TLR-5 ligand conjugated with Per a 10 and T cell peptides potentiates Treg/Th1 response through PI3K/mTOR axis.

Novel strategies are needed to enhance efficacy of immunotherapy for allergic diseases. The present study evaluates role of conjugating flagellin with allergen, Per a 10 and its T cell epitopes in DC mediated polarization. Fusion proteins (FPer a 10, FPT1, FPT2 and FPT3) were sub-cloned, expressed and purified to homogeneity. FlaA fused proteins showed TLR-5 binding in TLR5+HEK293 cells as observed by enhanced NF-κB dual luciferase assay and IL-8 secretion in culture supernatant. Fusion proteins upregulate co-stimulatory molecules CD80, CD83 and CD86 on BMDCs and enhanced IL-6, IL-ß, IL-12p70 and IL-10 secretion. Inhibition studies suggest fusion proteins mediate IL-10 secretion via activation of PI3K/mTOR/NF-κB pathway. FPT1 and FPT3 deviate Th2 cytokine milieu towards Th1/Treg by upregulating the expression of IFN-γ, IL-12p70, IL-10 and TGF-ß in BMDCs co-culture with allergen specific T cells. Flow cytometric examination of BMDC:T cell co-culture revealed CD4+ T cell differentiation towards Th1/Treg by FPT1 and FPT3. Thus, our results indicate that flagellin fused with T cell peptides are efficient immunomodulators and may possess potential for allergy therapeutics.

Immuno-informatic Prediction of B and T cell Epitopes of Cysteine Protease Allergen from Phaseolus vulgaris with Cross-reactive Potential and Population Coverage.

BACKGROUND: In-silico mapping of epitopes by immune-informatics has simplified the efforts towards understanding antigen-antibody interactions. The knowledge of allergen epitopes may help in advancing the diagnosis and therapy of allergic diseases. OBJECTIVE: This study was intended to identify B and T cell epitopes of cysteine protease allergen of Phaseolus vulgaris. METHODS: Modeller 9v20 software was used for the generation of three-dimensional model of cysteine protease and quality assessment was performed using SAVES webserver and other in silico software. Linear and conformational B and T cell epitopes were predicted via immuno-informatics based computational servers. Epitopes were synthesized and their immunoreactivity was analyzed using specific IgE ELISA with food allergy positive patient's sera. Cellular immune response of peptides was determined through basophil activation assay. Consurf and SDAP (property distance) were used to examine the evolutionary conservancy and potential cross-reactivity of predicted epitopes. MSA based positional conservancy between HDM allergen epitopes and predicted peptides was also established using IEDB epitope database. Finally, population coverage for each promiscuous T cell epitope was predicted using IEDB population coverage analysis tool. RESULTS: Cysteine protease structure was derived by homology modeling and combination of bioinformatic tools predicted three B- and three T-cell peptides by consensus method and validated computationally. ELISA with kidney bean sensitive patient's sera showed higher IgE binding of B-cell peptides as compared to T-cell or control peptides. Epitope conservancy revealed B-cell epitopes being upto 95% conserved in comparison to variable T-cell epitopes (upto 69%). B-cell peptides were crossreactive with homologous allergens based on PD values. Structural comparison of cysteine protease with Der p 1 and Der f 1 showed similar epitopic regions, validating the prediction accuracy of epitopes. Promiscuous T-cell epitopes binding to broad-spectrum class-II MHC alleles demonstrated the distribution of T-cell peptides world-wide (30-98%) and in Asian population (99%). CONCLUSION: The current approach can be applied for identification of epitopes. Analysis of crossreactive and widely-distributed specific epitopes of allergen and knowledge about their interactive surfaces will help in understanding of food allergy and related immune responses.

The role of immunoinformatics in the development of T-cell peptide-based vaccines against Mycobacterium tuberculosis.

INTRODUCTION: Tuberculosis (TB) is a major health problem worldwide. The BCG, the only authorized vaccine to fight TB, shows a variable protection in the adult population highlighting the need of a new vaccine. Immunoinformatics offers a variety of tools that can predict immunogenic T-cell peptides of Mycobacterium tuberculosis (Mtb) that can be used to create a new vaccine. Immunoinformatics has made possible the identification of immunogenic T-cell peptides of Mtb that have been tested in vitro showing a potential for using these molecules as part of a new TB vaccine. AREAS COVERED: This review summarizes the most common immunoinformatics tools to identify immunogenic T-cell peptides and presents a compilation about research studies that have identified T-cell peptides of Mtb by using immunoinformatics. Also, it is provided a summary of the TB vaccines undergoing clinical trials. EXPERT OPINION: In the next few years, the field of peptide-based vaccines will keep growing along with the development of more efficient and sophisticated immunoinformatic tools to identify immunogenic peptides with a greater accuracy.

Utilization of Feline ELISpot to Evaluate the Immunogenicity of a T Cell-Based FIV MAP Vaccine.

