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Background: Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in infants and is a risk factor for the development of asthma. Allergic asthmatics are more susceptible to RSV infection and viral exacerbation., Methods: Since the effectiveness of corticosteroids in treating RSV infection has been controversial, we tested fluticasone propionate (FP) and salmeterol (Sal) alone versus FP plus Sal (FPS) on RSV-induced airway inflammation. Mice were sensitized and challenged with ovalbumin (OVA) and infected with RSV. Following infection they were treated with FP, Sal, or FPS intranasally and airway hyperreactivity (AHR), inflammation and RSV titers were examined., Results: The group treated with FPS showed significantly lower AHR compared to the group treated with FP or Sal alone. The group treated with FP alone showed slightly decreased (non-significant) AHR compared to controls. Treatment with FPS resulted in significant decreases in the percentage of eosinophils and neutrophils in bronchoalveolar lavage fluid and in lung pathology compared to FP or Sal. FP alone decreased eosinophils but not neutrophils or lymphocytes, while Sal alone decreased eosinophils and neutrophils but not lymphocytes. FPS treatment of mice infected with RSV in the absence of allergen sensitization resulted in a 50% decrease of RSV titer in the lung and a reduction in neutrophils compared to FP or Sal., Conclusion: Together, these results indicate that fluticasone in combination with salmeterol is a more effective treatment for decreasing airway hyperreactivity and inflammation than either of them alone in allergen-sensitized, RSV-infected mice.

Among pollen allergens, grass pollen allergens are some of the most frequent contributors to allergic symptoms. Substantial progress has been made since the 1960s in the identification and characterization of the grass allergens. Members of this group belong to the Poaceae family, and have been classified into 13 distinct groups based on their structure, and their biological and immunologic properties. The major contributors to allergy and, hence, most studied among the grass allergens, are those belonging to groups 1 and 5. This review is focused on the structure and immunobiology of the grass allergens and highlights how recent advances in the field have contributed to superior diagnosis and immunotherapy for allergy to grass pollens.

Respiratory syncytial virus (RSV) infection is considered a risk factor for bronchial asthma; however, the synergy between allergen sensitization and RSV infection in the development of pulmonary inflammation and asthma has been controversial. In this study the effects of primary and recurrent RSV infection on allergic asthma were examined in a group of control, RSV-infected, Dermatophagoides farinae (Df) allergen-sensitized, and Df allergen-sensitized plus RSV-infected BALB/c mice. Primary RSV infection in Df-sensitized mice transiently increases airway responsiveness, which is accompanied by increases in eosinophilic infiltration, the expression of ICAM-1, and macrophage inflammatory protein-1alpha (MIP-1alpha) in the lung tissue. A secondary RSV infection persistently enhances airway responsiveness in Df-sensitized mice, with a concomitant increase in MIP-1alpha and RSV Ag load in lung tissues. Bulk cultures of thoracic lymph node mononuclear cells demonstrate that acute RSV infection augments both Th1- and Th2-like cytokines, whereas secondary and tertiary infections shift the cytokine profile in favor of the Th2-like cytokine response in Df-sensitized mice. The elevated total serum IgE level in the Df-sensitized mice persists following only RSV reinfection. Thus, recurrent RSV infections in Df-sensitized mice augment the synthesis of Th2-like cytokines, total serum IgE Abs, and MIP-1alpha, which are responsible for persistent airway inflammation and hyperresponsiveness, both of which are characteristics of asthma.

The interaction between mite allergen sensitization and respiratory syncytial virus (RSV) infection at the level of cytokine mRNA expression was examined in a murine model in the present study. Primary RSV infection enhances expression of inflammatory cytokines such as IL-6, IFN-gamma, and eotaxin in the lung and upregulates the expression of Th2-like cytokines IL-10 and IL-13 in the spleen in BALB/c mice. Mite antigen-sensitized and RSV-infected (ASRSV) mice show enhanced (P < 0.05) total serum IgE compared to antigen-sensitized mice. However, the levels of viral mRNA in the lung tissues are comparable between RSV-infected and ASRSV mice. It is concluded that compartmentalization of cytokine expression following RSV infection plays a role in the augmentation of Th2-like and IgE antibody response to RSV.

