Basal serum IgE levels and HLA antigen frequencies in allergic subjects

We have studied the genetics of human immune response utilizing IgE-mediated allergic response as a model system. Tests performed on unrelated allergic Caucasian subjects revealed a weak association between responsiveness to ragweed pollen allergen Ra3 and HLA-A2 (p=0.04). Most importantly, we observed a relationship between the frequency of HLA-A2 and total serum IgE level in people allergic to Ra3. This frequency was significantly higher in atypical Ra3-sensitive people with low IgE levels of 16–127 U/ml (where over 90 percent possessed A2) than in those with more typical high IgE levels. We postulate that a majorIr-Ra3+ allele of anIr-Ra3 locus is associated with HLA-A2. Given the natural limiting conditions of exposure to Ra3, we suggest that only thisIr-Ra3+ allele will permit IgE antibody synthesis against Ra3 in allergic subjects with limiting low IgE levels. However, the expression of minorIr-Ra3+ alleles (of the same or differentIr loci) not associated with A2 may be apparent in Ra3 responders having high IgE levels.Preliminary findings were presented at the Federation Meetings, Atlantic City, 1975 and at the Birth Defects Conference, Kansas City, 1975.     

Immune responsiveness to Ambrosia artemishfolia (short ragweed) pollen allergen Amb a VI (Ra6) is associated with HLA-DR5 in allergic humans

The relationship between HLA type and specific immune responsiveness toward ultrapure Ambrosia artemisiifolia (short ragweed) pollen allergen Amb a VI (Ra6) was explored in a genetic-epidemiologic study of groups of 116 and 81 Caucasoid subjects who were skin-test \ positive (ST^–) toward common environmental allergens. Specific immune responsiveness to Amb a VI was assessed by measuring serum IgE and IgG antibodies (Abs) by double Ab radioimmunoassay in both ST^– groups. Significant associations were found between IgE Ab responsiveness to Amb a VI and the possession of HLA-DR5; P values for the two groups were, respectively, 7 × 10^–7 and 1 × 10^–3 by nonparametric analyses, and 4 × 10^–11 and 5 × 10^–8 by parametric analyses. The levels of significance for the associations between HLA-DR5 and IgG Ab responsiveness were highly dependent on the extent of ragweed immunotherapy (Rx) within the patient group; by parametric statistics, the associations were 10^–11 for the group that had received relatively little Rx and 2 × 10^–3 for the group that had received more intensive Rx. These results provide further striking evidence for the existence of specific HLA-linked human Ir genes involved in responsiveness toward inhaled allergens and illustrate the usefulness of the allergy model in studies of the genetic basis of human immune responsiveness. Extension of these studies to investigation of structure-function relationships involved in antigen recognition by Ia molecules and the T-cell receptor will lead to a better understanding of human susceptibility toward immunologic diseases.Abbreviations used in this paper Ab antibody - Amb a VI Amb a V, new IUIS nomenclature for Ambrosia artemisiifolia pollen allergens nos. 6 and 5 (short ragweed Ra6 and Ra5) (Marsh et al. 1986b) - Lol p II, III new IUIS nomenclature for Lolium perenne pollen allergens II and III (perennial rye grass, Rye II and Rye III) (Marsh et al. 1986b) - BBS borate-buffered physiologic saline - BSA bovine serum albumin - DARIA double-antibody radioimunoassay - Ia immune-associated - PAGE polyacrylamide gel electrophoresis - RIST radioimmunosorbent test - Rx immunotherapy - SDS sodium dodecyl sulfate - ST skin test     

Human allospecific TLCs generated against HLA antigens associated with DRl through DRw8