The prototype and the commercial dual-subtype feline immunodeficiency virus (FIV) vaccines conferred protection against homologous FIV strains as well as heterologous FIV strains from the vaccine subtypes with closely related envelope (Env) sequences. Such protection was mediated by the FIV neutralizing antibodies (NAbs) induced by the vaccines. Remarkably, both prototype and commercial FIV vaccines also conferred protection against heterologous FIV subtypes with highly divergent Env sequences from the vaccine strains. Such protection was not mediated by the vaccine-induced NAbs but was mediated by a potent FIV-specific T-cell immunity generated by the vaccines (Aranyos et al., Vaccine 34: 1480-1488, 2016). The protective epitopes on the FIV vaccine antigen were identified using feline interleukin-2 (IL-2) and interferon-γ (IFNγ) ELISpot assays with overlapping FIV peptide stimulation of the peripheral blood mononuclear cells (PBMC) from cats immunized with prototype FIV vaccine. Two of the protective FIV peptide epitopes were identified on FIV p24 protein and another two protective peptide epitopes were found on FIV reverse transcriptase. In the current study, the multiple antigenic peptides (MAPs) of the four protective FIV peptides were combined with an adjuvant as the FIV MAP vaccine. The laboratory cats were immunized with the MAP vaccine to evaluate whether significant levels of vaccine-specific cytokine responses can be generated to the FIV MAPs and their peptides at post-second and post-third vaccinations. The PBMC from vaccinated cats and non-vaccinated control cats were tested for IL-2, IFNγ, and IL-10 ELISpot responses to the FIV MAPs and peptides. These results were compared to the results from CD4+ and CD8+ T-cell proliferation to the FIV MAPs and peptides. Current study demonstrates that IL-2 and IFNγ ELISpot responses can be used to detect memory responses of the T cells from vaccinated cats after the second and third vaccinations.

Recombinant allergen and peptide-based approaches for allergy prevention by oral tolerance.

Several studies conducted in animal models for immunologically-mediated hypersensitivity diseases have shown that oral administration of antigens early in life can prevent the development of specific humoral and cellular immune responses and thus hypersensitivity reactions to the respective antigens. Such data were also obtained in models for Immunoglobulin E (IgE)-associated allergy, the most common hypersensitivity disease affecting more than 25% of the population. Based on data obtained in animal models for allergy several clinical intervention studies have been conducted in children to study if oral administration of materials containing allergens or allergen-derived peptides early in life can prevent the subsequent development of allergy. In this article we argue that oral tolerance induction could be a potent way to prevent allergy and may be even improved regarding efficacy provided that well-defined allergen molecules and/or allergen-derivatives were used in optimized dose regimens and periods of intervention. The knowledge regarding the molecular and immunological characteristics of allergens which has been achieved in the last decades is a prerequisite for such a treatment. In fact, defined recombinant allergens/allergen derivatives and allergen-derived synthetic peptides from the most common allergen sources are now available for targeted intervention. Moreover, molecular allergy diagnosis allows deciphering the disease-causing relevant allergens for different regions in the world allowing composing cocktails of tolerogens according to the needs of populations from different parts of the world. Furthermore, it is suggested to use defined allergen molecules and epitopes in the analysis of clinical tolerance studies. This will allow understanding if clinical unresponsiveness is due to true immunological tolerance or to other mechanisms such as induction of blocking antibodies or cellular immunomodulation. Using molecularly defined tolerogens it can now be explored if oral tolerance induction is a powerful strategy to prevent IgE-associated allergy.

New vaccines for Mammalian allergy using molecular approaches.

Allergen-specific immunotherapy (SIT) offers a disease specific causative treatment by modifying the allergen-specific immune response allowing tolerance to higher doses of allergen and preventing progression of allergic diseases. It may be considered in patients allergic to furry animals. Current mammalian allergy vaccines are still prepared from relatively poorly defined allergen extracts and may induce immediate and late phase side effects. Although the mechanisms of SIT are still not fully understood, the more recent approaches report different strategies to reduce both allergen-specific IgE as well as T cell reactivity. The availability of recombinant allergens and synthetic peptides from the mammalian species has contributed to formulating new allergy vaccines to improve SIT for furry animal allergy. The majority of studies have focused on the major cat allergen Fel d 1 due to its extensive characterization in terms of IgE and T cell epitopes and to its dominant role in cat allergy. Here we review the most recent approaches, e.g., synthetic peptides, recombinant allergen derivatives, different hypoallergenic molecules, and recombinant allergens coupled to virus-like particles or immunomodulatory substances as well as strategies targeting the allergen to Fcγ receptors and the MHC class II pathway using a new route for administration. Many of the new vaccines hold promise but only a few of them have been investigated in clinical trials which will be the gold standard for evaluation of safety and efficacy in allergic patients.