Biomedical subjects

A Sette

Publications and source records attributed to A Sette.

At least 55 records · Page 3Linked to original sources

PubMed2000

Identification of five different Patr class I molecules that bind HLA supertype peptides and definition of their peptide binding motifs.

We have sequenced the Pan troglodytes class I (Patr) molecules from three common chimpanzees and expressed them as single molecules in a class I-deficient cell line. These lines were utilized to obtain purified class I molecules to define the peptide binding motifs associated with five different Patr molecules. Based on these experiments, as well as analysis of the predicted structure of the B and F polymorphic MHC pockets, we classified five Patr molecules (Patr-A*0101, Patr-B*0901, Patr-B*0701, Patr-A*0602, and Patr-B*1301) within previously defined supertype specificities associated with HLA class I molecules (HLA-A3, -B7, -A1, and -A24 supertypes). The overlap in the binding repertoire between specific HLA and Patr class I molecules was in the range of 33 to 92%, depending on the particular Patr molecule as assessed by the binding of HIV-, hepatitis B virus-, and hepatitis C virus-derived epitopes. Finally, live cell binding assays of nine chimpanzee-derived B cell lines demonstrated that HLA supertype peptides bound to Patr class I molecules with frequencies in the 20-50% range.

Alleles↗

PubMed2000

Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are characterized by early peaks of viraemia that decline as strong cellular immune responses develop. Although it has been shown that virus-specific CD8-positive cytotoxic T lymphocytes (CTLs) exert selective pressure during HIV and SIV infection, the data have been controversial. Here we show that Tat-specific CD8-positive T-lymphocyte responses select for new viral escape variants during the acute phase of infection. We sequenced the entire virus immediately after the acute phase, and found that amino-acid replacements accumulated primarily in Tat CTL epitopes. This implies that Tat-specific CTLs may be significantly involved in controlling wild-type virus replication, and suggests that responses against viral proteins that are expressed early during the viral life cycle might be attractive targets for HIV vaccine development.

AIDS Vaccines↗

PubMed2000

HLA-DR-promiscuous T cell epitopes from Plasmodium falciparum pre-erythrocytic-stage antigens restricted by multiple HLA class II alleles.

Previously, we identified and established the antigenicity of 17 CD8+ T cell epitopes from five P. falciparum Ags that are restricted by multiple common HLA class I alleles. Here, we report the identification of 11 peptides from the same Ags, cicumsporozoite protein, sporozoite surface protein 2, exported protein-1, and liver-stage Ag-1, that bind between at least five and up to 11 different HLA-DR molecules representative of the most common HLA-DR Ags worldwide. These peptides recall lymphoproliferative and cytokine responses in immune individuals experimentally immunized with radiation-attenuated Plasmodium falciparum sporozoites (irradiated sporozoites) or semi-immune individuals naturally exposed to malaria in Irian Jaya or Kenya. We establish that all peptides are recognized by individuals of each of the three populations, and that the frequency and magnitude of helper T lymphocyte responses to each peptide is influenced by the intensity of exposure to P. falciparum sporozoites. Mean frequencies of lymphoproliferative responses are 53.2% (irradiated sporozoites) vs 22.4% (Kenyan) vs 5.8% (Javanese), and mean frequencies of IFN-gamma responses are 66.3% (irradiated sporozoites) vs 27.3% (Kenyan) vs 8. 7% (Javanese). The identification of HLA class II degenerate T cell epitopes from P. falciparum validates our predictive strategy in a biologically relevant system and supports the potential for developing a broadly efficacious epitope-based vaccine against malaria focused on a limited number of peptide specificities.

Adolescent↗

PubMed2000

Induction of AIDS virus-specific CTL activity in fresh, unstimulated peripheral blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen.

The observed role of CTL in the containment of AIDS virus replication suggests that an effective HIV vaccine will be required to generate strong CTL responses. Because epitope-based vaccines offer several potential advantages for inducing strong, multispecific CTL responses, we tested the ability of an epitope-based DNA prime/modified vaccinia virus Ankara (MVA) boost vaccine to induce CTL responses against a single SIVgag CTL epitope. As assessed using both 51Cr release assays and tetramer staining of in vitro stimulated PBMC, DNA vaccinations administered to the skin with the gene gun induced and progressively increased p11C, C-->M (CTPYDINQM)-specific CD8+ T lymphocyte responses in six of six Mamu-A*01+ rhesus macaques. Tetramer staining of fresh, unstimulated PBMC from two of the DNA-vaccinated animals indicated that as much as 0.4% of all CD3+/CD8alpha+ T lymphocytes were specific for the SIVgag CTL epitope. Administration of MVA expressing the SIVgag CTL epitope further boosted these responses, such that 0.8-20.0% of CD3+/CD8alpha+ T lymphocytes in fresh, unstimulated PBMC were now Ag specific. Enzyme-linked immunospot assays confirmed this high frequency of Ag-specific cells, and intracellular IFN-gamma staining demonstrated that the majority of these cells produced IFN-gamma after peptide stimulation. Moreover, direct ex vivo SIV-specific cytotoxic activity could be detected in PBMC from five of the six DNA/MVA-vaccinated animals, indicating that this epitope-based DNA prime/MVA boost regimen represents a potent method for inducing high levels of functionally active, Ag-specific CD8+ T lymphocytes in non-human primates.

