Biomedical subjects

Søren Buus

Publications and source records attributed to Søren Buus.

At least 19 recordsLinked to original sources

PubMed2006

Identification of an HLA-A*0201 restricted Bcl2-derived epitope expressed on tumors.

A large number of human tumor-associated antigen-derived peptides have been identified that are recognized by CTLs in a MHC-I restricted fashion. The apoptosis inhibitory protein Bcl2 is overexpressed in many human cancers as part of their neoplastic phenotype. Since inhibition or loss of Bcl2 expression might impair tumor growth and survival, this protein may serve as a rational target for vaccine-induced CTL responses. By Western blot technique, we screened a panel of established human tumor cell lines for proteins involved in the apoptotic process. Two of eight tumor cell lines, a B lymphoma (Loukes) and a colon carcinoma (CCL220) cell line showed increased Bcl2 protein expression whereas the majority of tumor cell lines expressed proapoptotic proteins. Neither fibroblasts nor peripheral blood mononuclear cells showed Bcl2 expression. An HLA-A*0201 restricted CTL epitope was deduced in silica from the amino acid sequence of the Bcl2 protein and its binding affinity for HLA-A*0201 was confirmed using a biochemical binding assay. We here demonstrate that the 9-mer peptide Bcl2(85-93) induces specific CTL reactivity in immunized C57-A2K(b) or -A2D(b) tg mice. These Bcl2(85-93) specific CTLs react with and lyse Bcl2-expressing human colon carcinoma CCL220 cells which have been transfected with a chimeric HLA-A*0201/H2-K(b) DNA construct similar to that expressed in the transgenic mice. Based on these observations, we suggest that Bcl2(85-93) may be a target for immune therapy.

Amino Acid Sequence↗

PubMed2006

Structure of HLA-A*1101 in complex with a hepatitis B peptide homologue.

A high-resolution structure of the human MHC-I molecule HLA-A*1101 is presented in which it forms a complex with a sequence homologue of a peptide that occurs naturally in hepatitis B virus DNA polymerase. The sequence of the bound peptide is AIMPARFYPK, while that of the corresponding natural peptide is LIMPARFYPK. The peptide does not make efficient use of the middle E pocket for binding, which leads to a rather superficial and exposed binding mode for the central peptide residues. Despite this, the peptide binds with high affinity (IC50 of 31 nM).

Amino Acid Sequence↗

PubMed2006

Crystal structures of two peptide-HLA-B*1501 complexes; structural characterization of the HLA-B62 supertype.

MHC class I molecules govern human cytotoxic T cell responses. Their specificity determines which peptides they sample from the intracellular protein environment and then present to human cytotoxic T cells. More than 1100 different MHC class I proteins have been found in human populations and it would be a major undertaking to address each of these specificities individually. Based upon their peptide binding specificity, they are currently subdivided into 12 supertypes. Several of these HLA supertypes have not yet been described at the structural level. To support a comprehensive understanding of human immune responses, the structure of at least one member of each supertype should be determined. Here, the structures of two immunogenic peptide-HLA-B*1501 complexes are described. The structure of HLA-B*1501 in complex with a peptide (LEKARGSTY, corresponding to positions 274-282 in the Epstein-Barr virus nuclear antigen-3A) was determined to 2.3 A resolution. The structure of HLA-B*1501 in complex with a peptide (ILGPPGSVY) derived from human ubiquitin-conjugating enzyme-E2 corresponding to positions 91-99 was solved to 1.8 A resolution. Mutual comparisons of these two structures with structures from other HLA supertypes define and explain the specificity of the P2 and P9 peptide anchor preferences in the B62 HLA supertype. The P2 peptide residue binds to the B-pocket in HLA-B*1501. This pocket is relatively large because of the small Ser67 residue located at the bottom. The peptide proximal part of the B-pocket is hydrophobic, which is consistent with P2 anchor residue preference for Leu. The specificity of the B-pocket is determined by the Met45, Ile66 and Ser67 residues. The apex of the B-pocket is hydrophilic because of the Ser67 residue. The P9 peptide residue binds to the F-pocket in HLA-B*1501. The residues most important for the specificity of this pocket are Tyr74, Leu81, Leu95, Tyr123 and Trp147. These residues create a hydrophobic interior in the F-pocket and their spatial arrangement makes the pocket capable of containing large, bulky peptide side chains. Ser116 is located at the bottom of the F-pocket and makes the bottom of this pocket hydrophilic. Ser116, may act as a hydrogen-bonding partner and as such is a perfect place for binding of a Tyr9 peptide residue. Thus, based on structure information it is now possible to explain the peptide sequence specificity of HLA-B*1501 as previously determined by peptide binding and pool sequencing experiments.