Prophylactic vaccination has recently emerged as a major paradigm toward the prevention and therapy of allergies and asthma; however, the immunological basis of this approach remains to be elucidated. We examined the potential and mechanism of prophylaxis of allergic response in B6D2F1 mice with a multi-epitopic recombinant allergen, rKBG8.3 (MERA-8.3), which represents a major group of allergens of grass pollens, used herein as a model of MERA vaccine. Vaccination (subcutaneous) with soluble MERA-8.3, prior to immunization with the MERA-8.3 in alum, led to suppression of the IgE antibody response and a concomitant increase in IgG2a antibody response specific to the MERA-8.3 in a dose-dependent manner. Analysis of cytokine patterns in spleen and lymph node cells revealed a marked decrease of interleukin-2 (IL-2) and IL-4 production and to a lesser extent a decrease of interferon-gamma (IFN-gamma) synthesis, resulting in an increased ratio of IFN-gamma: IL-4 in vaccinated-immunized mice compared with untreated-immunized control mice. Furthermore, splenocytes of mice treated with the MERA-8.3 alone proliferated to MERA-8.3 in vitro with reduced capacity compared with the splenocytes of MERA-8.3-alum immunized mice, owing to a markedly reduced level of IL-2 production in the former. Collectively, these results suggest that vaccination with the MERA-8.3 induces T-cell anergy, which is pivotal to deviation of specific immunity from Th2- to Th1-like, and may serve as an important approach to prevention and therapy of allergic disorders.

The role of host genetic and adjuvant factors in the induction of immune responses to a major recombinant Kentucky bluegrass allergen was examined utilizing five strains of mice and two different adjuvants. Analysis of the recombinant allergen-specific antibodies induced in these strains indicated that the antibodies of various isotypes were differentially regulated. In terms of IgE antibody response, BDF1 and DBA/2 were characterized as high responder, whereas BALB/C, CBA/J and C57BL/6 were intermediate and SJL was a low responder. In different strains, both dextran sulfate (DS) and complete Freund's adjuvant (CFA), as adjuvants, induced recombinant allergen-specific IgE antibodies of similar titer, however, CFA induced higher IgG2a and lower IgM antibodies compared to DS. Further, analysis of T cell proliferative responses of the splenocytes of different strains demonstrated that these strains varied also in their capacity to respond to synthetic peptides. Furthermore, utilizing a panel of synthetic peptides corresponding to the recombinant allergen, we demonstrated that the antibodies induced by the recombinant allergen with CFA in different strains vary with respect to their epitope specificity. In the BDF1 strain, compared to DS, CFA as adjuvant induced recombinant allergen-specific antibodies of additional peptide specificity. Taken together, these results suggest that both host genetic background and adjuvants govern the fine specificity of antibodies produced against this recombinant Kentucky bluegrass allergen.

The B- and T-cell epitopes of a recombinant grass allergen, rKBG60, were delineated using a set of overlapping synthetic peptides. Direct binding by enzyme-linked immunosorbent assay (ELISA) utilizing serum pools led to the identification of 13 murine immunoglobulin-, and nine to 13 human IgG- and five to seven human IgE-reactive overlapping peptides. Of the peptides which bound to human IgE antibodies, all but three peptides bound to human and/or murine IgG antibodies. Furthermore, eight out of 12 synthetic peptides induced antigen-specific antibodies in mice, suggesting that these peptides contained epitopes that recognized and/or induced T cells. These results, in conjunction with cross-recognition of different peptides at the C-terminus of rKBG60 by antibodies to neighbouring or non-overlapping peptides suggest that the C-terminus of this antigen represents a dominant antigenic and allergenic site. Peripheral blood mononuclear cell (PBMC) proliferation studies using these synthetic peptides for 13 grass allergic individuals indicated that seven potential human T-cell epitopes exist on this allergen. Taken together, the results demonstrate that multiple B- and T-cell epitopes exist on this major group of grass allergens, the majority of which are localized at the C-terminus of this antigen.

