective antigens. In all cases an increase in IgG1 and IgG2a antibody titres was seen, with maximum levels being reached later (weeks 6-7) in infected rats than in immunized ones (weeks 4-5). No IgG2b or IgG2c responses were detected in any of the groups studied.     

The reactogenicity and immunological efficacy of a Klebsiella vaccine in donors

Klebsiella vaccine, when injected subcutaneously to donors, proved to be faintly reactogenic and safe. The injection of the vaccine had no effect on changes in the morphological composition of peripheral blood and on liver function. In persons with the initially low content of IgG an increase in this characteristic was observed after immunization. No changes in the synthesis of IgE occurred in healthy donors under the influence of immunization. The vaccine was shown to be immunogenic when introduced according to immunization schedules comprising 3 and 5 injections, the titer of Klebsiella antibodies increasing 3- to 5-fold.     

冻干甲型肝炎-腮腺炎联合疫苗猴体安全性及免疫原性研究试验

为了观察冻干甲型肝炎-腮腺炎联合疫苗免疫恒河猴后的安全性及免疫原性,用静脉注射和丘脑注射的方式接种疫苗,观察恒河猴的临床症状、体征、生化、免疫学反应以及脑和肝组织的病理变化。结果未见临床症状和体征的异常改变,ALT正常,抗-HAV、抗流行性腮腺炎病毒的抗体在观察期内持续阳性,脑组织和肝组织无病毒性肝炎和腮腺炎病毒引起的病理性改变。因此该冻干甲型肝炎堋;腺炎联合疫苗抗原问无干扰,具有良好的安全性及免疫原性。     

Prevention of allergic asthma by vaccination with transgenic rice seed expressing mite allergen: induction of allergen-specific oral tolerance without bystander suppression

This study tested the feasibility of oral immunotherapy for bronchial asthma using a newly developed subunit vaccine in which a fragment (p45-145) of mite allergen (Der p 1) containing immunodominant human and mouse T cell epitopes was encapsulated in endoplasmic reticulum-derived protein bodies of transgenic (Tg) rice seed. Allergen-specific serum immunoglobulin responses, T cell proliferation, Th1/Th2 cytokine production, airway inflammatory cell infiltration, bronchial hyper-responsiveness (BHR) and lung histology were investigated in allergen-immunized and -challenged mice. Prophylactic oral vaccination with the Tg rice seeds clearly reduced the serum levels of allergen-specific IgE and IgG. Allergen-induced CD4(+) T cell proliferation and production of Th2 cytokines in vitro, infiltration of eosinophils, neutrophils and mononuclear cells into the airways and BHR were also inhibited by oral vaccination. The effects of the vaccine were antigen-specific immune response because the levels of specific IgE and IgG in mice immunized with Der f 2 or ovalbumin were not significantly suppressed by oral vaccination with the Der p 1 expressing Tg rice. Thus, the vaccine does not induce nonspecific bystander suppression, which has been a problem with many oral tolerance regimens. These results suggest that our novel vaccine strategy is a promising approach for allergen-specific oral immunotherapy against allergic diseases including bronchial asthma.     

Formulation with CpG oligodeoxynucleotides prevents induction of pulmonary immunopathology following priming with formalin-inactivated or commercial killed bovine respiratory syncytial virus vaccine

