T-cell epitope conservation across allergen species is a major determinant of immunogenicity.

BACKGROUND: Patients with pollen allergies are frequently polysensitized. Pollens contain epitopes that are conserved across multiple species. OBJECTIVE: We sought to demonstrate that cross-reactive T cells that recognize conserved epitopes show higher levels of expansion than T cells recognizing monospecific epitopes because of more frequent stimulation. METHOD: RNA was sequenced from 9 pollens, and the reads were assembled de novo into more than 50,000 transcripts. T-cell epitopes from timothy grass (Phleum pratense) were examined for conservation in these transcripts, and this was correlated to their ability to induce T-cell responses. T cells were expanded in vitro with P pratense-derived peptides and tested for cross-reactivity to pollen extracts in ELISpot assays. RESULTS: We found that antigenic proteins are more conserved than nonimmunogenic proteins in P pratense pollen. Additionally, P pratense epitopes that were highly conserved across pollens elicited more T-cell responses in donors with grass allergy than less conserved epitopes. Moreover, conservation of a P pratense peptide at the transcriptomic level correlated with the ability of that peptide to trigger T cells that were cross-reactive with other non-P pratense pollen extracts. CONCLUSION: We found a correlation between conservation of peptides in plant pollens and their T-cell immunogenicity within P pratense, as well as their ability to induce cross-reactive T-cell responses. T cells recognizing conserved epitopes might be more prominent because they can be stimulated by a broader range of pollens and thereby drive polysensitization in allergic donors. We propose that conserved peptides could potentially be used in diagnostic or immunomodulatory approaches that address the issue of polysensitization and target multiple pollen allergies.

Previously undescribed grass pollen antigens are the major inducers of T helper 2 cytokine-producing T cells in allergic individuals.

T cells play an important role in the pathogenesis of allergic diseases. However, the proteins considered as potential immunogens of allergenic T-cell responses have traditionally been limited to those that induce IgE responses. Timothy grass (TG) pollen is a well-studied inhaled allergen for which major IgE-reactive allergens have also been shown to trigger T helper 2 (Th2) responses. Here we examined whether other TG pollen proteins are recognized by Th2 responses independently of IgE reactivity. A TG pollen extract was analyzed by 2D gel electrophoresis and IgE/IgG immunoblots using pooled sera from allergic donors. Mass spectrometry of selected protein spots in combination with de novo sequencing of the whole TG pollen transcriptome identified 93 previously undescribed proteins for further study, 64 of which were not targeted by IgE. Predicted MHC binding peptides from the previoulsy undescribed TG proteins were screened for T-cell reactivity in peripheral blood mononuclear cells from allergic donors. Strong IL-5 production was detected in response to peptides from several of the previously undescribed proteins, most of which were not targeted by IgE. Responses against the dominant undescribed epitopes were associated with the memory T-cell subset and could even be detected directly ex vivo after Th2 cell enrichment. These findings demonstrate that a combined unbiased transcriptomic, proteomic, and immunomic approach identifies a greatly broadened repertoire of protein antigens targeted by T cells involved in allergy pathogenesis. The discovery of proteins that induce Th2 cells but are not IgE reactive may allow the development of safer immunotherapeutic strategies.

Development of a DNA vaccine designed to induce cytotoxic T lymphocyte responses to multiple conserved epitopes in HIV-1.

Epitope-based vaccines designed to induce CTL responses specific for HIV-1 are being developed as a means for addressing vaccine potency and viral heterogeneity. We identified a set of 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 to develop such a vaccine. Based on peptide-binding studies and phenotypic frequencies of HLA-A2, HLA-A3, and HLA-B7 allelic variants, these epitopes are predicted to be immunogenic in greater than 85% of individuals. Immunological recognition of all but one of the vaccine candidate epitopes was demonstrated by IFN-gamma ELISPOT assays in PBMC from HIV-1-infected subjects. The HLA supertypes of the subjects was a very strong predictor of epitope-specific responses, but some subjects responded to epitopes outside of the predicted HLA type. A DNA plasmid vaccine, EP HIV-1090, was designed to express the 21 CTL epitopes as a single Ag and tested for immunogenicity using HLA transgenic mice. Immunization of HLA transgenic mice with this vaccine was sufficient to induce CTL responses to multiple HIV-1 epitopes, comparable in magnitude to those induced by immunization with peptides. The CTL induced by the vaccine recognized target cells pulsed with peptide or cells transfected with HIV-1 env or gag genes. There was no indication of immunodominance, as the vaccine induced CTL responses specific for multiple epitopes in individual mice. These data indicate that the EP HIV-1090 DNA vaccine may be suitable for inducing relevant HIV-1-specific CTL responses in humans.