Pollen from 10 agricultural plant species was surveyed for the presence of proteins crossreactive with group I, group IV and group IX allergens. Barley (Hordeum vulgare), maize (Zea mays), rye (Secale cerale), triticale (xTriticosecale cereale), oats (Avena sativa), Canola (Brassica napus) and sunflower (Helianthus annus) pollens contained numerous allergen cognate proteins. Northern blot analysis of barley pollen RNA revealed the presence of group I and group IX allergen transcripts. The barley pollen cDNA hvp9742, and three other cloned allergens: phlenum protense (Phl p) V, Phl p Va and Lolium perenne (Lol p) 1b, were demonstrated to have extensive nucleotide and amino acid sequence similarity to the Poa p IX isoallergens. It was concluded that hvp9742 represents a Poa p IX isoallergen homologue expressed by barley pollen, and was therefore designated Hor v IX. It is further shown that the most highly conserved domains of all seven proteins, including Hor v IX, map to previously defined Poa p IX antibody binding epitopes.

To evaluate the use of recombinant allergens for the diagnosis of grass pollen allergies, we examined the levels of IgE antibodies specific for grass pollen as by immunoassays. SDS-PAGE analysis of two batches of Kentucky bluegrass pollen extracts demonstrated that there was considerable variability in allergen content of extracts, which in turn affected quantitation of specific IgE antibodies by different immunoassay procedures. Furthermore, the levels of IgE antibodies in human sera specific for a recombinant grass pollen allergen, rKBG8.3, were examined by enzyme immunoassay. The results demonstrated that quantitation of IgE antibodies specific for even one single allergen may be used to discriminate sera of allergic individuals with respect to IgE specific for grass pollen in general. A positive correlation, r = .82, was found for IgE binding of the recombinant allergen and the crude extracts of grass pollens. It is concluded from these results that a single recombinant allergen or a combination of a few major recombinant allergens can substitute the crude extract for in vitro as well as in vivo diagnostic purposes.

Cross-reactivities between the major grass pollen allergens, at the level of T-cell recognition was examined employing several Lolium perenne I (Lol p I)-specific human T-cell clones. Nine of these Lol p I-specific T-cell clones exhibited cross-recognition of the recombinant Poa pratensis IX (Poa p IX) allergen, rKBG7.2, indicating that these two major antigens of a grass pollen share T-cell epitopes. Furthermore, proliferative responses of two other T-cell clones demonstrated that individual allergens of diverse grass pollens also possess common T-cell epitopes. Examination of the T-cell receptor (TcR) V alpha genes of these T-cell clones indicated that these cloned cells utilized distinct J alpha genes and that nine out of 10 clones possessed V alpha 13 gene. Furthermore, sequence comparisons of several allergenic molecules indicated that this cross-reactivity may be due to the presence of epitope(s) with structure(s) similar to the major T-cell epitope of Poa p IX allergens. Taken together, these results suggest for the first time that the major grass pollen allergens share cross-reacting T-cell epitope(s), and that this cross-reactivity is due to the structural homologies among allergens and restricted usage of TcR V alpha genes.

Ag-specific IgE and IgG antibody responses to a recombinant Ag-allergen chimeric (RAAC) protein were examined in B6D2F1 mice. The RAAC protein consisted of the truncated beta-galactosidase (beta-gal), linked at its C terminus to a polypeptide representing the conserved region of the recombinant Kentucky Bluegrass allergen encoded by the cDNA clone KBG8.3(rKBG8.3). Immunization of the mice with the RAAC protein in dextran sulfate as adjuvant led to the differential production of antibodies to the two constituents of RAAC protein with respect to their isotypic classes. Most of the antibody responses to both sets of determinants on the fusion protein were IgG1. In addition, the allergenic polypeptide of RAAC protein induced IgE antibodies, whereas the beta-gal elicited IgG2a antibodies. The same pattern of antibody isotypes was produced when the individual components, rKBG8.3 and beta-gal were separately used for immunization with dextran sulfate. On the other hand, immunization of mice with either RAAC or the beta-gal in CFA induced primarily a IgG response, and no IgE antibodies; however, under the same conditions of immunization rKBG8.3 induced IgG1 antibodies and also low levels of IgE antibodies. In contrast, the RAAC and the beta-gal induced IgG2a antibodies, whereas rKBG8.3 induced no detectable IgG2a antibodies. Furthermore, high titers of IgE antibodies were induced by the rKBG8.3 and not by the RAAC protein in dextran sulfate after the mice had been immunized twice with the same polypeptide in CFA. It is inferred from these results that the induction of isotype-specific immune responses in the animals with the same genetic background is dependent upon the Ag in question as well as the adjuvant; the latter, however, influences the magnitude but does not determine the isotype of the immune responses.