Binding Sites↗

PubMed2006

The effect of a therapeutic dendritic cell-based cancer vaccination depends on the blockage of CTLA-4 signaling.

Dendritic cells (DCs) were pulsed with the H-2K(b) binding OVA(257-264)-peptide (SIINFEKL), and used as one single-injection vaccine in combination with anti-CTLA-4 monoclonal antibody (mAb) to treat mice inoculated 3 days previously with 3x10(5) E.G7-OVA lymphoma cells. Neither DC vaccination nor CTLA-4 blockage alone prevented tumor growth in tumor challenged mice. In contrast, the combination of one vaccination and injection of anti-CTLA-4 mAb lead to rejection or retarded tumor growth in more than 60% of the mice. The OVA-transgene or the SIINFEKL-epitope was not lost in the progressing tumors of vaccinated mice, however, the highest degree of anti-SIINFEKL reactivity of host CTLs in an IFN-gamma ELISPOT assay was found only in mice showing complete tumor rejection. Vaccinated mice having rejected E.G7-OVA tumors were capable of rejecting subsequent challenges with 1x10(6) E.G7-OVA tumor cells, and later on these mice even rejected wild-type EL-4 tumor cells indicating that tumor epitope spreading takes place during the process of vaccination-induced E.G7-OVA rejection. In agreement with these observations, mice having rejected E.G7-OVA tumors showed long lasting CTL memory in spleen and bone marrow towards both the SIINFEKL-peptide and other EL-4-derived tumor rejecting epitopes.

Animals↗

PubMed2006

Conflicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope.

Cytotoxic T lymphocytes (CTLs) are critical for the control of human immunodeficiency virus, but containment of virus replication can be undermined by mutations in CTL epitopes that lead to virus escape. We analyzed the evolution in vivo of an immunodominant, HLA-A2-restricted CTL epitope and found two principal, diametrically opposed evolutionary pathways that exclusively affect T cell-receptor contact residues. One pathway was characterized by acquisition of CTL escape mutations and the other by selection for wild-type amino acids. The pattern of CTL responses to epitope variants shaped which variant(s) prevailed in the virus population. The pathways notably influenced the amount of plasma virus, as patients with efficient CTL selection had lower plasma viral loads than did patients without efficient selection. Thus, viral escape from CTL responses does not necessarily correlate with disease progression.

Adult↗

PubMed2006

Effect of masker-fringe onset asynchrony on overshoot (L).

The study examines how overshoot is influenced by masker-signal onset asynchrony when the masker contains frequencies above or below the signal frequency. Masked thresholds were measured for a 2-ms tone at 5 kHz. The measurements were made in a reference condition with a narrow center-band (CB) noise masker (4590-5464 Hz), and in conditions with either a low-fringe (1900-4590 Hz) or a high-fringe noise band (5500-11 000 Hz) added to the CB. The signal always came on 2 ms after the onset of the CB. The time interval, between fringe and signal onsets varied from -98 ms (signal onset before fringe onset) to +502 ms (signal onset after fringe onset). Results show that overshoot is largest, 8-11 dB, for a high fringe with onset occurring between 8 ms before the signal onset and 12 ms after it. Thus, pronounced overshoot is observed even when the high fringe is gated on after the signal's offset. Low fringes produce no more than 4 dB of overshoot, much less than high fringes produce. The finding of pronounced overshoot elicited by a high fringe presented shortly after the end of the signal suggests that overshoot is governed, at least in part, by central mechanisms.

