Functional T-cell Epitopes Research Articles

Humanized Recombinant Immunotoxin Targeting hCG Demonstrates Therapeutic Potential for Advanced Stage Difficult to Treat Cancers

We report the development of an immunotherapeutic molecule, a humanized immunotoxin, for treating hCG-expressing advanced-stage cancers. PiPP, a high-affinity anti-hCG monoclonal antibody, is used in the immunotoxin for ‘homing’ hCG-positive cancer cells. The deimmunized (DI) form of α-Sarcin, a fungal-origin toxin that lacks functional T-cell epitopes, is used in the design to ensure minimal immunogenicity of the immunotoxin for repetitive use in humans. A single-chain variable fragment (scFv) of PiPP was constructed by linking the humanized VH and VL regions of the antibody. The scFv part of the antibody was further linked to the toxin α-Sarcin (DI) at the gene level and expressed as a recombinant protein in E. coli. The immunotoxin was purified from the bacterial cell lysate and analyzed for binding and cytotoxicity to hCG-secreting colorectal and pancreatic cancer cells. The results showed that the scFv(PiPP)-Sarcin immunotoxin was able to bind to colorectal and pancreatic cancer cells and killed approximately 85% of the cells. In vivo testing of the immunotoxin produced results similar to those of in vitro testing against colorectal adenocarcinoma-induced tumors. This immunotoxin could be a promising immunotherapeutic agent for treating colorectal, pancreatic and other terminal-stage hCG-expressing cancers.

The Parallel Presentation of Two Functional CTL Epitopes Derived from the O and Asia 1 Serotypes of Foot-and-Mouth Disease Virus and Swine SLA-2*HB01: Implications for Universal Vaccine Development.

Foot-and-mouth disease virus (FMDV) poses a significant threat to the livestock industry. Through their recognition of the conserved epitopes presented by the swine leukocyte antigen (SLA), T cells play a pivotal role in the antiviral immunity of pigs. Herein, based on the peptide binding motif of SLA-2*HB01, from an original SLA-2 allele, a series of functional T-cell epitopes derived from the dominant antigen VP1 of FMDV with high binding capacity to SLA-2 were identified. Two parallel peptides, Hu64 and As64, from the O and Asia I serotypes, respectively, were both crystallized with SLA-2*HB01. Compared to SLA-1 and SLA-3, the SLA-2 structures showed the flexibility of residues in the P4, P6, and P8 positions and in their potential interface with TCR. Notably, the peptides Hu64 and As64 adopted quite similar overall conformation when bound to SLA-2*HB01. Hu64 has two different conformations, a more stable 'chair' conformation and an unstable 'boat' conformation observed in the two molecules of one asymmetric unit, whereas only a single 'chair' conformation was observed for As64. Both Hu64 and As64 could induce similar dominant T-cell activities. Our interdisciplinary study establishes a basis for the in-depth interpretation of the peptide presentation of SLA-I, which can be used toward the development of universal vaccines.

Fusion of foreign T-cell epitopes and addition of TLR agonists enhance immunity against Neospora caninum profilin in cattle.

We demonstrated recently that immunization with recombinant Neospora caninum profilin (rNcPRO) induces limited protection and a regulatory T-cell response in mice. The aim of this study was to evaluate the immune response elicited by rNcPRO in cattle and assess a strategy to enhance its immunogenicity, combining the addition of T-cell epitopes and immune modulators. We developed a chimeric recombinant profilin fused to functional T-cell epitopes present in the N-terminal sequence of vesicular stomatitis virus (VSV) glycoprotein G (rNcPRO/G). Groups of three cattle were immunized with two doses (2weeks apart) of rNcPRO or rNcPRO/G formulated with alum hydroxide or a nanoparticulated soya-based adjuvant enriched with Toll-like receptor (TLR) 2 and TLR9 agonists, aimed to tackle the MyD88 pathway (AVECplus). rNcPRO induced only a primary immune response (IgM mediated), while antibodies in rNcPRO/G-vaccinated animals switched to IgG1 after the booster. The vaccine formulated with rNcPRO/G and AVECplus improved the production of systemic IFN-γ and induced long-term recall B-cell responses. Overall, our study provides data supporting the use of T-cell epitopes from VSV glycoprotein G and TLR agonists to enhance and modulate immunity to peptide antigens in bovines, particularly when using small proteins from parasites for which immune responses are usually feeble.