Antibody binding epitopes of a recombinant Poa p IX allergen were delineated using recombinant DNA and solid-phase peptide synthesis procedures. The full-length cDNA clone KBG60 and its four overlapping recombinant fragments, KBG60.1, KBG60.2, KBG8.3 and KBG10 which spanned the entire molecule were synthesized in E. coli with aid of the plasmid expression vector, pWR590.1. The antigenic and allergenic sites of these recombinant proteins were analyzed by ELISA using human IgE and murine IgG antibodies. It was thus demonstrated that although the epitopes were found on all the fragments tested, the majority of these were located on a C-terminal fragment, rKBG8.3. Furthermore, synthetic peptides were also employed to identify the epitopes of rKBG60 protein. The use of antisera raised against native KBG pollen extract and the recombinant KBG8.3 protein to scan a total of 56 overlapping deca-penta peptides, covering the entire rKBG60 protein, revealed that 10 positive peptides involved in the antibody-binding site(s). Taken together, the results of these studies indicate that rKBG60 protein possesses at least 10 antibody binding epitopes.

In this study, recombinant Poa pratensis (Poa p) IX allergens were examined for their in vivo allergenicity and antigenicity. Immunization of mice with a fusion protein (FP) comprising beta-galactosidase and recombinant KBG8.3 (rKBG8.3) allergen induced high titres of both IgG and IgE antibodies. By contrast, immunization with rKBG60.2, which represents the N-terminal fragment of rKBG8.3, induced only IgG antibodies. The IgE antibody titre specific to Kentucky Bluegrass (KBG) was significantly higher than that to beta-galactosidase. Moreover, KBG-specific IgE antibodies showed no apparent decrease in their titres until 60 days after immunization, whereas the beta-galactosidase-specific IgE antibodies disappeared after 40 days. The antibodies induced with rKBG8.3 in mice were capable of inhibiting the binding of human IgE antibodies to KBG pollen allergens, which indicated that rKBG8.3-specific murine antibodies recognized epitopes similar to those recognized by human IgE antibodies. Analysis of allergenic cross-reactivities of rKBG8.3 with components from five other species of grass pollens revealed that IgE antibodies induced by this allergen are capable of binding in vivo to components from other grass pollens. These results suggest that the mouse may serve as a model for the manipulation of IgE responses to recombinant allergens or their chemically modified derivatives.

The high-level expression and purification of Poa p IX recombinant grass pollen allergens were examined utilizing a modified pGEX plasmid, designated as pGEX 2T-1. This vector permits frame-1 ligation of lambda gt11 cDNA inserts and cleavage of the recombinant allergenic protein from the fusion partner glutathione S-transferase. The expression of the fusion proteins in water-soluble form varied among the transformants of the same bacterial strain and also between different host strains. Purification of the fusion proteins by affinity chromatography employing glutathione agarose gel revealed that proteases in the bacterial lysate bound to the gel and were co-eluted with the fusion proteins. These proteases, which specifically degraded the recombinant proteins to varying degrees, were inhibited by both of the inhibitors, phenylmethylsulfonyl fluoride and aprotinin. Cleavage by thrombin of the fusion proteins indicated that the structure of the individual protein affected the thrombin accessibility to the cleavage site. Increased concentration of thrombin partly compensated this effect, but resulted in a broader specificity of the enzyme. By contrast, cleavage of the fusion protein when it was still attached to the glutathione gel was convenient and led to purification of the product devoid of proteolytic activity. Since almost all the recombinant allergens have been cloned in lambda gt11 vector, the pGEX 2T-1 vector reported herein will facilitate the synthesis, purification of the corresponding allergenic proteins or their peptides in soluble and biologically active forms.