Acoustic Stimulation↗

PubMed2006

Identification of MHC class I H-2 Kb/Db-restricted immunogenic peptides derived from retinal proteins.

PURPOSE: To identify H-2 Kb/Db-binding immunogenic peptides derived from retinal proteins. METHODS: Computer-based prediction was used to identify potentially H-2 Kb/Db-binding peptides derived from the interphotoreceptor retinol-binding protein (IRBP), soluble retinal antigen (S-antigen), recoverin, phosducin, and pigment epithelium-derived factor (PEDF). The affinity of the peptides was analyzed by their abilities to upregulate the expression of major histocompatibility complex (MHC) class I on TAP-deficient cells (RMA-S cells) with flow cytometry. C57BL/6 mice were immunized subcutaneously, with individual peptides in incomplete Freund's adjuvant (IFA). Eight days after immunization, splenocytes were isolated for cytotoxic T-lymphocyte (CTL) analysis. A 51chromium-release assay was used to detect specific CTL reactivity generated in the cultures. Eyes were enucleated for histopathological analysis on day 21 after immunization with IRBP or IRBP and the immunogenic peptides. RESULTS: All the 21 predicted peptides were found to upregulate expression of H-2 Kb/Db on RMA-S cells. Five peptides, the two IRBP-derived peptides IRBP89-96 and IRBP(101-108), and the three PEDF-derived peptides, PEDF389-397, PEDF139-147, and PEDF272-279, induced specific CTL responses in vivo, whereas the remaining 16 peptides, including 5 IRBP-derived peptides, 5 S-antigen-derived peptides, 1 recoverin-derived peptide, 1 phosducin-derived peptide, and 4 PEDF-derived peptides, did not induce specific CTL reactivity. The immunogenic peptides alone did not induce inflammation in the eyes, but they could enhance severity of uveitis induced by IRBP. CONCLUSIONS: Five of 21 H-2 Kb/Db-binding retinal protein-derived peptides were found to be immunogenic, suggesting that these peptides could function as autoantigenic epitopes in the development of inflammatory eye diseases, such as uveitis.

Animals↗

PubMed2005

Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology.

Controlled and oriented immobilisation of proteins for biosensor purposes is of extreme interest since this provides more efficient sensors with a larger density of active binding sites per area compared to sensors produced by conventional immobilisation. In this paper oriented coupling of a major histocompatibility complex (MHC class I) to a sensor surface is presented. The coupling was performed using light assisted immobilisation--a novel immobilisation technology which allows specific opening of particular disulphide bridges in proteins which then is used for covalent bonding to thiol-derivatised surfaces via a new disulphide bond. Light assisted immobilisation specifically targets the disulphide bridge in the MHC-I molecule alpha(3)-domain which ensures oriented linking of the complex with the peptide binding site exposed away from the sensor surface. Structural analysis reveals that a similar procedure can be used for covalent immobilisation of MHC class II complexes. The results open for the development of efficient T cell sensors, sensors for recognition of peptides of pathogenic origin, as well as other applications that may benefit from oriented immobilisation of MHC proteins.

Adsorption↗

PubMed2005

High-resolution structure of HLA-A*1101 in complex with SARS nucleocapsid peptide.

