Allergen-Specific IgE and IgG4 as Biomarkers for Immunologic Changes during Subcutaneous Allergen Immunotherapy.

(1) Background: Biomarkers of efficacy for subcutaneous immunotherapy (SCIT) on allergic rhinitis have not been evaluated in details. The present study aims to assess the relevance of measuring of sIgE, sIgG4 and IgE/IgG4 ratio during SCIT in patients with allergic rhinitis; (2) Methods: 20 patients, 13 men and 7 women aged 19 to 58 years, with clinically manifested seasonal and perennial allergic rhinitis were studied. At the initiation and in the end of the three-year course of SCIT serum allergen-specific IgE and IgG4 were measured with ImmunoCAP system. The sIgE/sIgG4 ratio was calculated as a biomarker for immunologic effectiveness; (3) Results: There was a significant increase of sIgG4 antibodies (p < 0.05), while at the end of SCIT for the sIgE levels no significant changes were seen (p > 0.05). Moreover, 90% of patients showed a decrease of the IgE/IgG4 ratio; (4) Conclusions: In most of treated patients with AR, SCIT with Bulgarian allergen products leads to clear immunological changes. After a 3-year of SCIT there is a significant increase in allergen specific IgG4 levels and both decrease of sIgE and IgE/IgG4 ratio. sIgE, sIgG4 and IgE/IgG4 ratio can be used as a substantial biomarker for predicting immunological effectiveness of SCIT.

Cellular and humoral systemic and mucosal immune responses stimulated in volunteers by an oral polybacterial immunomodulator "Dentavax".

The oral polybacterial immunomodulator Dentavax (D), composed of killed cells from Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Candida albicans and Lactobacillus acidophilus and their lysates was created for immunoprophylaxis and therapy of oral mucosa and parodont inflammations. The stimulating effect of the preparation was evaluated in twelve volunteers immunized for 10 consecutive days. On days 7, 14, 21, 28 and 49 after the last immunization peripheral blood (PB) lymphocyte subsets, T lymphocyte activation and PB phagocytic activity, were studied by flow cytometry. PB lymphocyte proliferative responses to PHA, rIL-2, LPS and D were evaluated radiometrically. The production of TNF-alpha in supernatants of in vitro stimulated lymphocytes and specific IgA, IgM and IgG antibodies in serum and saliva was determined by ELISA. Ultrastructural morphologic changes in T and B lymphocyte populations were also investigated. Although no significant changes in the levels of basic lymphocyte subsets were detected, the early/late (CD57+/CD57-) CD8 T effectors ratio was increased at the end of the studied period, as were the percentage of PHA-responding (CD69+) T cells and PB phagocytizing cells. The most prominent lymphoprolipherative responses were measured upon costimulation with LPS+D and PHA+D on day 21. Electron-microscopic studies demonstrated a significant effect of D on both T and B cell activity. TNF-alpha concentration increased progressively from day 7 till the end of the investigation. Maximal concentrations were observed after stimulation with D and LPS. An increased level of specific salivary and serum antibodies against the components of D was found, with highest levels between days 7 and 21. Specific secretory IgA predominated in saliva as compared to IgM and IgG. Our results demonstrate the stimulating effect of Dentavax on PB lymphocyte functional activity and the specific humoral systemic and mucosal immunity.

Suppression of allergen-specific B lymphocytes by chimeric protein-engineered antibodies.

House dust mites Dermatophagoides pteronyssinus (Dpt) are among the most frequent causes of allergy symptoms in Europe. Der p 1 is one of the major allergenic compounds of Dpt and the pathological Der p 1-specific B cells play a key role as producers of allergen-binding antibodies. The selective elimination of these cells by artificial protein molecules which inhibit the production of Dpt-recognizing IgE antibodies is a perspective therapeutic goal of allergy. A protein engineered chimeric molecule has been constructed, which binds Der p 1-specific B cells via their BCR and suppresses selectively the production of anti-Der p 1 antibodies via CR1. The synthetic peptide Der p 1 p52-71 and an anti-CD35 monoclonal antibody were used for the construction of Der p 1 chimera. The functional effects of engineered antibodies were analyzed in vitro using PBMCs from allergy patients. Significant inhibition of allergen-specific proliferation and reduction of Der p 1-IgE antibody production were observed after treatment of PBMCs from allergic patients with Der p 1-peptide chimera. Culturing of these PBMCs in the presence of the chimeric molecule increased the percentage of apoptotic (Annexin V-positive) B lymphocytes, but not T lymphocytes.