We have studied the complexity and fine specificity of the HLA-D region using a panel of T lymphocyte clones generated against alloantigens associated with HLA-DR1 through DRw8. After extensive testing in population studies, 89 clones were tested in proliferation assays with 14 families. Segregation patterns were analyzed for haplotype associations by calculating sequential lod scores to test the likelihood that genes encoding epitopes detected by TLCs were linked to HLA genes. Four general categories were identified: (1) clonal responses that segregated with the same HLA-D region haplotype in all informative pedigrees; (2) clonal responses that segregated with HLA in all pedigrees but not always with the same haplotype; (3) clonal responses that segregated with HLA in some families but failed to segregate in others or produced equivocal results; (4) clonal responses that did not segregate with HLA haplotypes.Abbreviations used in this paper cpm counts per minute - DNV double normalized value - EBV Epstein-Barr virus - FCS fetal calf serum - HLA human MHC - HTC homozygous typing cell - LCL lymphoblastoid cell line - MHC major histocompatibility complex - MLC mixed lymphocyte culture - mAb monoclonal antibody - PBL peripheral blood lymphocyte - PLT primed lymphocyte typing - T-max maximized T test analysis - TCGF T-cell growth factor - TLC T-lymphocyte clone     

Genetic and immunological characterization of naturally occurring recombinant B3 rats

The B-stock population of rats was bred for homozygosity at the loci controlling coat color. In this process, theAg-B1 andAg-B3 haplotypes became fixed in Hardy-Weinberg equilibrium. Extensive immunization and absorption studies showed that the specificities in the B-stock rats homozygous for theAg-B1 haplotype were the same as those found in the inbred F344 strain (Ag-B1), and that the specificities in the rats homozygous for theAg-B3 haplotype were the same as those found in the inbred BN (Ag-B3) strain. A homozygous line derived from the rats carrying theAg-B3 haplotype (B3) has the mixed lymphocyte reactivity and antibody responsiveness to poly (Glu⁵²Lys³³Tyr¹⁵) characteristic of the inbred strains in theAg-B4 group. Thus, it represents a naturally occurring recombination between the loci controlling MLR and immune responsiveness, on the one hand, and those controlling the Ag-B antigens on the other. Antibody responsiveness segregated with theAg-B3 haplotype in crosses between the B3 homozygotes and the low responder BUF and M520 strains; hence, this recombination is a stable one. There was no linkage of antibody formation or haplotype to coat color. The finding of a strain with a naturally occurring recombination in the major histocompatibility complex between the loci controlling mixed lymphocyte reactivity and the Ag-B histocompatibility antigens provides evidence for the separateness of these loci. Since the portion of the genetically determined mechanism controlling antibody responsiveness which is linked to the MHC was that characteristic of the MLR type, it too must lie outside the region defined by the serological specificities of theAg-B haplotype.     

HLA-D gene studies in relation to immune responsiveness to a grass allergen Lol p III

The grass pollen allergen Lol p III (M _r11 000) is a well-characterized antigen that has been found useful in immunogenetic studies of human immune responsiveness. Since immune responsiveness to this allergen is associated with HLA-DR3, we investigated whether there was any sequence in the HLA-D region that would render a person susceptible [antibody (Ab)-positive] to the allergen. By sequence-specific oligonucleotide (SSO) slot-blot and sequence analyses of polymerase chain reaction (PCR)-amplified genomic DNA from Lol p III responder and nonresponder subjects, Ab responsiveness was found to be strongly associated with the sequence Glu-Tyr-Ser-Thr-Ser (EYSTS), present in the first polymorphic regions of DRI polypeptide chains of DR3, DR11 (split of DR5), and DRw6. Of the 41 grass-allergic subjects investigated, 19 had the EYSTS sequence, of whom 18 (95%) were Lol p III immunoglobulin G (IgG) Ab responder; among the 22 EYSTS^- subjects, ten were Lol p III responders (P=0.0001, relative risk=21.6). No such association was found with any polymorphic sequences in othe DR chains,or in DQI and DQI chains. These findings suggest that the EYSTS sequence is important in the presentation of an epitope of Lol p III; other sequence(s) may be involved in the presentation of othe epitope(s). To our knowledge, this is the first demonstration of a strong association between a specific HLA sequence and immune responsiveness to a well-defined antigen. The paper shows that presence of the EYSTS sequence classifies subjects as Lol p III responders in 18/19 cases.     

Regulation of IgE activity in inhalational tolerance via formation of IgG anti-IgE/IgE immune complexes

Background

Allergic asthma is an inflammatory disorder of the airways that results from inappropriate production of IgE against harmless, environmental antigens. Sequestration of free IgE using humanized IgG anti-IgE is an effective therapy for asthma and other atopic disorders. However, the status of free IgE in subjects who have naturally developed immune tolerance to inhaled antigens has not been well studied.