The structure of the human MHC-I molecule HLA-A*1101 in complex with a nonameric peptide (KTFPPTEPK) has been determined by X-ray crystallography to 1.45 A resolution. The peptide is derived from the SARS-CoV nucleocapsid protein positions 362-370 (SNP362-370). It is conserved in all known isolates of SARS-CoV and has been verified by in vitro peptide-binding studies to be a good to intermediate binder to HLA-A*0301 and HLA-A*1101, with IC50 values of 70 and 186 nM, respectively [Sylvester-Hvid et al. (2004), Tissue Antigens, 63, 395-400]. In terms of the residues lining the peptide-binding groove, the HLA-A*1101-SNP362-370 complex is very similar to other known structures of HLA-A*1101 and HLA-A*6801. The SNP362-370 peptide is held in place by 17 hydrogen bonds to the alpha-chain residues and by nine water molecules which are also tightly bound in the peptide-binding groove. Thr6 of the peptide (Thr6p) does not make efficient use of the middle (E) pocket. For vaccine development, there seems to be a potential for optimization targeted at this position. All residues except Thr2p and Lys9p are accessible for T-cell recognition.

Amino Acid Sequence↗

PubMed2005

An integrative approach to CTL epitope prediction: a combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions.

Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have also been developed for predicting the antigen-processing steps preceding MHC class I binding, including proteasomal cleavage and transporter associated with antigen processing (TAP) transport efficiency. Here, we use a dataset obtained from the SYFPEITHI database to show that a method integrating predictions of MHC class I binding affinity, TAP transport efficiency, and C-terminal proteasomal cleavage outperforms any of the individual methods. Using an independent evaluation dataset of HIV epitopes from the Los Alamos database, the validity of the integrated method is confirmed. The performance of the integrated method is found to be significantly higher than that of the two publicly available prediction methods BIMAS and SYFPEITHI. To identify 85% of the epitopes in the HIV dataset, 9% and 10% of all possible nonamers in the HIV proteins must be tested when using the BIMAS and SYFPEITHI methods, respectively, for the selection of candidate epitopes. This number is reduced to 7% when using the integrated method. In practical terms, this means that the experimental effort needed to identify an epitope in a hypothetical protein with 85% probability is reduced by 20-30% when using the integrated method. The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php.

ATP-Binding Cassette Transporters↗

PubMed2005

A novel system for continuous protein refolding and on-line capture by expanded bed adsorption.

A novel two-step protein refolding strategy has been developed, where continuous renaturation-bydilution is followed by direct capture on an expanded bed adsorption (EBA) column. The performance of the overall process was tested on a N-terminally tagged version of human beta2-microglobulin (HAT-hbeta2m) both at analytical, small, and preparative scale. In a single scalable operation, extracted and denatured inclusion body proteins from Escherichia coli were continuously diluted into refolding buffer, using a short pipe reactor, allowing for a defined retention and refolding time, and then fed directly to an EBA column, where the protein was captured, washed, and finally eluted as soluble folded protein. Not only was the eluted protein in a correctly folded state, the purity of the HAThbeta2m was increased from 34% to 94%, and the product was concentrated sevenfold. The yield of the overall process was 45%, and the product loss was primarily a consequence of the refolding reaction rather than the EBA step. Full biological activity of HAT-hbeta2m was demonstrated after removal of the HAT-tag. In contrast to batch refolding, a continuous refolding strategy allows the conditions to be controlled and maintained throughout the process, irrespective of the batch size; i.e., it is readily scalable. Furthermore, the procedure is fast and tolerant toward aggregate formation, a common complication of in vitro protein refolding. In conclusion, this system represents a novel approach to small and preparative scale protein refolding, which should be applicable to many other proteins.

Adsorption↗

PubMed2004

Changes in protein expression in p53 deleted spontaneous thymic lymphomas.