Adjuvants, Immunologic↗

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↗

PubMed2000

Vaccination to treat persistent viral infection.

Persistent infections caused by such agents as the human immunodeficiency virus, hepatitis B virus, Epstein-Barr virus, etc., present formidable medical problems. A defining characteristic of these infections is that anti-viral cytotoxic T lymphocytes (CTL) may be lost or, if present, fail to clear the infection. Here we report a vaccination strategy which was successful in generating lytic CTL in persistently infected mice. Vaccination with an immunodominant CTL epitope derived from the nucleoprotein of lymphocytic choriomeningitis virus (LCMV) delivered in the form of a lipopeptide incorporating a universal CD4 helper epitope successfully induced lytic MHC-restricted CTL in mice persistently infected with LCMV since birth. However, induction of such CTL did not eliminate the virus, most likely because the CTL were generated at low frequencies and had 2 to 3 logs lower affinity than CTL generated in uninfected mice inoculated with the vaccine. Both CTL populations from either uninfected or persistently infected mice produced significant and similar amounts of interferon-gamma and IL-6. Vaccine-induced low-affinity CTL were still inadequate at complete removal of the virus when combined with LCMV-specific CD4 helper T lymphocytes. Thus, our results establish that CTL can be generated in persistently infected mice and that a crucial factor for clearing viral infection is the affinity of the CTL.

Adoptive Transfer↗

PubMed2000

4-1BB costimulation is required for protective anti-viral immunity after peptide vaccination.

Peptide vaccination induces T cell activation and cytotoxic T cell development. In an effort to understand what factors can improve immune responses to peptide vaccination, the role of 4-1BB (CD137) costimulation was examined, since 4-1BB has been shown to promote T cell responses in other systems. 4-1BBL-deficient (-/-) and wild-type (+/+) mice were immunized with a lipidated lymphocytic choriomeningitis virus (LCMV) peptide NP396-404. Analysis of peptide-specific responses early after immunization by CTL assay, intracellular IFN-gamma staining, and IFN-gamma enzyme-linked immunospot assay (ELISPOT) indicated that CD8 T cell responses were reduced 3- to 10-fold in the absence of 4-1BB costimulation. Moreover, when agonistic anti-4-1BB Ab was given, CD8 T cell responses in 4-1BBL-/- mice were augmented to levels similar to those in 4-1BBL+/+ mice. Two months after immunization, 4-1BBL+/+ mice still had epitope-specific cells and were protected against viral challenge, demonstrating that peptide vaccination can induce long-term protection. In fact, 70% of CD8 T cells were specific for the immunizing peptide after viral challenge, demonstrating that strong, epitope-specific CD8 T cell responses are generated after peptide vaccination. In contrast, peptide-immunized 4-1BBL-/- mice had fewer epitope-specific cells and were impaired in their ability to resolve the infection. These results show that immunization with a single LCMV peptide provides long term protection against LCMV infection and point to costimulatory molecules such as 4-1BB as important components for generating protective immunity after vaccination.

Animals↗

PubMed2000

Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses.

Linear carbohydrate-peptide constructs based on the 13 amino acid nonnatural pan DR epitope (PADRE) and carbohydrate B cell epitopes are demonstrated to be potent immunogens. These data support our belief that PADRE should be considered as an alternative to more complex carriers for use in prophylaxis and therapeutic vaccines. Two model carbohydrate-PADRE glycoconjugates were used to demonstrate that PADRE could effectively provide T cell help for carbohydrate-specific Ab responses. Conjugates of PADRE covalently linked to the human milk oligosaccharide, lacto-N-fucopentose II or a dodecasaccharide derived from Salmonella typhimurium O-Ag induced high titer IgG Ab responses in mice, which were comparable to glycoconjugates employing human serum albumin (HSA) as the carrier protein. Different adjuvants, in combination with PADRE conjugates, allowed for the modulation of the isotype profile with alum supporting an IgG1 profile; QS-21 an IgG2a, 2b profile, while an alum/QS-21 mixture generated a balanced IgG1/IgG2b isotype profile. As defined by binding to synthetic glycoconjugates, dodecasaccharide-specific Abs exhibited fine specificity similar to protective polyclonal Ab responses previously reported for dodecasaccharide-protein conjugates. The same Abs bound to intact S. typhimurium cells, suggesting that biologically relevant specificities were produced. The affinity of the dodecasaccharide-specific Abs was further shown to be comparable to that of a well-characterized, high affinity monoclonal anti-carbohydrate Ab recognizing the same epitope.