Commercial killed bovine respiratory syncytial virus (K-BRSV) and formalin-inactivated BRSV (FI-BRSV) tend to induce Th2-type immune responses, which may not be protective and may even be detrimental during subsequent exposure to the virus. In this study we assessed the ability of CpG oligodeoxynucleotides (ODNs) to aid in the generation of effective and protective BRSV-specific immune responses. Mice were immunized subcutaneously with FI-BRSV formulated with CpG ODN, Emulsigen (Em), CpG ODN and Em, or non-CpG ODN and Em. Two additional groups were immunized with K-BRSV or K-BRSV and CpG ODN. After two vaccinations, the mice were challenged with BRSV. FI-BRSV induced Th2-biased immune responses characterized by production of serum immunoglobulin G1 (IgG1) and IgE, as well as interleukin-4 (IL-4), by in vitro-restimulated splenocytes. Formulation of FI-BRSV with CpG ODN, but not with non-CpG ODN, enhanced serum IgG2a and IFN-gamma production by splenocytes, whereas serum IgE was reduced. Although the immune response induced by K-BRSV was not as strongly Th2 biased, the addition of CpG ODN to this commercial vaccine also resulted in a more Th1-type response. Furthermore, the addition of CpG ODN to the BRSV vaccine formulations resulted in enhanced neutralizing antibody responses. Significant production of IL-5, eotaxin, and eosinophilia was observed in the lungs of FI-BRSV- and K-BRSV-immunized mice. However, IL-5 and eotaxin levels, as well as the number of eosinophils, were decreased in the mice vaccinated with the CpG ODN-formulated vaccines. Finally, when formulated with CpG ODN, both FI-BRSV and K-BRSV significantly reduced virus production after challenge with BRSV.     

Measurement of IgG blocking antibodies by interference in the radioallergosorbent test

We previously found that sera of patients immunized with ragweed pollen extract contained a factor that interfered with the binding of IgE antibodies to solid-phase allergens in the radioallergosorbent test (RAST). We now describe an assay, RAST interference, to measure this factor, and we present evidence that the factor is IgG blocking antibody. Sera from immunized allergic patients were heated at 56 degrees C for 4 hr to destroy heat-labile Fc determinants on IgE and were tested for their ability to prevent binding of additional IgE antibody to solid-phase allergens in the RAST. Eight of 10 sera from allergic immunized patients gave RAST interference dose-response curves that did not differ from the arbitrary standard. The factor causing interference showed specificity for the immunizing antigen, was heat-stable, eluted from Sephadex G-200 in the 7S peak, was present only in sera of immunized patients, and rose after initiation of immunization. These results indicated that RAST interference can be used to measure IgG blocking antibodies with the same reagents employed for the measurement of IgE antibodies, provided the antiserum to IgE is specific for the heat-labile FC determinants on IgE.     

Antigenicity and immunogenicity of a multiple peptidic construction of the Schistosoma mansoni Sm-28 GST antigen in rat, mouse, and monkey. 1. Partial protection of Fischer rat after active immunization

Among the schistosome proteins characterized as vaccine candidates, an Ag of 28 kDa (Sm-28-GST) has received considerable attention. It was shown to be antigenic in humans and protective in mice, rats, hamsters, and baboons. Synthetic peptides derived from its sequence have been used to characterize the immune response to the molecule and one of these, comprising aminoacids 115-131 has been shown to incorporate both T and B cell recognition sites in a variety of experimental models. An octameric ("octopus") construction of the 115-131 peptide has been synthesized and its antigenicity and immunogenicity have been examined. The octopus construct is immunogenic in rats, mice and baboons in the presence of CFA (for rodents) and Bacille-Calmette-Guérin vaccine (for primates) as adjuvants. This clearly indicates that the construction allowed the conservation of the immune sites of the cognate protein. Moreover, anti-octopus sera from immunized Fischer rats were able to mediate platelet-, macrophage-, and eosinophil-dependent cytotoxicity toward schistosomula. Rats immunized with the 115-131 octopus were partially protected against a challenge infection with Schistosoma mansoni cercariae and this was paralleled by an increased level of IgG and more importantly, of IgE Sm-28-GST-specific antibodies.     

Suppression of IgE antibody production in SJL mice. V. Effect of irradiation and adult thymectomy on the suppression of IgE antibody production in SJL mice

Anti-DNP IgE antibody production was low and transient in SJL mice which were immunized with 1 microgram DNP-Nb and 1 mg A1(OH)3. The immunized SJL mice were irradiated (60-540 R) 1 day after challenge. A dose higher than 180 R induced enhancement of anti-DNP IgE antibody production as compared to nonirradiated control mice, suggesting the existence of irradiation-sensitive suppressor cells. Anti-DNP IgE antibody production was suppressed when immunized and irradiated SJL mice were injected with spleen cells from adult-thymectomized SJL mice. The donors of the spleen cells were thymectomized 2 or 4 months previously, and this suggests that the suppressor cells from unprimed mice are long-living T cells.     

