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G Ishioka

Publications and source records attributed to G Ishioka.

9 recordsLinked to original sources
PubMed2002

Optimizing vaccine design for cellular processing, MHC binding and TCR recognition.

In this review we describe the methods and processes that our group have developed while aiming to test and design multiepitope vaccines for infectious diseases and cancer. Testing the performance of vaccines composed of epitopes restricted by human leukocyte antigen (HLA) molecules is accomplished by in vitro antigenicity assays, as well as in vivo immunogenicity assays in HLA transgenics. The efficiency by which multiepitope vaccines are processed is optimized by spacer residues, which are designed to facilitate generation by natural processing of the various class I- and class II-restricted epitopes. Methods and strategies to test and optimize HLA binding affinity, patient coverage from the vaccine construct, and TCR recognition of HLA/epitope complexes are also discussed.

Epitopes↗
PubMed2000

Characterization of an in situ IFN-gamma ELISA assay which is able to detect specific peptide responses from freshly isolated splenocytes induced by DNA minigene immunization.

An in situ IFN-gamma ELISA assay has been developed and optimized for both freshly isolated and peptide-restimulated splenocytes. This assay is based on the ELISPOT assay, but utilizes a soluble chromagen, making it readily adaptable to high-throughput analysis. We show that in both the primary and restimulation assays this technique is more sensitive than either a traditional supernatant ELISA or the 51Cr-release assay, in that responses are observed in the in situ ELISA that are not detectable in these other assays. On a per-cell basis, the sensitivity of the in situ ELISA is approximately one IFN-gamma secreting cell/10(4) plated cells. The in situ IFN-gamma ELISA was utilized to describe the kinetics of the IFN-gamma response to DNA vaccination with pMin.1. For freshly isolated splenocytes, the peak response for all the peptides tested was observed from 10 to 12 days after immunization, with responses seen to some peptides as early as 7 days. When a 6-day in vitro peptide restimulation step was added, responses were seen for all the peptides tested after 7 days of in vivo immunization. This data demonstrates that a single intramuscular administration of a DNA vaccine can induce T-cell responses that can be detected in freshly isolated splenocytes.

Amino Acid Sequence↗
PubMed1997

The hepatitis B virus-specific CTL responses induced in humans by lipopeptide vaccination are comparable to those elicited by acute viral infection.

We have previously described the development of a lipopeptide-based vaccine, Theradigm-HBV, capable of inducing CTL responses in humans. This vaccine incorporates the HLA-A2.1-restricted CTL epitope hepatitis B core Ag 18-27, linked to the universal helper T lymphocyte (HTL) epitope tetanus toxoid (TT) 830-843. Herein, we report the results of a phase I trial designed to examine the effects of Theradigm-HBV dose and regimen on the magnitude and duration of the memory CTL response. A total of four injections of up to 5 mg/dose were found to be a safe and effective means of generating substantial memory CTL responses. Precursor frequency analysis demonstrated CTL responses of similar magnitude to those previously observed in patients who successfully cleared hepatitis B virus infection and to influenza-specific memory CTL responses induced by natural exposure to influenza virus. Theradigm-HBV induced CTL responses that persisted for more than 9 months after the last injection. HTL responses were associated with significant CTL responses, and sustained HTL activity was necessary for development of persistent memory CTL activity. These results represent the first demonstration of the importance of HTL activity for development of long-lived memory CTL responses in humans. In conclusion, our results show that lipopeptides safely induce specific CTL activity in humans of such magnitude and persistence as to be of potential therapeutic significance.

Acute Disease↗
PubMed1995

Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection. I. Induction of a primary cytotoxic T lymphocyte response in humans.

Our goal is to use peptide epitopes that are recognized by cytotoxic T lymphocytes (CTL) as immunogens for the development of prophylactic and therapeutic vaccines with chronic hepatitis B virus (HBV) infection being our first therapeutic target. Because most CTL peptide epitopes are poor immunogens, we specifically modified them by covalently attaching two additional components: a T helper peptide epitope and two lipid molecules. Using the murine influenza virus CTL epitope NP 147-155 as a model system, we found this construct to be highly immunogenic, and a single injection resulted in memory CTL induction that persisted for > 1 yr. Based on the animal studies, a vaccine was designed and tested for both safety and its ability to induce a primary CTL response in normal subjects. The three vaccine components included HBV core antigen peptide 18-27 as the CTL epitope, tetanus toxoid peptide 830-843 as the T helper peptide, and two palmitic acid molecules as the lipids. A dose escalation trial (5, 50, and 500 micrograms) carried out in 26 normal subjects showed that the vaccine was safe and able to induce a primary HBV-specific CTL response. A dose-response curve was observed and five out of five subjects responded to the 500-micrograms dose.

Adolescent↗
PubMed1994

A single TCR antagonist peptide inhibits experimental allergic encephalomyelitis mediated by a diverse T cell repertoire.