By the use of high-resolution two-dimensional gel electrophoresis and computerized image analysis we investigated and compared the expression of cellular proteins from p53 positive (+/+) mouse thymocytes, p53-/- thymocytes before neoplastic transformation, and from cell lines derived from two spontaneous p53-/- thymic lymphomas, SM5 and SM7. A total of around 1500 proteins were detected on individual gels. Only changes in protein expression by a factor of 2 or more were considered. In the thymic lymphoma cells 3-5% of the proteins were found to be differentially regulated when compared with the protein expression in p53+/+ and p53-/- thymocytes. Only a minority (13 proteins) of the quantitatively changed proteins were common for the two thymic lymphoma cell lines, suggesting that the p53 deficiency mainly results in genetic dysfunctions which are individual for a given tumor. Two of the detected proteins increased their expression levels by more than 10 times from the p53+/+ to the p53-/- thymocytes and these high expression levels were also found in thymic lymphomas. The two proteins were identified by mass spectrometry as acidic ribosomal phosphoprotein P0 and a 33-kDa protein with a primary structure containing motifs of the glyoxalase-bleomycin resistance protein family (MDR) as deduced from the cDNA.

Animals↗

PubMed2004

Vaccination with p53-peptide-pulsed dendritic cells, of patients with advanced breast cancer: report from a phase I study.

Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumour-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer vaccine. Our preclinical studies have shown that wild-type p53-derived HLA-A2-binding peptides are able to activate human T cells and that the generated effector T cells are cytotoxic to human HLA-A2+, p53+ tumour cells. In this phase I pilot study, the toxicity and efficacy of autologous dendritic cells (DCs) loaded with a cocktail of three wild-type and three modified p53 peptides are being analysed in six HLA-A2+ patients with progressive advanced breast cancer. Vaccinations were well tolerated and no toxicity was observed. Disease stabilisation was seen in two of six patients, one patient had a transient regression of a single lymph node and one had a mixed response. ELISpot analyses showed that the p53-peptide-loaded DCs were able to induce specific T-cell responses against modified and unmodified p53 peptides in three patients, including two of the patients with a possible clinical benefit from the treatment. In conclusion, the strategy for p53-DC vaccination seems safe and without toxicity. Furthermore, indications of both immunologic and clinical effect were found in heavily pretreated patients with advanced breast cancer. An independent clinical effect of repeated administration of DCs and IL-2 can not of course be excluded; further studies are necessary to answer these questions.

Adult↗

PubMed2004

Definition of supertypes for HLA molecules using clustering of specificity matrices.

Major histocompatibility complex (MHC) proteins are encoded by extremely polymorphic genes and play a crucial role in immunity. However, not all genetically different MHC molecules are functionally different. Sette and Sidney (1999) have defined nine HLA class I supertypes and showed that with only nine main functional binding specificities it is possible to cover the binding properties of almost all known HLA class I molecules. Here we present a comprehensive study of the functional relationship between all HLA molecules with known specificities in a uniform and automated way. We have developed a novel method for clustering sequence motifs. We construct hidden Markov models for HLA class I molecules using a Gibbs sampling procedure and use the similarities among these to define clusters of specificities. These clusters are extensions of the previously suggested ones. We suggest splitting some of the alleles in the A1 supertype into a new A26 supertype, and some of the alleles in the B27 supertype into a new B39 supertype. Furthermore the B8 alleles may define their own supertype. We also use the published specificities for a number of HLA-DR types to define clusters with similar specificities. We report that the previously observed specificities of these class II molecules can be clustered into nine classes, which only partly correspond to the serological classification. We show that classification of HLA molecules may be done in a uniform and automated way. The definition of clusters allows for selection of representative HLA molecules that can cover the HLA specificity space better. This makes it possible to target most of the known HLA alleles with known specificities using only a few peptides, and may be used in construction of vaccines. Supplementary material is available at http://www.cbs.dtu.dk/researchgroups/immunology/supertypes.html.

Amino Acid Motifs↗

PubMed2004

Improved prediction of MHC class I and class II epitopes using a novel Gibbs sampling approach.