鸭源致病性大肠杆菌Ⅰ型菌毛pilA基因的原核表达及重组蛋白对强毒攻击的免疫保护作用

根据GenBank中人源大肠杆菌pilA基因序列,用OLIGO6.0设计PCR引物,从鸭源致病性大肠杆菌GH1.2中扩增到pilA基因并将其克隆至pMD18-T载体,经PCR、酶切和DNA测序鉴定后,将鸭源致病性大肠杆菌pilA基因正向插入原核表达载体pET-32a( )的BamHⅠ和HindⅢ位点间,成功构建了重组表达质粒pET-32a-pilA。重组表达质粒pET-32a-pilA转化表达宿主菌BL21(DE3),用IPTG诱导,表达出了大小约为36kD的pilA重组蛋白。表达产物用镍柱亲和层析纯化,与等量弗氏佐剂混合制备pilA重组蛋白疫苗,分别在1日龄、8日龄时两次对雏鸭进行免疫,二免后2周测定鸭血清中的ELISA抗体效价,并以109PFU同源菌株GH1.2攻毒,根据攻毒后鸭的死亡率、E.coli分离率和各组织器官的病变等级来判定pilA重组蛋白的免疫保护效果。结果pilA重组蛋白免疫鸭的血清中ELISA抗体效价为1∶12800,全菌灭活苗免疫组的血清ELISA抗体效价为1∶200;同源菌株攻毒后,pilA重组蛋白免疫保护组鸭的死亡率、E.coli分离率和各组织器官的病变程度均比攻毒对照组下降且差异显著或极显著,与全菌灭活苗免组比较差异不显著。表明pilA重组蛋白对同源菌株GH1.2的感染具有一定的保护效果。     

Relationships between cell-mediated immunity and the IgE antibody response: II. Delayed hypersensitivity and antibody production to DNP-Ascaris conjugates

Rats of the W/F strain were immunized with DNP-Ascaris conjugates using complete Freund's adjuvant (CFA), Al(OH)₃ gel (alum), or B. pertussis vaccine as adjuvants. Cell-mediated immunity was assessed by lymphotoxin in vitro and by delayed hypersensitivity in vivo. IgE and IgG antibody determinations were made on serum pools obtained at various times during the primary and secondary responses. Although delayed hypersensitivity appeared earlier than lymphotoxin, these two parameters correlated during the primary but not during the secondary response. The discrepancies suggested that different cells may be responsible for these two phenomena. Antibody production was influenced by the adjuvant used. CFA led to IgG antibody responses to both hapten and carrier but not to IgE antibody production. The use of B. pertussis resulted in both IgE and IgG antibody production. In the case of alum, anti-hapten antibodies appeared during the primary response while anti-carrier antibodies of both IgE and IgG classes were detected after booster. The results indicated that cell-mediated immunity, IgE, and IgG antibodies appeared independently in an ordered, temporal sequence, and that these responses were not mutually exclusive but were under strong modulatory influences of the various adjuvants used.     

T cell-independent regulation of IgE antibody production induced by surface-linked liposomal antigen

Control of IgE Ab production is important for the prevention of IgE-related diseases. However, in contrast to the existing information on the induction of IgE production, little is known about the regulation of the production of this isotype, with the exception of the well-documented mechanism involving T cell subsets and their cytokine products. In this study, we demonstrate an alternative approach to interfere with the production of IgE, independent of the activity of T cells, which was discovered during the course of an investigation intended to clarify the mechanism of IgE-selective unresponsiveness induced by surface-coupled liposomal Ags. Immunization of mice with OVA-liposome conjugates induced IgE-selective unresponsiveness without apparent Th1 polarization. Neither IL-12, IL-10, nor CD8(+) T cells participated in the regulation. Furthermore, CD4(+) T cells of mice immunized with OVA-liposome were capable of inducing Ag-specific IgE synthesis in athymic nude mice immunized with alum-adsorbed OVA. In contrast, immunization of the recipient mice with OVA-liposome did not induce anti-OVA IgE production, even when CD4(+) T cells of mice immunized with alum-adsorbed OVA were transferred. In the secondary immune response, OVA-liposome enhanced anti-OVA IgG Ab production, but it did not enhance ongoing IgE production, suggesting that the IgE-selective unresponsiveness induced by the liposomal Ag involved direct effects on IgE, but not IgG switching in vivo. These results suggest the existence of an alternative mechanism not involving T cells in the regulation of IgE synthesis.     