An efficient T-cell epitope discovery strategy using in silico prediction and the iTopia assay platform

Functional T-cell epitope discovery is a key process for the development of novel immunotherapies, particularly for cancer immunology. In silico epitope prediction is a common strategy to try to achieve this objective. However, this approach suffers from a significant rate of false-negative results and epitope ranking lists that often are not validated by practical experience. A high-throughput platform for the identification and prioritization of potential T-cell epitopes is the iTopiaTM Epitope Discovery SystemTM, which allows measuring binding and stability of selected peptides to MHC Class I molecules. So far, the value of iTopia combined with in silico epitope prediction has not been investigated systematically. In this study, we have developed a novel in silico selection strategy based on three criteria: (1) predicted binding to one out of five common MHC Class I alleles; (2) uniqueness to the antigen of interest; and (3) increased likelihood of natural processing. We predicted in silico and characterized by iTopia 225 candidate T-cell epitopes and fixed-anchor analogs from three human tumor-associated antigens: CEA, HER2 and TERT. HLA-A2-restricted fragments were further screened for their ability to induce cell-mediated responses in HLA-A2 transgenic mice. The iTopia binding assay was only marginally informative while the stability assay proved to be a valuable experimental screening method complementary to in silico prediction. Thirteen novel T-cell epitopes and analogs were characterized and additional potential epitopes identified, providing the basis for novel anticancer immunotherapies. In conclusion, we show that combination of in silico prediction and an iTopia-based assay may be an accurate and efficient method for MHC Class I epitope discovery among tumor-associated antigens.

Characterization of immunologic properties of a second HLA-A2 epitope from a granule protease in CML patients and HLA-A2 transgenic mice

AbstractThe serine proteases, neutrophil elastase (HNE) and proteinase 3 (PR3), are aberrantly expressed in human myeloid leukemias. T-cell responses to these proteins have been correlated with remission in patients with chronic myeloid leukemia (CML). Human PR3/HNE-specific CD8+ T cells predominantly recognize a nonameric HLA-A2–restricted T-cell epitope called PR1 which is conserved in both Ags. However, CML patients have CD8+ T cells in peripheral blood recognizing an additional HLA-A2 epitope termed PR2. To assess immunologic properties of these Ags, novel recombinant vaccinia viruses (rVV) expressing PR3 and HNE were evaluated in HLA-A2 transgenic (Tg) mice (HHDII). Immunization of HHDII mice with rVV-PR3 elicited a robust PR3-specific CD8+ T-cell response dominated by recognition of PR2, with minimal recognition of the PR1 epitope. This result was unexpected, because the PR2 peptide has been reported to bind poorly to HLA. To account for these findings, we proposed that HHDII mice negatively selected PR1-specific T cells because of the presence of this epitope within murine PR3 and HNE, leading to immunodominance of PR2-specific responses. PR2-specific splenocytes are cytotoxic to targets expressing naturally processed PR3, though PR1-specific splenocytes are not. We conclude that PR2 represents a functional T-cell epitope recognized in mice and human leukemia patients. These studies are registered at www.clinicaltrials.gov as NCT00716911.

Characterisation of the membrane affinity of an isoniazide peptide conjugate by tensiometry, atomic force microscopy and sum-frequency vibrational spectroscopy, using a phospholipid Langmuir monolayer model

Tensiometry, sum-frequency vibrational spectroscopy, and atomic force microscopy were employed to assess the cell penetration ability of a peptide conjugate of the antituberculotic agent isoniazide. Isoniazide was conjugated to peptide (91)SEFAYGSFVRTVSLPV(106), a functional T-cell epitope of the immunodominant 16 kDa protein of Mycobacterium tuberculosis. As a simple but versatile model of the cell membrane a phospholipid Langmuir monolayer at the liquid/air interface was used. Changes induced in the structure of the phospholipid monolayer by injection of the peptide conjugate into the subphase were followed by tensiometry and sum-frequency vibrational spectroscopy. The drug penetrated lipid films were transferred to a solid support by the Langmuir-Blodgett technique, and their structures were characterized by atomic force microscopy. Peptide conjugation was found to strongly enhance the cell penetration ability of isoniazide.