Adjuvants, Immunologic↗

PubMed2000

Melan-A/MART-1(51-73) represents an immunogenic HLA-DR4-restricted epitope recognized by melanoma-reactive CD4(+) T cells.

The human Melan-A/MART-1 gene encodes an HLA-A2-restricted peptide epitope recognized by melanoma-reactive CD8(+) cytotoxic T lymphocytes. Here we report that this gene also encodes at least one HLA-DR4-presented peptide recognized by CD4(+) T cells. The Melan-A/MART-1(51-73) peptide was able to induce the in vitro expansion of specific CD4(+) T cells derived from normal DR4(+) donors or from DR4(+) patients with melanoma when pulsed onto autologous dendritic cells. CD4(+) responder T cells specifically produced IFN-gamma in response to, and also lysed, T2.DR4 cells pulsed with the Melan-A/MART-1(51-73) peptide and DR4(+) melanoma target cells naturally expressing the Melan-A/MART-1 gene product. Interestingly, CD4(+) T cell immunoreactivity against the Melan-A/MART-1(51-73) peptide typically coexisted with a high frequency of anti-Melan-A/MART-1(27-35) reactive CD8(+) T cells in freshly isolated blood harvested from HLA-A2(+)/DR4(+) patients with melanoma. Taken together, these data support the use of this Melan-A/MART-1 DR4-restricted melanoma epitope in future immunotherapeutic trials designed to generate, augment, and quantitate specific CD4(+) T cell responses against melanoma in vivo.

Amino Acid Sequence↗

PubMed2000

Conserved MHC class I peptide binding motif between humans and rhesus macaques.

Since the onset of the HIV pandemic, the use of nonhuman primate models of infection has increasingly become important. An excellent model to study HIV infection and immunological responses, in particular cell-mediated immune responses, is SIV infection of rhesus macaques. CTL epitopes have been mapped using SIV-infected rhesus macaques, but, to date, a peptide binding motif has been described for only one rhesus class I MHC molecule, Mamu-A*01. Herein, we have established peptide-live cell binding assays for four rhesus MHC class I molecules: Mamu-A*11, -B*03, -B*04, and -B*17. Using such assays, peptide binding motifs have been established for all four of these rhesus MHC class I molecules. With respect to the nature and spacing of crucial anchor positions, the motifs defined for Mamu-B*04 and -B*17 present unique features not previously observed for other primate species. The motifs identified for Mamu-A*11 and -B*03 are very similar to the peptide binding motifs previously described for human HLA-B*44 and -B*27, respectively. Accordingly, naturally processed peptides derived from HLA-B*44 and HLA-B*27 specifically bind Mamu-A*11 and Mamu-B*03, respectively, indicating that conserved MHC class I binding capabilities exist between rhesus macaques and humans. The definition of four rhesus MHC class I-specific motifs expands our ability to accurately detect and quantitate immune responses to MHC class I-restricted epitopes in rhesus macaques and to rationally design peptide epitope-based model vaccine constructs destined for use in nonhuman primates.

Alleles↗

PubMed2000

Immunogenicity of self antigens is unrelated to MHC-binding affinity: T-cell determinant structure of Golli-MBP in the BALB/c mouse.

The 'classical' myelin basic protein (MBP) exons belong to a much larger unit, termed the 'Golli-MBP' gene. Here we have examined the T-cell determinant structure of the Golli protein region in the BALB/c mouse. Golli p10-24, which was shown to have the strongest affinity for I-A(d), could not induce T-cell activation. Paradoxically, the poorer binding, overlapping p5-19 was effective at inducing T-cell proliferation. Thus, immunogenicity is not necessarily related to the MHC-binding affinity of self-peptides. In addition, MBP: p151-168-specific T cell clones responded only poorly to J37, a Golli-MBP protein, while MBP: 59-76-specific clones responded well to J37.

Amino Acid Sequence↗

PubMed2000

Human memory CTL response specific for influenza A virus is broad and multispecific.