Opsonizing activity of sera from animals immunized with pertussis vaccine

Opsonizing activity of guinea pig blood serum containing mercaptoethanol-resistant pertussis antibodies was studied in vitro on a model of microorganism ingestion by the mononuclears of the guinea pig peritoneal exudate. There were revealed distinct differences in the serum activity depending on the phagocytosis object. The blood serum of hyperimmunized rabbits stimulated the ingestion of Bordetella pertussis by mononuclears of guinea pigs--normal and immunized with pertussis vaccine. The blood sera of hyperimmunized guinea pigs and of mice immunized with pertussis vaccine twice displayed opsonins to B. pertussis. The blood sera of animals immunized with pertussis vaccine inhibited the staphylococcus ingestion by the peritoneal exudate mononuclears of guinea pigs, both normal and those immunized with pertussis vaccine.     

Delayed-type hypersensitivity and in vitro lymphocyte response in guinea pigs immunized with a live varicella vaccine

The relation of delayed type hypersensitivity (DTH) assessed by the Varicella-Zoster virus (VZV) skin test and lymphocyte transformation (LTF) with VZV antigen was investigated in guinea pigs immunized with live varicella vaccine virus, or heat-inactivated vaccine virus. Guinea pigs immunized with live varicella vaccine virus showed positive DTH and LTF responses to viral antigen as well as a neutralizing (NT) antibody response, while those immunized with heat-inactivated vaccine virus showed only an NT antibody response of the same degree as that to live vaccine virus. These results show the reliability of the skin test in assessing cell-mediated immunity (CMI) to VZV and the advantage of the live varicella vaccine over the inactivated one in immunizing guinea pigs.     

Effect of myeloma IgE injections on passive and active cutaneous anaphylaxis in rats

The ability of injected rat IgE myeloma protein IR162 to inhibit passive and active cutaneous anaphylaxis in Lewis rats was investigated. IgE injected i.p. 24 hr before the sensitization with IgE anti-ovalbumin (OVA) completely inhibited both IgE- and IgG2a-induced passive cutaneous anaphylactic (PCA) reactions at a dose (2.5 mg/100 g body weight) that resulted in peak serum concentrations of 150 micrograms IgE IR162/ml. Peak IgE IR162 serum concentrations of 20 to 60 micrograms/ml inhibited the PCA reaction in approximately 50% of the rats. Intracutaneous injection of a mixture of myeloma IgE and anti-OVA IgE in a ratio of 100:1 or more also inhibited the PCA reaction. In contrast, the PCA reaction was not inhibited by seven daily doses of IgE beginning 24 hr after passive sensitization. Likewise, the cutaneous anaphylactic reaction elicited in rats 14 days after immunization with OVA and Bordetella pertussis was not prevented by daily injections of myeloma IgE despite a 1000- to 3000-fold excess of the myeloma IgE to anti-OVA IgE serum concentration. The data demonstrate that parenteral administration of myeloma IgE inhibits the PCA reaction only when given before passive sensitization and does not prevent cutaneous anaphylaxis in actively immunized rats. Because myeloma IgE failed to inhibit anaphylactic reactions in actively immunized rats, it is questionable whether administering human IgE-derived synthetic peptides or recombinant DNA-produced IgE fragments will be able to prevent allergic diseases by blocking the IgE Fc receptors on mast cells.     