Methods

C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) for 7 days to induce allergic airway disease (AAD) or 6 weeks to induce a state of local inhalational tolerance (LIT). Serum from AAD or LIT mice, diluted to achieve equivalent levels of total OVA-specific IgE, was used to sensitize rat basophil leukemia cells for allergen-mediated degranulation. Levels of degranulation were measured in relation to serum concentrations of free IgE and IgG anti-IgE/IgE immune complexes.

Results

Serum from AAD animals induced a greater degree of basophil degranulation than serum from LIT animals. These results correlated with higher levels of free IgE in AAD animals, whereas LIT mice demonstrated a significant increase in IgG anti-IgE/IgE immune complexes relative to their diseased counterparts.

Conclusions

Sequestration of free IgE by naturally occurring IgG anti-IgE may aid in the development of immune tolerance against inhaled allergens. The decrease in bioavailability of free IgE may, in turn, contribute to the overall reduction of asthma symptoms via a mechanism that mimics the therapeutic effects of humanized IgG anti-IgE.

     

The roles of interleukin-1 and interleukin-1 receptor antagonist in antigen-specific immune responses

Despite evidence that interleukin (IL)-1 promotes the proliferation of some T helper 2 (Th2) cell clones in vitro, the physiological role of IL-1 in the regulation of antigen-specific immune responses remains undefined. Using a liposome-DNA delivery system, we transiently expressed IL-1 receptor antagonist (IL-1Ra) to suppress IL-1 functions at the site of the antigen-specific primary immune response. Our data indicate, for the first time, that IL-1Ra downregulates antigen-specific IL-4 and IgE responses, with concomitant enhancement of interferon- and IgG2a responses in vivo. In addition, IL-1 can promote Th2 development in an IL-4-independent manner in vitro. Thus, the balance between endogenous IL-1 and IL-1Ra during the primary immune response can be an important factor in determining the antigen-specific effector function of T cells.     

IgE and IgG are synergistic in antigen-mediated release of thromboxane from human lung macrophages.

The mechanism of IgE-mediated release of thromboxane A2 from human lung macrophages has been studied using a monoclonal chimeric human/mouse IgE antibody and its specific antigen. The cells could be sensitized at 37 degrees C but not at 4 degrees C by incubation with IgE, and released a significant amount of thromboxane A2 (TXA2), measured as the stable hydrolysis product TXB2, in response to an anti-chimeric IgE antibody. In contrast, stimulation of IgE-sensitized macrophages with the specific antigen produced less than 10% of this response. A similar time course for the release of TXB2 and the formation of inositol monophosphate in the presence of LiCl was observed. Cleavage of the Fc domain of the anti-chimeric IgE antibody substantially eliminated its capacity to stimulate IgE-sensitized cells. However, the weak or undetectable response to chimeric IgE plus specific antigen was substantially potentiated by an antigen-specific chimeric IgG antibody. IgG-sensitized macrophages did not respond to antigen challenge by the release of TXB2. Preincubation of the cells with a monoclonal antibody against the low affinity receptor for IgE (Fc epsilon RII/CD23) did not prevent IgE sensitization. We conclude that cell-bound IgE antibody cannot induce the release of TXB2 but has fixed antigen which then must interact with specific IgG antibody and IgG receptors to induce mediator release.     

The transition from specific to nonspecific desensitization in human basophils

Human basophils can be desensitized to IgE-mediated stimuli either specifically (to the desensitizing antigen only) or nonspecifically (to all antigens). It has been suggested that the specificity of desensitization depends on the number of membrane-bound, antigen-specific IgE antibody molecules per basophil. We have varied the number of IgE antibody molecules/basophil by passive sensitization of mixed leukocyte preparations with increasing concentrations of purified IgE anti-penicillin (BPO) antibody. The cells were then desensitized with penicillin-human serum albumin (BPO-HSA). Desensitization was specific (lack of response to BPO-HSA only) with 1000 specific antibody molecules/basophil, and increasingly nonspecific (greater than 70% desensitization to rechallenge with anti-IgE and ragweed antigen E as well as lack of response to BPO-HSA) as the number of antibody molecules was increased to 14,000. This formally established that the number of specific IgE antibody molecules/basophil determines the mode of desensitization.     