We reported previously the primary structure of three full-length cDNA clones that encode a new group of IgE-binding proteins of Kentucky bluegrass (KBG) pollen, designated as Poa p IX. In the present study we have further characterized the cloned Poa p IX proteins, identified the corresponding proteins in KBG pollen extract, and determined their antigenic relationships with other known grass pollen allergens. A recombinant IgE-binding polypeptide rKBG7.2 that represents the C-terminal fragment, conserved in Poa p IX proteins, appeared to contain epitopes unique to these proteins and served as an immunosorbent for the isolation of the corresponding human IgE antibodies. On two-dimensional PAGE blots these IgE antibodies bound selectively to five distinct KBG pollen proteins with molecular mass 28 to 34 kDa and isoelectric point greater than 9.5. These proteins differ in size and charge from known allergens, but are very similar to those of the recombinant Poa p IX proteins. The rKBG3.1, which represents the N-terminal region of the Poa p IX clone KBG31, as well as the corresponding natural allergens were shown to possess epitopes that crossreact with the acidic group V allergens of Timothy. Comparison of amino acid sequences of recombinant Poa p IX proteins with those of Lol p I isoallergens revealed no significant sequence similarities. In contrast, partial homology was demonstrated between the N-terminal sequences of these proteins and the Phl p V proteins. Our results confirm that the Poa p IX clones represent a distinct and major group of allergens of KBG pollen, and demonstrate structural similarities and antigenic cross-reactivities among different groups of allergenic proteins in grass pollens.

A recombinant peptide of Kentucky bluegrass (KBG) pollen was synthesized as a fusion protein (FP) in Escherichia coli by recombinant DNA procedures and was compared with its natural counterparts with respect to its allergenic properties. The FP was demonstrated to bind to the IgE antibodies (Abs) of greater than or equal to 95% of 55 individual sera examined. A positive correlation (r = 0.90) was observed between the levels of IgE Abs corresponding to the FP and the grass-pollen extract(s). With sera of five allergic patients, the IgE binding of three different protein preparations were compared, namely, KBG pollen proteins, 27 to 35 kd gel-purified pollen proteins, and the FP. Results indicated that about 50% of the total IgE binding of KBG pollen proteins was due to the IgE Abs specific to FP. Comparison of the above protein preparations with respect to their abilities to specifically stimulate murine popliteal lymph node cells in vitro indicated that the total pollen proteins stimulated the highest proliferation of lymph node cells. Interestingly, the FP supported higher proliferation of lymph node cells than the gel-purified proteins. Collectively, these results suggest that the recombinant peptide constitutes a major allergenic constituent of grass pollens and may be of diagnostic and therapeutic value.

The allergenic and antigenic cross-reactivities between a major recombinant Poa pratensis (Poa p) IX allergen, rKBG8.3, and its corresponding proteins of different grass pollens were examined. Immunoblotting of the proteins of thirteen different grass pollens using anti-rKBG8.3 antibodies indicated that Poa p IX-like proteins are present in ten other grass pollens, albeit in variable amounts and polymorphic forms. These proteins ranged in size from 20 to 88 kDa in different grass pollens. The percent relative binding determined for each grass pollen extract using allergic human sera showed a significant correlation (r = 0.891) with that of anti-rKBG8.3 antiserum. Moreover, there was a strong association (r = 0.901) between the Kentucky bluegrass extract and rKBG8.3 with respect to their inhibition of the binding of human IgE antibodies to allergens in grass pollen extracts. Taken together, these results suggest that the allergenic and antigenic epitopes of the Poa p IX-related proteins in some but not all grass pollens are similar in structure and specificities. It is concluded that the group IX allergens constitute a major family of homologous proteins in several grass pollens.