Polybacterial immunomodulator Respivax restores the inductive function of innate immunity in patients with recurrent respiratory infections.

Respivax (BulBio-NCIPD Ltd.) is an oral polybacterial immunomodulator intended for treatment and prevention of non-specific respiratory tract infections. We studied for the first time its effects on the inductive mechanisms of innate immunity, in the course of 3-month immunoprophylaxis of 25 patients with recurrent and chronic respiratory infections. The expression of pattern-recognition receptors on peripheral blood (PB) monocytes and plymorphonuclear cells (PMNs), the antigen-presenting and co-stimulatory potential of peripheral blood monocytes and dendritic cells; and the stimulated Th1/Th2 cytokine production were determined by flow cytometry. As compared to healthy controls, patients were characterized with down-regulation of TLR2 and TLR4/CD14 complex on PB monocytes (p<0.01), decreased share of CD14+CD16+ DCs precursors (p<0.01), decreased CD86 expression on PB DCs (p<0.05) and a Th2 shift of cytokine profile. Respivax modulated differentially the surface expression of pattern-recognition receptors on PB monocytes, increasing TLR2 and CD14 without affecting TLR4 expression. Further on, Respivax enhanced the differentiation of mature CD86(high) dendritic cells (DCs). Importantly, Respivax promoted a Th1 shift of cytokine profile and restored the Th1/Th2 cytokine balance without pro-inflammatory effects. Noteworthy, Th1/Th2 ratios in the patient's group correlated positively with the levels of TLR2 (R=0.5, p<0.001) and CD14 expression (R=0.4, p<0.05). We conclude that Respivax treatment restores the inductive function of innate immunity at three key levels: antigen recognition and presentation, co-stimulation of naïve T cells, and Th1/Th2 balance. This results, at least in part, from a differential modulation effect on the expression of pathogen-recognition receptors.

Design of immunogenic peptides from Mycobacterium tuberculosis genes expressed during macrophage infection.

In vitro diagnosis of MTB-infection uses MTB-proteins coded for by genes of the region of differentiation 1 (RD1) of the MTB genome. This study wants to test if proteins preferentially expressed during MTB-intracellular growth might provide new targets for the diagnosis of MTB-infection. To this end seventy-five multiepitopic HLA-promiscuous MTB-peptides were designed by quantitative implemented peptide-binding motif analysis from 3 MTB-protein genes expressed in activated human macrophages (MA), 4 genes expressed during growth in non-activated human macrophages (MN-A), 12 housekeeping genes (HKG) and 6 genes of the RD1 region (RD1) as control. ELISpot for IFN-was performed to measure the responses of PBMCs deriving from 45 patients affected by active tuberculosis and 34 controls. In active-TB patients, the mean response to RD1-derived peptides was higher than that to either MA (p<0.01), MN-A (p<0.008) or HKG (p<0.01) derived peptides. In TST-positive subjects all selected peptides elicited significant IFN-T-cell responses (p<0.02 compared to TST-negatives), but without differences between the subgroups. Further, T-cell responses to RD1 peptides were lower in the 23 active-TB treated patients than in the untreated ones (p<0.01). The response to MA peptides in treated active-TB was higher than when untreated (p<0.01). These results demonstrate that the use of in vitro models of MTB-intracellular infection to select MTB gene products for further in silico and in vitro assessment of their immunogenicity have the potential to identify novel antigens amenable to the design of new tools for diagnosis and monitoring of tuberculosis.