A differential effect of IgM and IgG antibodies on the blastogenic response of lymphocytes to rubella virus

Peripheral blood lymphocytes of rabbits immunized with live rubella vaccine respond to rubella virus antigens in tissue culture with increased DNA synthesis as measured by incorporation of ³H-thymidine. This reaction can be inhibited by rubella antibody. A dose dependent effect was observed when antibodies in whole serum were mixed with virus prior to addition to lymphocyte cultures. When antisera were fractionated and their individual immunoglobulins tested, a paradoxical effect was obtained. Immune IgG although it was highly effective in neutralizing the virus was incapable of inhibiting the lymphocyte response and at times caused an increased response. In contrast, immune IgM which was less efficient in neutralizing virus caused significant suppression of the blastogenic reaction. By themselves these results might have signified that IgG and IgM antibodies have different specificities or different binding properties with respect to viral surface antigens. However, immune complexes consisting of virus and IgM reduced response of both rubella immune and normal rabbit lymphocytes to PHA. This nonspecific inhibitory action required a specific step of antigen and IgM antibody interaction and normal IgM-virus mixtures or mixtures of anti-rubella IgM and poliovirus or influenza virus did not suppress lymphocyte response to PHA. Anti-rubella IgG complexed with rubella virus did not suppress the PHA response. The IgG function was apparently limited to neutralization of the infectivity of rubella virus whereas the major role of IgM was manifested through its suppressive effect on lymphocyte reactions.     

Brugia pahangi: immunologic evaluation of the differential susceptibility of filarial infection in inbred Lewis rats

After inoculating inbred Lewis rats with infective larvae of Brugia pahangi, we found consistently that patent infection was established in only about two-thirds of experimental animals. To determine which aspects of the host response were responsible for this differential susceptibility to infection, we evaluated all animals with respect to blood leukocyte levels, antifilarial IgG and IgE antibody production, and specific lymphocyte responses to mitogens and filarial antigens during the first 50 weeks of infection. In the critical “prepatent” period of infection, each of these responses developed in an essentially identical fashion in the infected and resistant groups except for the production of specific IgE antibodies. While those animals destined to resist infection developed specific IgE antibodies between Weeks 5 and 8, none of the animals that became microfilaremic showed a similar IgE response. These findings suggest that the development of specific IgE antibodies played a role in protecting the animals from acquiring filarial infection.     

Control of IgE and IgG1 antibody production in mice.

The production of IgE and IgG1 was studied in untreated, thymectomized. splenectomized, anti-thymocyte serum-treated, or sublethally X-irradiated mice. Dinitrophenyl Ascaris and ovalbumin were used as antigens, and aluminum hydroxide was used as adjuvant. A suppression of IgE production was observed in adult thymectomized mice, although the kinetic pattern of the antibody response was the same as in control animals. IgG1 antibody production was not affected by thymectomy. Splenectomy did not change either IgE or IgG1 production. A single dose of rabbit anti-thymocyte serum (ATS) given 8 days after immunization inhibited IgE antibody production. The effect of ATS was dose dependent and also varied with the amount of antigen used, the immune response to high doses being more susceptible to the effect of ATS. No alteration in IgG1 production was caused by ATS even when IgE antibody formation was completely inhibited. When preceding immunization, sublethal irradiation enhanced IgE antibody formation and partially suppressed IgG1 production; applied after immunization, irradiation caused an enhancement of IgE production which was inversely proportional to the interval elapsed between the two procedures. On the other hand, the IgG1 antibody production was fairly resistant to the same treatment. The results suggest a clearcut separation between the mechanisms regulating IgE and IgG1 production in mice.     