Class I restricted cytotoxic T-lymphocyte (CTL) responses are thought to be focused against few immunodominant epitopes. In humans, an often quoted example of such narrow focus is the influenza A (FLU) matrix 58-66 specific memory CTL activity, detectable in HLA-A2 individuals as a result of natural infection. Herein, we analyzed the repertoire of memory, FLU-specific CTLs in A2 and A11 positive individuals. Eighteen A2.1 binding peptides, derived from the FLU-Puerto Rico/8/34 (PR8) isolate, elicited CTL activity in A2. 1/Kb transgenic mice upon direct immunization. These peptides were also tested for their capacity to recall memory CTL responses from peripheral blood mononuclear cells (PBMC) of human A2.1 donors. Besides the known dominant M1.58 peptide, 5 new epitopes (PA.46, PA. 225, PB1.413, NA.75 and M1.59) were identified. Similarly, eleven, A11-binding, FLU-PR8 peptides, which were immunogenic in HLA-A11/Kb transgenic mice, were assayed for induction of recall CTL responses using peripheral blood lymphocytes from a cohort of A11-positive donors. Eight different peptides (NP.188, NP.342, HA.63(,) HA.149, HA.450, M1.13, M1.178, and M2.70) induced memory CTL activity. Several of these peptides were found to be highly conserved amongst different FLU isolates, and also capable of binding multiple A2 and A11 supertype molecules. Finally, 37 HLA-B7 binding peptides were also identified. In conclusion, a previously unappreciated breadth of FLU-specific, memory CTL responses in humans was revealed. The relevance of these findings to the design of multiepitope vaccines is discussed.

Animals↗

PubMed2000

Supermotif peptide binding and degeneracy of MHC: peptide recognition in an EBV peptide-specific CTL response with highly restricted TCR usage.

We have investigated the presentation and CTL recognition of an HLA A*1101-restricted CTL peptide epitope AVFDRKSDAK (AVF)(3), derived from the EBV nuclear antigen (EBNA) 4, in the context of alleles belonging to the A3-supertype, A*0101, 0301, 1101, 3101, 3301, and 6801. The peptide binds to a A*6801 molecule as efficiently as to A*1101. The A*6801:AVF complex is recognized by some A*1101-restricted AVF- specific CTL clones. However, A*6801-positive (A*6801+) EBV-transformed lymphoblastoid cell lines (LCLs) are not killed by the same effectors. Furthermore, two A*6801+ donors did not mount an AVF-specific CTL response in vitro and lacked detectable AVF-specific effectors. Thus, this epitope is either subdominant, or non-immunogenic in the context of A*6801. These characteristics correlate with low stability of this MHC:peptide complex in living cells. We also demonstrate that a highly conserved AVF-specific TCR that dominates the AVF-specific CTL response in the majority of A*1101+ individuals recognizes the A*6801 molecule as a crossreactive alloantigen. Therefore, deletion of AVF-specific T cells may contribute to the non-immunogenicity or subdominance of the peptide in A*6801+ individuals.

Alleles↗

PubMed2000

Antigen recognition.

Explore the source record for details and available documents.

Antigen Presentation↗

PubMed2000

Structure-based prediction of binding peptides to MHC class I molecules: application to a broad range of MHC alleles.

Specific binding of antigenic peptides to major histocompatibility complex (MHC) class I molecules is a prerequisite for their recognition by cytotoxic T-cells. Prediction of MHC-binding peptides must therefore be incorporated in any predictive algorithm attempting to identify immunodominant T-cell epitopes, based on the amino acid sequence of the protein antigen. Development of predictive algorithms based on experimental binding data requires experimental testing of a very large number of peptides. A complementary approach relies on the structural conservation observed in crystallographically solved peptide-MHC complexes. By this approach, the peptide structure in the MHC groove is used as a template upon which peptide candidates are threaded, and their compatibility to bind is evaluated by statistical pairwise potentials. Our original algorithm based on this approach used the pairwise potential table of Miyazawa and Jernigan (Miyazawa S, Jernigan RL, 1996, J Mol Biol 256:623-644) and succeeded to correctly identify good binders only for MHC molecules with hydrophobic binding pockets, probably because of the high emphasis of hydrophobic interactions in this table. A recently developed pairwise potential table by Betancourt and Thirumalai (Betancourt MR, Thirumalai D, 1999, Protein Sci 8:361-369) that is based on the Miyazawa and Jernigan table describes the hydrophilic interactions more appropriately. In this paper, we demonstrate how the use of this table, together with a new definition of MHC contact residues by which only residues that contribute exclusively to sequence specific binding are included, allows the development of an improved algorithm that can be applied to a wide range of MHC class I alleles.