Elimination of a Human T-cell Region in Staphylokinase by T-cell Screening and Computer Modeling

Staphylokinase is a potent highly fibrin-selective thrombolytic agent, but it induces a humoral immune response in most treated patients. Staphylokinase-specific T-lymphocytes can be found in normal healthy individuals, from whom a large panel of staphylokinase-specific T-cells were cloned. The staphylokinase amino acid sequence 71-87 was widely recognized, as it induced proliferation of T-cell clones isolated from 90% of the donors. Computer modeling of this area, threaded as 11-mer peptides within the peptide-binding groove of the major HLA-DR alleles, indicated two putative partially overlapping binding sequences. The region-(71-87)-specific T-cell clones recognized either one or the other minimal peptide, confirming that both sequences could be functional T-cell epitopes. Furthermore, to guide the mutagenesis to eliminate T-cell reactivity, the contribution of each residue to the HLA-DR-anchoring and T-cell receptor exposure was evaluated for both binding motifs. Computer calculations combined with functional assays resulted in the design of staphylokinase-variants, including 2 to 4 amino acid substitutions in the region 71-87. These variants were no longer recognized by the region-(71-87)-specific T-cell clones, and importantly no new staphylokinase-variant-specific cellular immune response could be measured.

Adoptive transfer of CD4+ T cells specific for subunit A of Helicobacter pylori urease reduces H. pylori stomach colonization in mice in the absence of interleukin-4 (IL-4)/IL-13 receptor signaling.

Protection in the murine model of Helicobacter pylori infection may be mediated by CD4+ T cells, but the mechanism remains unclear. To better understand how protection occurs in this model, we generated and characterized H. pylori urease-specific CD4+ T cells from BALB/c mice immunized with Salmonella enterica serovar Typhimurium expressing H. pylori urease (subunits A and B). The CD4+ T cells were found to be specific for subunit A (UreA). Upon antigen-specific stimulation, expression of interleukin 4 (IL-4), IL-10, gamma interferon (IFN-gamma), and tumor necrosis factor alpha was induced. Immunocytochemical analysis showed that the majority of cells produced IFN-gamma and IL-10. Adoptive transfer of the UreA-specific CD4+ T cells into naive syngeneic recipients led to a threefold reduction in the number of bacteria in the recipient group when compared to that in the nonrecipient group. Stomach colonization was also reduced significantly after transfer of these cells into patently infected mice. Adoptive transfer of UreA-specific CD4+ T cells into IL-4 receptor alpha chain-deficient BALB/c mice indicated that IL-4 and IL-13 were not critical in the control of bacterial load. In addition, synthetic peptides were used to identify three functional T-cell epitopes present in subunit A which were recognized by the UreA-specific T cells. Analysis of H. pylori-specific cellular immune responses in recipient challenged and nonrecipient infected mice indicated a strong local restriction of the response in infected animals. The implications of these findings for the mechanism of protection and the development of peptide-based vaccination are discussed.

Temporal Loss of Nef-Epitope CTL Recognition following Macaque Lipopeptide Immunization and SIV Challenge

To address the subtle interactions between antiviral cytotoxic T-cell (CTL) immune responses and the evolution of viral quasispecies variants in vivo, we performed a longitudinal study in a simian immunodeficiency virus (SIV)-infected rhesus macaque that had a long experimental SIV infection before developing simian AIDS. Before being infected with SIV, this animal was immunized with a mixture of seven lipopeptides derived from SIV Nef and Gag proteins and showed a bispecific antiviral CTL response directed toward Nef 169–178 and 211–225 peptides. After SIV infection, CTL activity against the Nef 169–178 epitope was no longer detectable, as assessed from peripheral blood mononuclear cells stimulated by autologous SIV. CTL activity against the 211–225 epitope was lost after 3 months, and an additional CTL response to the amino acids 112–119 Nef epitope emerged. Analysis of the Nef proviral sequence revealed the presence of immune escape variants first in the 211–225 epitope and much later in the 112–119 epitope. In contrast, epitope 169–178 showed only two mutations among all viral sequencing performed. We conclude that in this macaque, bispecific CTL exerted a strong selective pressure and escape virus mutants finally emerged. We identified CTL recognizing a conserved Nef epitope 112–119 (SYKLAIDM), essential for viral replication, which could be associated with a prolonged AIDS-free period. These results stress the importance of the induction of broader multispecific CTLs directed against highly conserved and functional T-cell epitopes by vaccination, with the aim of keeping HIV infection in check.