IgE responses in human filariasis. IV. Parallel antigen recognition by IgE and IgG4 subclass antibodies

Immediate hypersensitivity responses are highly modulated in filariasis, and with few exceptions, the majority of infected individuals do not develop allergic manifestations. One possible mechanism for this modulated responsiveness could involve the high levels of IgG "blocking antibodies" shown to be present in filariasis and other chronic helminth infections. When immunoblot analyses were done to analyze the immunoglobulin (Ig) E and IgG antibody responses of patients simultaneously, remarkable similarity in the patterns of antigen binding was observed. In this study, the four IgG subclasses were analyzed in a similar manner in relation to IgE. The results clearly demonstrate that IgG4 was primarily responsible for this "parallel" recognition that was seen previously between IgG and IgE antibodies. These results lend additional support to the possibility that IgG4 may play an important role in modulating IgE-mediated allergic responses in vivo.     

Role of theHLA complex in the antibody response to malaria under natural conditions

Antibodies toP. falciparum antigens and to the EB virus antigens, VCA and EBNA, were determined soon after the end of the major rainy season in 140 Africans living in 3 villages at varying altitudes in northeastern Tanzania. Also, their peripheral blood mononuclear cells were stored in liquid nitrogen and subsequently used for HLA phenotype determination of serologically determined antigens coded by genes at theA andB loci and for cell culture with mitogens (PHA, Con-A PWM) and with allogeneic cells in the mixed leukocyte reaction. A strong correlation was found between the presence of high titres of immunofluorescent antibody to falciparum antigens and the combination of A2 with AW30 in the same individuals. Individuals having one or the other of these specificities, but not both, did not have unusually high titres (P=0.0005). Individuals having the combination A2 and BW17 also tended to have higher than average antibody titres to falciparum antigens, but the difference was not statistically significant (P=0.09). The data suggests that individuals with A2 and AW30 may haveHLA-associated genes having the function ofIr genes and that these genes interact from the trans position to affect the capacity to make antibodies to malarial antigens. Thus, these genes may confer a survival advantage for individuals exposed to malaria. In the cell culture studies there were no correlations between responses and with IFA titres toP. falciparum, except for an inverse association between responses to PWM and level of IFA titre. This suggests that the B cell response to mitogens is impaired in individuals with strong responses to malarial antigens. There was no association between any of the cell culture responses and the HLA phenotypes of the cell donors.Abbreviations used in this paper P. Plasmodium - EB Epstein-Barr - VCA virus capsid antigen - EBNA Epstein-Barr nuclear antigen - PHA phytohemagglutinin - Con-A concanavalin-A - PWM pokeweed mitogen - IFA immunofluorescent antibody - Ir immune response - EA early antigen - MLR mixed leukocyte reaction - EAIMVBD East African Institute for Malaria and Vector-Borne Diseases - MEM minimal essential medium - EBV Epstein-Barr virus - AMMN alpha medium minus nucleosides - equal to or less than - equal to or greater than - RGM reciprocal geometric mean     

Involvement of both HLA and Ig heavy chain haplotypes in human IgA deficiency

Immunoglobulin-A deficiency (IgA-D) is the most common human Ig class deficiency with an estimated frequency of approximately 1 in 500 in the Swedish population. We investigated the immunoglobulin heavy chain constant region gene segments (IGHC) in 103 individuals with IgA-D and the immunoglobulin heavy chain variable region gene segments (IGHV) in 20 of these, in order to identify a possible molecular basis of the defect. No deletions of IGHV gene segments of the VH2, VH5, and VH6 families or the IGHG genes were observed. In the IGHC, there were, however, differences in the restriction fragment length polymorphism frequencies of IGHG genes where the Bam HI haplotype H2 [IGHGP, 10 kilobases (kb), IGHG2, 25 kb; and IGHG4, 9.0 kb] was overrepresented. The mean serum levels of IgG4 and IgE were significantly lower in individuals (both IgA-D subjects and healthy controls) homozygous for the H2 haplotype than in individuals homozygous for the H1 haplotype (IGHGP, 8.8 kb, IGHG2, 13.5 kb, and IGHG4, 9.4 kb). IgA-D subjects homozygous for HLADQB1*0201 (DQw2), a marker that has previously been reported to show a strong association with IgA deficiency, showed a similar reduction of serum levels of IgG4 and IgE as compared with DQB1*0201 negative IgA-D subjects. These findings suggest that the two loci found to be associated with IgA deficiency may act via a common pathway. Address correspondence and offprint requests to: P. G. Olsson     

High Antibody Responses against Plasmodium falciparum in Immigrants after Extended Periods of Interrupted Exposure to Malaria

Background

Malaria immunity is commonly believed to wane in the absence of Plasmodium falciparum exposure, based on limited epidemiological data and short-lived antibody responses in some longitudinal studies in endemic areas.

