Kiyoshi Miwa

Identification of multiple HIV-1 CTL epitopes presented by HLA-B*5101 molecules

We attempted to identify and characterize HIV-1 CTL epitopes presented by HLA-B51 which is associated with a slow progression to AIDS. HLA-B*5101 stabilization assay showed that 33 out of 172 HIV-1 peptides carrying HLA-B*5101 anchor residues bound to HLA-B*5101. Seven peptides were suggested as HIV-1 CTL epitopes presented by HLA-B*5101 because the specific CTL was induced for these peptides in PBMC from three HIV-1 seropositive individuals carrying HLA-B51 by stimulation with HLA-B*5101 binding peptides. Analysis of these epitopes using the specific CTL clones confirmed that six of seven HIV-1 peptides are epitopes presented by HLA-B*5101. Three epitopes presented by HLA-B*5101 are highly conserved among the clade B strain, suggesting that the specific CTL for these epitopes might play an important role in recognition of HIV-1 infected cells. These epitopes will be useful to analyze CTL responses in HIV-1 infected individuals.

Expression of Escherichia coli promoters in Brevibacterium lactofermentum using the shuttle vector pEB003

Abstract A novel promoter probing vector for examining gene expression in a glutamic acid-producing bacterium Brevibacterium lactofermentum was constructed. This plasmid pEB003, a shuttle vector able to replicate either in B. lactofermentum or Escherichia coli , carries a promoter-less chloramphenicol acetyltransferase (CAT) gene as the index gene for probing promoter strength. By using pEB003, we demonstrated that the E. coli promoters, tac, trp and lac UV5 promoters, worked effectively in B. lactofermentum . As evaluated by the CAT activity, the relative strength of the promoters was the same in B. lactofermentum as in E. coli . Thus, B. lactofermentum seems to have a transcriptional machinery that is similar to that of E. coli .

Identification and characterization of a cytotoxic T cell epitope of hepatitis C virus presented by HLA-B*3501 in acute hepatitis

In order to clarify the role of cytotoxic T lymphocytes (CTL) in hepatitis C virus (HCV) infection, an HLA-B35-restricted cytotoxic T cell epitope of HCV was identified using a strategy called reverse immunogenetics. Twenty-eight of 53 HCV peptides carrying two anchor residues were selected as HLA-B*3501 binding peptides. These peptides were used to induce the specific cytotoxic T cells in peripheral blood lymphocytes from a patient with acute hepatitis C. Six HLA-B*3501 binding peptides induced the peptide-specific CTL. One (HPNIEEVAL) of five peptides was confirmed as the epitope by the specific T cell clones. A sequence identical to the epitope was detected in isolates of the virus from the patient and a strong CTL response to this epitope was observed in the acute phase of hepatitis C but not in the recovery phase. The specific CTL for this epitope were not detected in peripheral blood lymphocytes from patients with chronic hepatitis C. Together these results suggest that the CTL specific for this epitope have an important role in the elimination of the virus in the patient.

Role of strong anchor residues in the effective binding of 10-mer and 11-mer peptides to HLA-A^*2402 molecules.

The binding capacity of one-hundred-and-seventy-two 8-mer to 11-mer peptides carrying HLA-A24 anchor residues to HLA-A*2402 molecules was analyzed by using a HLA class I stabilization assay. Most (76.2%) of these peptides bound to HLA-A*2402 molecules. These results confirmed previous findings that Tyr and Phe at P2 as well as Phe, Trp, Ile, and Leu at the C-terminus were main anchor residues for HLA-A*2402. Tyr at P2 was a stronger anchor residue than Phe, while bulky aromatic hydrophobic residues Phe and Trp at the C-terminus are stronger anchors than aliphatic hydrophobic residues Ile and Leu. These results were also supported by an analysis using a panel of mutated 9-mer peptides at P2 and P9. Taken together, these results suggest that HLA-A*2402 molecules have deep B- and F-pockets because they favor peptides carrying bulky aromatic hydrophobic residues at P2 and the C-terminus. The affinity of 8-mer peptides was significantly lower than that of 9-mer to 11-mer peptides, while there was no difference in affinity between 9-mer, 10-mer, and 11-mer peptides. The affinity of peptides carrying bulky aromatic hydrophobic residues at the C-terminus was higher than that of peptides carrying aliphatic hydrophobic residues in each of the 8-mer to 11-mer peptides, though the greatest difference in affinity was observed in 11-mer peptides. The strong interaction of side chains of these anchor residues with the corresponding pockets may permit the effective binding of 10-mer and 11-mer peptides to HLA-A*2402 molecules.