Evaluation of the reactogenicity and immunogenic potency of combined vaccine "Bubo-Kok" in the immunization of children against diphtheria, tetanus, pertussis and viral hepatitis B

Combined vaccine "Bubo-Kok" is characterized by safety and high immunological activity. The number of postvaccinal reactions in children aged 1 and 2 years, immunized with vaccine "Bubo-Kok", was not statistically different from those in groups of children immunized with adsorbed DPT vaccine, as well with such vaccine in combination with vaccine against hepatitis B. After the completion of the primary course of immunization 100% of children had protective antibody titers against diphtheria, tetanus and hepatitis B. Antibody titers against pertussis, equal to or exceeding protective titers, were found in more than 70% of immunized children. The immunogenic potency of vaccine "Bubo-Kok" with respect to all its components was not inferior to that of adsorbed DPT vaccine and vaccine against hepatitis B, when introduced simultaneously in different areas of the body. Vaccine "Bubo-Kok" successfully passed state trials and was recommended for registration.     

Trypanosoma cruzi: homocytotrophic antibody response in mice immunized with unrelated antigens.

The homocytotropic antibody response to unrelated antigens of mice with acute or chronic infection with Trypanosoma cruzi was studied. The production of IgG1 and IgE antibodies was observed in animals immunized with ovalbumin. The levels of IgG1 and IgE antibody were determined by passive cutaneous anaphylaxis. There was a depression in both IgG1 and IgE when infection and immunization were simultaneous. This depression was more intense when the animals were immunized 3 days after infection. A depression of IgG1 but not of IgE was observed in the animals with chronic infection and in the animals infected during the course of the humoral antibody response (12 days after immunization). It is suggested that a loss of T-cell regulatory mechanism may explain these results.     

Aluminum hydroxide associated to Schistosoma mansoni 22.6 kDa protein abrogates partial protection against experimental infection but not alter interleukin-10 production

The need to develop a vaccine against schistosomiasis led several researches and our group to investigate proteins from Schistosoma mansoni as vaccine candidates. Sm22.6 is a protein from S. mansoni that shows high identity with Sj22.6 and Sh22.6 (79 and 91%, respectively). These proteins are associated with high levels of IgE and protection to reinfection. Previously, we have shown that Sm22.6 induced a partial protection of 34.5% when used together with Freund's adjuvant and produced a Th0 type of immune response with interferon-g and interleukin-4. In this work, mice were immunized with Sm22.6 alone or with aluminum hydroxide adjuvant and high levels of IgG, IgG1, and IgG2a were measured. Unfortunately, no protection was detected. Since IL-10 is a modulating cytokine in schistosomiasis, we also observed a high level of this molecule in splenocytes of vaccinated mice. In conclusion, we did not observe the adjuvant effect of aluminum hydroxide associated with rSm22.6 in protective immunity.     

Surface-linked liposomal antigen induces ige-selective unresponsiveness regardless of the lipid components of liposomes

We have previously reported that antigen coupled with liposomes induced antigen-specific and IgE-selective unresponsiveness in mice. This antigen preparation was investigated for application in a novel vaccine protocol to induce minimal IgE synthesis. In this study, ovalbumin (OVA)-liposome conjugates were made using liposomes of four different lipid components, including unsaturated carrier lipid and three different saturated carrier lipids, after which the induction of anti-OVA antibody production was investigated in mice. All of the OVA-liposome conjugates induced IgE-selective unresponsiveness. The membrane fluidity of liposomes, as measured by detecting changes in the fluorescence polarization of a 1,6-diphenyl-1,3,5-hexatriene (DPH) probe located in the bilayers, was significantly higher in liposomes consisting of unsaturated carrier lipids than those of the other liposomes consisting of saturated carrier lipids. The highest titer of anti-OVA IgG was observed in mice immunized with OVA-liposomes made using liposomes consisting of unsaturated carrier lipids. In addition, among these OVA-liposomes, the one possessing the longest carbon chain induced the lowest IgG antibody production. These results suggest that the membrane fluidity of liposomes might affect the adjuvant effect of liposomes but not the induction of IgE-selective unresponsiveness in immunizations with surface-linked liposomal antigens.