Methods

A cross-sectional study was conducted among sub-Saharan African adults residing in Spain for 1 up to 38 years (immigrants) with clinical malaria (n=55) or without malaria (n=37), naïve adults (travelers) with a first clinical malaria episode (n=20) and life-long malaria exposed adults from Mozambique (semi-immune adults) without malaria (n=27) or with clinical malaria (n=50). Blood samples were collected and IgG levels against the erythrocytic antigens AMA-1 and MSP-1₄₂ (3D7 and FVO strains), EBA-175 and DBL-α were determined by Luminex. IgG levels against antigens on the surface of infected erythrocytes (IEs) were measured by flow cytometry.

Results

Immigrants without malaria had lower IgG levels than healthy semi-immune adults regardless of the antigen tested (P≤0.026), but no correlation was found between IgG levels and time since migration. Upon reinfection, immigrants with malaria had higher levels of IgG against all antigens than immigrants without malaria. However, the magnitude of the response compared to semi-immune adults with malaria depended on the antigen tested. Thus, immigrants had higher IgG levels against AMA-1 and MSP-1₄₂ (P≤0.015), similar levels against EBA-175 and DBL-α, and lower levels against IEs (P≤0.016). Immigrants had higher IgG levels against all antigens tested compared to travelers (P≤0.001), both with malaria.

Conclusions

Upon cessation of malaria exposure, IgG responses to malaria-specific antigens were maintained to a large extent, although the conservation and the magnitude of the recall response depended on the nature of the antigen. Studies on immigrant populations can shed light on the factors that determine the duration of malaria specific antibody responses and its effect on protection, with important implications for future vaccine design and public health control measures.     

Heritability of Antibody Isotype and Subclass Responses to Plasmodium falciparum Antigens

Background

It is important to understand the extent to which genetic factors regulate acquired immunity to common infections. A classical twin study design is useful to estimate the heritable component of variation in measurable immune parameters.

Methodology/Principal Findings

This study assessed the relative heritability of different plasma antibody isotypes and subclasses (IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE) naturally acquired to P. falciparum blood stage antigens AMA1, MSP1-19, MSP2 (two allelic types) and MSP3 (two allelic types). Separate analyses were performed on plasma from 213 pairs of Gambian adult twins, 199 child twin pairs sampled in a dry season when there was little malaria transmission, and another set of 107 child twin pairs sampled at the end of the annual wet season when malaria was common. There were significantly positive heritability (h ²) estimates for 48% (20/42) of the specific antibody assays (for the seven isotypes and subclasses to the six antigens tested) among the adults, 48% (20/42) among the children in the dry season and 31% (13/42) among the children in the wet season. In children, there were significant heritability estimates for IgG4 reactivity against each of the antigens, and this subclass had higher heritability than the other subclasses and isotypes. In adults, 75% (15/20) of the significantly heritable antigen-specific isotype responses were attributable to non-HLA class II genetic variation, whereas none showed a significant HLA contribution.

Significance

Genome-wide approaches are now warranted to map the major genetic determinants of variable antibody isotype and subclass responses to malaria, alongside evaluation of their impact on infection and disease. Although plasma levels of IgG4 to malaria antigens are generally low, the exceptionally high heritability of levels of this subclass in children deserves particular investigation.     

