Shyam S. Mohapatra

Mapping of antibody binding epitopes of a recombinant Poa p IX allergen

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.

Multiple B‐ and T‐cell epitopes on a major allergen of Kentucky Bluegrass pollen

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.

VACCINATION WITH A MULTI-EPITOPIC RECOMBINANT ALLERGEN INDUCES SPECIFIC IMMUNE DEVIATION VIA T-CELL ANERGY

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‐γ (IFN‐γ) synthesis, resulting in an increased ratio of IFN‐γ: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.

Immunologic characterization of a recombinant Kentucky (Poa pratensis) allergenic peptide

Abstract 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 ⩾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.

Modulation of allergen-specific antibody responses by T-cell-based peptide vaccine(s): principles and potential

Summary and concluding remarksExtensive studies of immunopathology of asthma have revealed the predominant role of T-cells in driving the inflammatory responses associated with bronchial asthma and the role of allergen-induced IgE responses in exacerbating the asthmatic responses. With recent advances in molecular immunoallergology, in general, and the role of T-cells in asthma in particular, it has become apparent that therapy invooving T-cell-based vaccines may provide a rational immunologic approach(es) for the treatment of specific allergen induced asthma. Three main approaches appear to have potential:1.Immunotherapy with allergen in combination with appropriate cytokines;2.Vaccinations with T-cell epitopic peptides; and3.Vaccinations with T-cell receptor peptides. It is to be noted that these therapeutic approaches are presently at the experimental stage. Given that asthma is a very complex disorder and each of these approaches has its own advantages and disadvantages, it is difficult at present to predict which of these would succeed. It is clear, however, that each of these immunologic approaches has sufficient potential to be useful either by itself or in combination with other therapies and/or in conjunction with pharmacotherapy, depending in the prognosis of asthma in individuals on a case by case basis.

Host Genetic and Adjuvant Factors Influence Epitope Specificity to a Major Recombinant Grass Allergen

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 Freunds 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.

Evidence for the existence of IL-4 and IFNγ secreting cells in the T cell repertoire of naive mice

Abstract The kinetics with which IgE responses develop in vivo following immunization of experimental animals indirectly support the existence of IL-4-secreting T cells as a normal component of the T cell repertoire. At the same time, studies of IL-4-secreting cell frequencies directly ex vivo have argued that T cells with the potential to become IL-4 secretors exist in vivo, in the form of precursors requiring stimulation and 4 – 12 days of culture as well as restimulation with mitogen or Ag before they become detectable as lymphokine-secreting cells. We demonstrate here that intravenous administration of low doses of anti-CD3 mAb 145-2C11 results in IL-4 production within 60 min of stimulation as demonstrated by Northern analysis of mRNA and a sensitive, selective bioassay (CT.4S cell proliferation) of biologically active IL-4 protein. Production of IL-4 is paralleled by IFNγ synthesis, displaying similar kinetics. These findings, consistent with the presence of mature cells capable of IL-4 and IFNγ synthesis in the T cell repertoire of naive mice, are supported by the observation that stimulation of spleen cells from naive mice with anti-CD3 mAb in vitro for 12 h also results in strong IL-4 and IFNγ mRNA and protein synthesis. The data support and extend those obtained through analysis of cytokine mRNA synthesis alone, thereby providing evidence that “fresh” T cells are indeed capable of producing IL-4 directly ex vivo and are consistent with the existence of IL-4-secreting cells as a normal component of the T cell repertoire of naive mice.

Molecular cloning of grass pollen allergens: progress and perspectives

SummaryIn this report the advances made in recent years as a result of application of recombinant DNA and allied technologies in relation to isolation, molecular characterization and synthesis of principal allergens—with particular emphasis on Kentucky Bluegrass (KBG,Poa pratensis) pollen allergens—has been reviewed. Screening of a cDNA library complementary to KBG pollen mRNA led to isolation of several cDNA clones. Hybridization studies using the labelled cDNAs as probes suggested the existence of at least three major classes of clones. The nucleotide sequences of several cDNAs encoding a major group of allergenic proteins were determined. Moreover, one cDNA clone, KBG7.2 was inserted in an expression plasmid to achieve high-level expression of the corresponding peptide. The recombinant peptide was shown to be comparable to its natural counterpart with regard to its ability to bind to human IgE and IgG antibodies of allergic patients, and to elicit passive cutaneous anaphylaxis (PCA) in rats. Furthermore, KBG 7.2 encoded peptide was shown to possess at least two IgE binding epitopes. About four T cell epitopes were predicted on this peptide. The results of these studies suggest that the recombinant allergens and/or allergenic peptides may be of diagnostic and therapeutic value.

Nanobiotechnology Medical Applications: Overcoming Challenges Through Innovation

Abstract Biomedical Nanotechnology (BNT) has rapidly become a revolutionary force that is driving innovation in the medical field. BNT is a subclass of nanotechnology (NT), and often operates in cohort with other subclasses, such as mechanical or electrical NT for the development of diagnostic assays, therapeutic implants, nano-scale imaging systems, and medical machinery. BNT is generating solutions to many conventional challenges through the development of enhanced therapeutic delivery systems, diagnostic techniques, and theranostic therapies. Therapeutically, BNT has generated many novel nanocarriers (NCs) that each express specifically designed physiochemical properties that optimize their desired pharmacokinetic profile. NCs are also being integrated into nanoscale platforms that further enhance their delivery by controlling and prolonging their release profile. Nano-platforms are also proving to be highly efficient in tissue regeneration when combined with the appropriate growth factors. Regarding diagnostics, NCs are being designed to perform targeted delivery of luminescent tags and contrast agents that enhance the NC -aided imaging capabilities and resulting diagnostic accuracy of the presence of diseased cells. This technology has also been advancing the ability for surgeons to practice true precision surgical techniques. Incorporating therapeutic and diagnostic NC-components within a single NC can facilitate both functions, referred to as theranostics, which facilitates real-time in vivo tracking and observation of drug release events via enhanced imaging. Additionally, stimuli-responsive theranostic NCs are quickly developing as vectors for tumor ablation therapies by providing a model that facilitates the location of cancer cells for the application of an external stimulus. Overall, BNT is an interdisciplinary approach towards health care, and has the potential to significantly improve the quality of life for humanity by significantly decreasing the treatment burden for patients, and by providing non-invasive therapeutics that confer enhanced therapeutic efficiency and safety

Nanotechnology platform for personalized cancer treatment

CS1 (also known as CD319, CRACC and SLAMF7) was identified as an NK cell receptor regulating immune functions. It is also expressed on B cells, T cells, Dendritic cells, NK-T cells, and monocytes. CS1 is overexpressed in multiple myeloma and makes it a target for immunotherapy. A humanized anti-CS1 antibody, Elotuzumab or Empliciti has shown promising results in clinical studies. This review focuses on the biology of CS1 in NK and other hematopoietic cells and multiple myeloma. Anti-CS1 mAb can activate natural cytotoxicity of NK cells as well as enhance ADCC (antibody-dependent cell-mediated cytotoxicity) and thus makes an effective target for immunotherapy of MM.