Detection and Characterization of Functional T-Cell Epitopes on the Structural Proteins VP2, VP3, and VP4 of Foot and Mouth Disease Virus O1 Campos

Foot and mouth disease virus (FMDV) is the cause of a widespread infectious disease affecting cloven-hoofed animals. It is controlled by vaccination with immune-inactivated virus grown in tissue culture. However, peptide vaccines represent a safer alternative to the current virus-inactivated immunogens. Their design requires the identification and evaluation of the sequences recognized by T- and B-lymphocytes. Four structural proteins, VP1, VP2, VP3, and VP4, comprise the viral capsid of the FMDV, but only VP1 has been extensively studied regarding the existence of relevant T-cell epitopes. Here, we utilize a murine model to present a functional T-cell epitope mapping on the complete sequences of VP2, VP3, and VP4 of FMDV O1 Campos. We used two in vitro assays to describe 13 amino acid sequences, each one of them including at least one T-cell epitope. The in vivo T-cell helper function of these sequences was studied in an adoptive cell-transfer assay in mice. Immunization experiments with a fusion peptide containing one of the sequences characterized were also done comparing the helper activity of this sequence with other T-cell epitopes included in the major immunogenic region of VP1.

Cell surface presentation of recombinant (poly-) peptides including functional T-cell epitopes by the AIDA autotransporter system

For the efficient surface presentation and release of virulence factors especially pathogenic Gram-negative bacteria have developed several distinct secretion mechanisms. An increasing number of pathogens in various species employs a mechanism denoted the ‘autotransporter’ pathway. This pathway is characterised by an outer membrane translocator module representing the C-terminal domain of the transported protein itself. An intriguing potential application of such systems involves the transport and surface expression of recombinant proteins or peptides, like e.g. the presentation of antigens for the generation of live oral vectors as vaccine carriers. Here we report on the incorporation of heterologous (poly-) peptides in permissive sites of the translocator module of the adhesin-involved-in-diffuse-adherence (AIDA) autotransporter system. We demonstrate the presentation of the B subunit of the heat labile enterotoxin of Escherichia coli (LTB) as well as of functional T-cell epitopes of Yersinia enterocolitica heat-shock protein 60 (Y-hsp60) on the surface of E. coli.

Cell surface presentation of recombinant (poly-) peptides including functional T-cell epitopes by the AIDA autotransporter system.

For the efficient surface presentation and release of virulence factors especially pathogenic Gram-negative bacteria have developed several distinct secretion mechanisms. An increasing number of pathogens in various species employs a mechanism denoted the 'autotransporter' pathway. This pathway is characterised by an outer membrane translocator module representing the C-terminal domain of the transported protein itself. An intriguing potential application of such systems involves the transport and surface expression of recombinant proteins or peptides, like e.g. the presentation of antigens for the generation of live oral vectors as vaccine carriers. Here we report on the incorporation of heterologous (poly-) peptides in permissive sites of the translocator module of the adhesin-involved-in-diffuse-adherence (AIDA) autotransporter system. We demonstrate the presentation of the B subunit of the heat labile enterotoxin of Escherichia coli (LTB) as well as of functional T-cell epitopes of Yersinia enterocolitica heat-shock protein 60 (Y-hsp60) on the surface of E. coli.

Different properties of T-cell epitopes within complementarity-determining regions 1 and 2 of idiotypic VH in B-lymphoma.