Genetic attachment of undecane peptides to ovomucoid third domain can suppress the production of specific IgG and IgE antibodies

An undecane peptide (Gly-Ser-Pro-Gly-Ile-Pro-Gly-Ser-Thr-Gly-Met) was genetically attached to the N-terminus of ovomucoid third domain (DIII) to investigate structural characteristics of linear IgE and IgG (B cell) epitopes in DIII with respect to modulation of the immune response towards antigenicity and allergenicity. Balb/c mice were sensitized with native DIII, wild type recombinant DIII, and recombinant modified DIII containing the extra amino acid stretch. The immune responses to the antigens were compared using enzyme-linked immunosorbent assay. Interestingly, specific IgE and IgG levels were suppressed when the modified DIII was used as antigen. This was further confirmed by synthesizing immunodominant IgE and IgG epitopes of DIII on cellulose acetate membrane (SPOTs) and probing them with antibodies raised against DIII antigens. Anti-recombinant wild type DIII anti-serum showed strong binding activities to immunodominant IgE and IgG epitopes, while anti-modified DIII serum did not show any significant binding to the IgE and IgG epitopes. Thus, it is clearly demonstrated that the amino acid stretch in DIII is masking the immune reactive epitope. Genetical attachment of peptides into DIII was found to be effective in reducing the production of specific IgE and IgG antibodies in mice.     

Engineered recombinant ovomucoid third domain can modulate allergenic response in Balb/c mice model

IgE-mediated allergic reactions to egg white are a serious health problem and ovomucoid being the dominant egg white allergen has been on focus in the past decade. Engineered hypoallergens with reduced reactivity for IgE antibodies are being examined to modulate the allergic response and develop prophylactic allergen vaccines. In this study, we evaluated the immunomodulatory effect of a genetic variant of the third domain of ovomucoid (GMFA) which showed reduced IgE binding with egg allergic patient's sera in comparison to the native form of the third domain of ovomucoid (DIII) in a murine model system. Balb/c mice were injected intraperitoneally with DIII and GMFA antigens. Allergen-specific serum IgG, IgG1, IgG2a, and IgE responses were evaluated using enzyme-linked immunosorbent assay. Splenocyte cytokine levels in the medium of the cultured cells were examined by ELISA and levels of IL-4, INF-gamma, and IL-12 (p70) cytokines were quantified. Neutralization with anti-IL-12 monoclonal antibody was assayed and cytokine levels with respect to GMFA mutant antigen stimulation were measured. GMFA mutant form was found to have significantly reduced levels of specific IgE when compared to the DIII suggesting a mutation-induced abrogation of the IgE binding epitope in mice. The increase in IgG2a levels in GMFA together with the decline of IgE and IgG1 points to a shift from a Th2 response to a Th1 dominated response. The cytokine profile showed a modulation of anti-allergic Th1 phenotype in GMFA from a proallergic Th2 response observed with DIII. Low levels of IL-4 and increased levels of INF-gamma and IL-12 were observed and anti-IL-12 monoclonal antibody restored the levels of IL-4 and suppressed levels of INF-gamma and IL-12 in the GMFA sensitized group. These results indicate that GMFA has a marked suppressive effect on the allergic response of ovomucoid and caused a shift towards a Th1 pathway, thereby modulating the Th1/Th2 cytokine balance and could be used as a potential hypoallergenic candidate for allergen-immunotherapy in the treatment of egg white allergy.     

Polysaccharides L900/2 and L900/3 isolated from Lactobacillus rhamnosus LOCK 0900 modulate allergic sensitization to ovalbumin in a mouse model

Here, we compared the abilities of polysaccharides L900/2 and L900/3, which were previously isolated from Lactobacillus rhamnosus LOCK 0900, to modulate the immune response to bystander antigens in a mouse model of ovalbumin (OVA) sensitization. In vivo, both polysaccharides reduced the levels of OVA‐specific IgE, IgE‐dependent basophil degranulation and IgG2a antibodies, but had no effect on the levels of OVA‐specific IgA or IgG1. Interestingly, both polysaccharides triggered recall cellular responses with distinct properties. L900/3 significantly suppressed the OVA‐induced upregulations of IL‐4, IL‐5, IL‐10 and IL‐13 in re‐stimulated spleen cells and mesenteric lymph nodes. Our findings support and expand on our previous in vitro studies by demonstrating that polymer L900/3 might modulate the Th1/Th2 balance and could be a promising candidate molecule for preventing allergic sensitization.