MOTIVATION: Prediction of which peptides will bind a specific major histocompatibility complex (MHC) constitutes an important step in identifying potential T-cell epitopes suitable as vaccine candidates. MHC class II binding peptides have a broad length distribution complicating such predictions. Thus, identifying the correct alignment is a crucial part of identifying the core of an MHC class II binding motif. In this context, we wish to describe a novel Gibbs motif sampler method ideally suited for recognizing such weak sequence motifs. The method is based on the Gibbs sampling method, and it incorporates novel features optimized for the task of recognizing the binding motif of MHC classes I and II. The method locates the binding motif in a set of sequences and characterizes the motif in terms of a weight-matrix. Subsequently, the weight-matrix can be applied to identifying effectively potential MHC binding peptides and to guiding the process of rational vaccine design. RESULTS: We apply the motif sampler method to the complex problem of MHC class II binding. The input to the method is amino acid peptide sequences extracted from the public databases of SYFPEITHI and MHCPEP and known to bind to the MHC class II complex HLA-DR4(B1*0401). Prior identification of information-rich (anchor) positions in the binding motif is shown to improve the predictive performance of the Gibbs sampler. Similarly, a consensus solution obtained from an ensemble average over suboptimal solutions is shown to outperform the use of a single optimal solution. In a large-scale benchmark calculation, the performance is quantified using relative operating characteristics curve (ROC) plots and we make a detailed comparison of the performance with that of both the TEPITOPE method and a weight-matrix derived using the conventional alignment algorithm of ClustalW. The calculation demonstrates that the predictive performance of the Gibbs sampler is higher than that of ClustalW and in most cases also higher than that of the TEPITOPE method.

Algorithms↗

PubMed2004

Identifying multiple tumor-specific epitopes from large-scale screening for overexpressed mRNA.

The rationale of a T-cell epitope-based approach to cancer treatment is primarily rooted in the hypothesis that CD8(+) cytotoxic T cells (CTLs) can be manipulated to specifically identify and kill cancer cells. A solid understanding of CTL specificity and activation is a fundamental requirement for tumor immunotherapy. The means to identify tumor-specific CTL epitopes and to monitor corresponding CTL responses are important enabling technologies. Recent advances in these enabling technologies include their ability to exploit genomic, transcriptomic and proteomic information. These advances constitute new opportunities, which will enable approaches to tumor immunotherapy that encompass both human diversity and tumor heterogeneity, increase the efficacy of tumor immunotherapy and potentially provide the opportunity for individualized therapy.

Epitopes, T-Lymphocyte↗

PubMed2004

The effects of window delay, delinearization, and frequency on tone-burst otoacoustic emission input/output measurements.

Tone-burst otoacoustic emissions (TBOAEs) are a potential tool for objectively examining cochlear activity in humans. However, their use requires knowledge of how the TBOAE input/output depends on measurement and analysis paradigms. The present experiment examined the effect of variations in response-window timing, response delinearization, and local changes in stimulus frequency on TBOAE input/output measurement. None of these experimental manipulations had a profound effect on TBOAE measurements as long as reasonable parameter choices were made. Nonetheless, judicial choice of the experimental parameters can optimize the assessment of BM I/O functions. It is concluded that the consistency of TBOAE I/O across the parameters tested makes it a viable tool to consider for examining human cochlear activity.

Acoustic Stimulation↗

PubMed2004

Spectral loudness summation and simple reaction time.

The present study evaluates the relation between loudness and simple reaction time (RT). Loudness matches between a narrowband noise (125 Hz wide) and a broadband noise (1500 Hz) were made at levels from near threshold to near 100 dB SPL. Over a similarly wide range of levels, RT to each of the noise bands was also measured. As reported often in previous loudness-matching studies, loudness summation depended strongly on level. With increasing SPL, the level difference between the noises needed to keep them equally loud first increased, to around 10 dB at moderate levels, and then decreased. Except for one listener, the RT data show the same pattern. The level difference needed to keep RT to the two noises the same first increased and then decreased. These results show that RT is closely related to loudness, but not to sensation level. If RT depended on sensation level, the level difference between the two noises needed to achieve equal RT would not change with SPL because the difference in sensation level between two sounds is a constant. Overall, the average results provide strong support for the contention that simple RT and loudness are closely related.

Adult↗