The idiotypic protein expressed by B-lymphoma cells can evoke an autologous idiotype-specific T-cell response in both animal studies and lymphoma patients. However, the locations within the idiotypic protein of the immune epitopes remains unclear. In this study, we determined the nature of any immune responses generated by peptides corresponding to the complementarity-determining region 1 (CDR1) and complementarity-determining region 2 (CDR2) sequences of idiotypic heavy chain variable region (VH) to identify the clinical potential of the peptides for immunotherapy of lymphoma. We found that a synthetic peptide corresponding to CDR1 sequence contains a functional T-cell epitope able to evoke a major histocompatibility complex (MHC) class II-dependent T-cell proliferative response and cytokine release without direct cytotoxicity for peptide-pulsed target or fresh autologous lymphoma cells. Although a peptide corresponding to CDR2 was able to generate MHC class I and class II dependent T-cell proliferative responses and cytokine release, T-cell epitopes within the peptide were cryptic in the context of intact idiotypic protein. Our results therefore suggest that the CDR1 sequence could be used for the immune targeting of B-lymphoma.

Immunogenicity and cross-reactivity with idiotypic IgA of VH CDR3 peptide in multiple myeloma.

Multiple myeloma idiotypic protein is clone-specific and therefore represents an ideal tumour antigen for immune targeting. In this study we determined whether a synthetic peptide corresponding to the autologous idiotypic VH CDR3 sequence could elicit peptide-specific immune responses in a patient with IgA myeloma. Not unlike B-cell lymphoma, the immune repertoire of the patient contained T cells capable of mounting proliferative and cytotoxic responses to antigen-presenting cells loaded with the CDR3 peptide. Furthermore, the T cells were also able to secrete interferon-gamma upon peptide rechallenge. Antigen recognition by peptide-primed T cells was MHC dependent and could be blocked by antibodies to both monomorphic MHC class I and class II molecules. These results therefore indicate the presence of T-cell epitopes on the VH CDR3 sequence. In addition, CDR3 peptide-primed T cells were also able to mount similar immune responses when rechallenged with the intact IgA idiotypic protein, suggesting that functional T-cell epitopes had been derived from the CDR3 sequence of the idiotypic protein. Our results therefore provide a new perspective to the immunogenicity of the idiotypic protein in myeloma.

Delineation of the minimal hepatitis B surface antigen-specific B- and T-cell epitope contained within an anti-idiotype-derived pentadecapeptide.

A pentadecapeptide (2F10 peptide) is capable of mimicking the group-specific "a" determinant of human hepatitis B surface antigen (HBsAg) at both the B- and the T-cell level. This peptide represents a sequence on the heavy-chain hypervariable region of a monoclonal "internal image" anti-idiotype (anti-id 2F10) that has partial sequence homology to the "a" determinant epitope of HBsAg. To identify the exact location of the B- and T-cell epitopes, four truncated peptides (peptides 1-4) were synthesized. Using these truncated peptides we have identified the minimal sequence (octapeptide 3) that represents a functional B- and T-cell epitope capable of generating HBsAg-specific antibodies and T cells. This to our knowledge represents the first example of a short peptide sequence functioning as both a B- and a T-cell epitope. We have also identified another T-cell epitope (2F10 peptide 4), but this peptide fails to elicit HBsAg-specific B cells and T cells. Thus, the 2F10 pentadecapeptide is composed of two nonoverlapping, functional T-cell epitopes only one of which is HBsAg specific. Since peptide 3 represents the complementarity-determining region and peptide 4 represents the framework region of the anti-id 2F10, we conclude that an 8-aa sequence from the complementarity-determining region of anti-id 2F10 is sufficient for the molecular mimicry of HBsAg. Finally, our experiments suggest that sequences flanking the minimal immunodominant epitope exert a considerable influence on the nature of antigenic processing that occurs and the resultant T-cell reactivity elicited.

Haplotype specific homology scanning algorithim to predict T‐cell epitopes from protein sequences

We present a homology scanning microcomputer program to predict functional T-cell epitopes within proteins. By taking into account particular human or mouse restriction elements the predictions are made haplotype-specific. The generality of this approach is confirmed by (i) identification of well-characterized immunogenic T-cell determinants in lysozyme (ii) search for potential T epitopes on unanalysed proteins like the human beta 2-adrenoreceptor (iii) modification of non-immunogenic peptide sequences in order to generate T-cell determinants.