Alleles↗

PubMed2000

A subdominant CD8(+) cytotoxic T lymphocyte (CTL) epitope from the Plasmodium yoelii circumsporozoite protein induces CTLs that eliminate infected hepatocytes from culture.

Previous studies indicated that the Plasmodium yoelii circumsporozoite protein (PyCSP) 57-70 region elicits T cells capable of eliminating infected hepatocytes in vitro. Herein, we report that the PyCSP58-67 sequence contains an H-2(d) binding motif, which binds purified K(d) molecules in vitro with low affinity (3, 267 nM) and encodes an H-2(d)-restricted cytotoxic T lymphocyte (CTL) epitope. Immunization of BALB/c mice with three doses of a multiple antigen peptide (MAP) construct containing four branches of amino acids 57 to 70 linked to a lysine-glycine core [MAP4(PyCSP57-70)] and Lipofectin as the adjuvant induced both T-cell proliferation and a peptide-specific CTL response that was PyCSP59-67 specific, H-2(d) restricted, and CD8(+) T cell dependent. Immunization with either DNA encoding the PyCSP or irradiated sporozoites demonstrated that this CTL epitope is subdominant since it is not recognized in the context of whole CSP immunization. The biological relevance of this CTL response was underlined by the demonstration that it could mediate genetically restricted, CD8(+)- and nitric-oxide-dependent elimination of infected hepatocytes in vitro, as well as partial protection of BALB/c mice against sporozoite challenge. These findings indicate that subdominant epitopes with low major histocompatibility complex affinity can be used to engineer epitope-based vaccines and have implications for the selection of epitopes for subunit-based vaccines.

Amino Acid Sequence↗

PubMed2000

Definition of five new simian immunodeficiency virus cytotoxic T-lymphocyte epitopes and their restricting major histocompatibility complex class I molecules: evidence for an influence on disease progression.

Simian immunodeficiency virus (SIV) infection of the rhesus macaque is currently the best animal model for AIDS vaccine development. One limitation of this model, however, has been the small number of cytotoxic T-lymphocyte (CTL) epitopes and restricting major histocompatibility complex (MHC) class I molecules available for investigating virus-specific CTL responses. To identify new MHC class I-restricted CTL epitopes, we infected five members of a family of MHC-defined rhesus macaques intravenously with SIV. Five new CTL epitopes bound by four different MHC class I molecules were defined. These included two Env epitopes bound by Mamu-A*11 and -B*03 and three Nef epitopes bound by Mamu-B*03, -B*04, and -B*17. All four restricting MHC class I molecules were encoded on only two haplotypes (b or c). Interestingly, resistance to disease progression within this family appeared to be associated with the inheritance of one or both of these MHC class I haplotypes. Two individuals that inherited haplotypes b and c separately survived for 299 and 511 days, respectively, while another individual that inherited both haplotypes survived for 889 days. In contrast, two MHC class I-identical individuals that did not inherit either haplotype rapidly progressed to disease (survived <80 days). Since all five offspring were identical at their Mamu-DRB loci, MHC class II differences are unlikely to account for their patterns of disease progression. These results double the number of SIV CTL epitopes defined in rhesus macaques and provide evidence that allelic differences at the MHC class I loci may influence rates of disease progression among AIDS virus-infected individuals.

Amino Acid Sequence↗

PubMed2000

["The memory effect" in healthy heart and transplanted heart. "Cor se ipse alit, se ipse movet, se ipse reget"].

The heart's memory, interpreted as information residue between a cardiac cycle and the subsequent one, is a very definite phenomenon (short- term memory) both in healthy subjects and in heart transplanted recipients. This "memory effect", we have observed by the autocorrelation and by the spectrum analysis of the values expressing the heartbeat acceleration variability (tachogram 24 h). The "memory effect" cannot be absolutely put down in heart transplanted recipients to interference or to the activity of the central and/or peripheral nervous system, because the heart transplanted is a denervated heart by definition. Moreover, an immediate regulating effect of hormonic factors in the short-term heartbeat regulation is not completely plausible. As already known, the heart transplanted responds to the emergency situation autonomously and autochthonously in proportion to the signals reaching it directly from the circle (vascular resistance, blood pressure, etc.). This implies the existence of functional memory that is likely to be inborn in the conduction system of the heart. Looking at the results of our research we can conclude: "cor se ipse alit, se ipse movet, se ipse reget" (it autonomously models itself to all the different modifications in the circle, it feeds itself as the organ propelling blood circulation and, if necessary, it carries on its kinetic activity autonomously).

Adult↗