Binding of nonamer peptides to three HLA-B51 molecules which differ by a single amino acid substitution in the A-pocket

The interaction between 9-mer peptides and HLA-B51 molecules was investigated by quantitative peptide binding assay using RMA-S cell expressing human β2-microglobulin and HLA-B51 molecules. Of 147 chemically synthesized 9-mer peptides possessing two anchor residues corresponding to the motif of HLA-B*5101 binding self-peptides, 27 paptides bound to HLA-B*5101 molecules. Pro and Ala at position 2 as well as Ile at position 9 were confirmed to be main anchor residues, while Gly at position 2 as well as Val, Leu, and Met at position 9 were weak anchor residues for HLA-B*5101. The A-pocket is suspected to have a critical role in peptide binding to MHC class I molecules because this pocket corresponds to the N-terminus of peptides and has a strong hydrogen bond formed by conserved Tyr residues. Further analysis of peptide binding to HLA-B*5102 and B*5103 molecules showed that a single amino acid substitution of Tyor for His at residue 171(B*5102) and that of Gly for Trp at residue 167 (B*5103) has a minimum effect in HLA-B51-peptide binding. Since previous studies showed that some HLA-B51 alloreactive CTL clones failed to kill the cells expressing HLA-B*5102 or HLA-B*5103, these results imply that the structural change of the A-pocket among HLA-B51 subtypes causes a critical conformational change of the epitope for TCR recognition rather than influences the interaction between peptides and MHC class I molecules.

A new non-protein enediyne antibiotic N1999A2: Unique enediyne chromophore similar to neocarzinostatin and DNA cleavage feature

Abstract The present NMR and X-ray structural studies demonstrated that the new antibiotic N1999A2 isolated from the broth filtrate of Streptomyces sp. AJ9493, possesses a novel 9-membered ring enediyne chromophore similar to neocarzinostatin, but is not chromoprotein. Of special interest is the fact that stable N1999A2 exists as enediyne chromophore alone as well as dynemicin A, esperamicin A 1 and calicheamicin γ 1 . The major difference between N1999A2 and neocarzinostatin chromophore is lack of the amino sugar in N1999A2. The antibiotic N1999A2 revealed more random DNA cutting profile than neocarzinostatin chromophore.

Induction of endogenous lymphokine-activated killer activity by combined administration of lentinan and interleukin 2

Lymphokine-activated killer activity in vivo (endogenous LAK activity) was found to be augmented by combined administration of lentinan, a beta (1-3) glucan with beta-1,6 branches, and interleukin 2 (IL-2). In contrast, addition of lentinan during culture in vitro did not augment LAK activity induced by IL-2. Surface marker analysis of endogenous LAK cells revealed that endogenous LAK cells induced by a combined administration of lentinan and IL-2 were all NK-type LAK cells, which express asialo-GM1 and lack T3, Thy-1 and Lyt2, whereas LAK cells generated in vitro were composed of both NK-type LAK and T-type LAK cells, which express T3 and Thy-1, and lack asialo-GM1. Furthermore, combined administration of lentinan and IL-2 was found to augment the endogenous LAK activity even in the tumor bearer, and show a substantial inhibition of tumor growth and a significant increase in survival rate in the C3H/HeN/MM46 system. Results of the present investigation offer a possible clinical application of a combination of lentinan and IL-2 for immunotherapy against cancer without detrimental side effects.