Previously, six T cell clones, which are specific for an encephalitogenic determinant of myelin proteolipid protein (PLP) peptide residues 139 to 151 (HSLGKWLGHPDKF), were derived from SJL mice and shown to use diverse TCR genes. To design TCR antagonist peptides that could interfere with the activation of these clones in vitro and inhibit experimental allergic encephalomyelitis (EAE) in vivo, we first determined the TCR and MHC contact residues of the encephalitogenic peptide. The analysis indicated that residues 144 (tryptophan) and 147 (histidine) were the TCR binding sites and that residues 145 (leucine) and 148 (proline) were important for MHC class II (IAs) binding. On the basis of this information, a peptide analogue (leucine 144/arginine 147), in which both of the major TCR contact residues were substituted, was synthesized. This analogue acts as a TCR antagonist for the panel of PLP 139-151-specific T cell clones, does not cause EAE by itself, blocks the induction of disease by the native 139-151 peptide, and prevents clinical disease progression if administered at the first signs of disease. Thus, although multiple TCR genes are used by PLP 139-151-specific clones, a single peptide analogue can interfere with the disease process. This approach should be feasible for designing peptide analogues that can be tested for therapeutic efficacy in human autoimmune diseases in which the pathogenic Ags are known and TCR use is diverse.

Amino Acid Sequence↗
PubMed1994

Antigen analogs/MHC complexes as specific T cell receptor antagonists.

Recent studies demonstrated that antigen analogs can act as powerful and specific inhibitors of T cell activation, leading to the formulation of the concept that antigen analog/MHC complexes may act as antagonists of the T cell receptor (TCR). TCR antagonism appears to be associated with engagement of the TCR below a crucial affinity threshold necessary for full T cell activation. Studies addressing the molecular mechanism of this effect suggest that TCR antagonists could act by interfering with membrane-related events (such as proper receptor clustering) that might precede intracellular signaling. Discovery of the TCR antagonism phenomenon also suggested a possible rational approach to antigen-specific immunointervention in allergies and autoimmune diseases. The feasibility of such an approach is now being actively investigated. Finally, TCR antagonist peptides may provide a useful tool to probe TCR-peptide/MHC interactions involved in the process of thymic education.

Animals↗
PubMed1992

DRB1*0301 molecules recognize a structural motif distinct from the one recognized by most DR beta 1 alleles.

The DR3-restricted peptide, MT 65 kDa 3-13, was used to develop a DR3-specific binding assay. The binding activity detected was strictly pH-dependent, in that it was optimal in the pH 4 to 5 range, and no activity was detected at neutral pH. By means of affinity chromatography purifications and the use of DR3-transfected fibroblasts, it was shown that no cross-reactivity exists at the level of DR52a molecules, thus allowing use of only partially purified DR3/DR52a mixtures in high throughput binding assays. The immunologic relevance of the assay established was also verified by examining the correlation between DR3 restriction and binding for a panel of DR-restricted peptides. When the structural requirements for peptide DR3 interactions were further examined by using panels of analogues of two different epitopes (Myo 132-151 and TT 830-843), it was found that DR3 molecules recognized a peptide motif distinct from the one recognized by the other major DR beta 1 alleles.

Alleles↗
PubMed1989

The role of dendritic cells as stimulators of minor lymphocyte-stimulating locus-specific T cell responses in the mouse. I. Differential capacity of dendritic cells to stimulate minor lymphocyte-stimulating locus-reactive T cell hybridomas and the primary anti-minor lymphocyte-stimulating locus mixed lymphocyte reaction.

The response of T cells to minor lymphocyte-stimulating locus (Mls) determinants remains poorly understood with respect to the antigenic determinants responsible for T cell stimulation and the types of APC capable of stimulating the response. In this report, we demonstrate that highly purified dendritic cells (DC) as well as B cells have the capacity to stimulate Mls-specific responses. Unseparated spleen cells, purified DC, resting B cells, and activated B cells were compared for their capacity to stimulate several Mls-reactive T cell hybridomas. Whereas the entire panel of Mls-reactive T cell hybridomas was stimulated strongly by unseparated spleen cells and activated B cells, the hybridomas responded only weakly to purified DC or resting B cells. Activation of resting B cells with either B cell stimulatory factor-1 (1 day pre-treatment) or LPS/dextran (2 or 3 day pre-treatment) greatly augmented their Mls-stimulatory capacity. In contrast, the Mls-stimulatory capacity of DC was not augmented by a 1-day pre-treatment with either B cell stimulatory factor-1 or supernatant from the DC-induced primary anti-Mls-MLR. In the primary anti-Mls-MLR, both purified DC and LPS/dextran-stimulated B blasts were found to elicit vigorous T cell proliferative responses. Much weaker responses were elicited by unseparated spleen cells. The stimulation of the primary anti-Mls-MLR by purified DC was further confirmed by producing Mls-specific T cell clones which were preferentially stimulated by DC. Autologous (Mlsb) DC were found to markedly enhance the primary anti-Mls-MLR response to small numbers of Mlsa B blasts. Thus, DC possess other "accessory cell" properties that augment the primary anti-Mls-MLR despite the predicted low level of Mls determinant expression on DC based on the results obtained with Mls-reactive hybridomas. Possible accessory cell properties of DC relevant to this phenomenon are discussed.

Animals↗