Binding of 8-mer to 11-mer peptides carrying the anchor residues to slow assembling HLA class I molecules (HLA-B*5101)

Abstract The binding of 303 8-mer to 11-mer peptides carrying the anchor residues at P2 and the C-terminus to HLA-B*5101 molecules was examined by a stabilization assay in which peptides were incubated with RMA-S-B*5101 cells at 26 °C for 3 h. Analysis of the binding of these peptides to HLA-B*5101 molecules showed that Pro and Ala at P2, and Ile, Val, and Leu at the C-terminus functioned as anchor residues, while Gly at P2 and Met at the C-terminus were weak anchors. Pro was a stronger anchor residue than Ala at P2, while Ile was the strongest anchor at the C-terminus. Among 8-mer to 11-mer peptides, the 9-mer peptides showed the strongest binding to HLA-B*5101 molecules. This is in contrast to our recent findings that 10-mer and 11-mer peptides bind to HLA-B*3501 molecules as effectively as 9-mer peptides. Since both HLA class I molecules have the same B-pocket and the binding peptides carry the same anchor residues, it is assumed that the structure of the F-pocket may restrict the length of binding peptides. The ability of HLA-B*5101 binding peptides to stabilize the HLA-B*5101 molecules was markedly lower than that of HLA-B*3501 binding peptides to stabilize the HLA-B*3501 molecules. It is known that HLA-B*5101 is a slow assembling molecule, while HLA-B*3501 assembles rapidly. The results imply that the slow assembling of HLA-B*5101 molecules results from the low affinity of peptides to HLA-B*5101 molecules.

Residue 116 determines the C-terminal anchor residue of HLA-B*3501 and -B*5101 binding peptides but does not explain the general affinity difference

Abstract HLA-B*3501 and -B*5101 molecules, which belong to the HLA-B5 cross-reactive group, bind peptides carrying similar anchor residues at P2 and the C-terminus, but differences are observed in the preference for a Tyr residue at the C-terminus and the affinity of peptides. A recent study of HLA-B*3501 crystal structure suggested that residue 116 on the floor of the F-pocket determines a preference for anchor residues at the C-terminus. In order to evaluate the role of the residue 116 in the peptide binding to both HLA-B*3501 and HLA-B*5101 molecules, we generated HLA-B*3501 mutant molecules carrying Tyr at residue 116 (B*3501–116Y) and tested the binding of a panel of nonamer peptides to the B*3501–116Y molecules by a stabilization assay with RMA-S transfectants expressing the mutant molecules. The substitution of Tyr for Ser at residue 116 markedly reduced the affinity of nonamer peptides carrying Tyr at P9, while it enhanced that of nonamer peptides carrying Ile and Leu at P9. On the other hand, the affinity of peptides carrying aliphatic hydrophobic residues at P9 to B*3501–116Y molecules was much higher than that to HLA-B*3501 and HLA-B*5101 molecules. These results indicate that residue 116 is critical for the structural difference of the F-pocket between HLA-B*3501 and HLA-B*5101 which determines the C-terminal anchor residues, while leaving other residues which differ between HLA-B*3501 and HLA-B*5101 may be responsible for the low peptide binding property of the latter.

Refined peptide HLA-B^*3501 binding motif reveals differences in 9-mer to 11-mer peptide binding

Abstract HLA-B*3501 is associated with subacute thyroiditis and fast progression of AIDS. An important prerequisite to investigate the T-cell recognition of HLA-B*3501-restricted antigens is the characterization of peptide-HLA-B*3501 interactions. In this study, peptide-HLA-B*3501 interactions were determined in quantitative peptide binding assays. The results were statistically analyzed to evaluate the influence of both anchor and nonanchor positions and the predictability of peptide binding. The binding data demonstrated that all anchor residues at position 2 and the C-terminus found in 9-mers functioned equally as anchors in 10-mers and 11-mers. These minimum requirements of peptide binding were refined by assessing positive and negative effects of nonanchor residues. Aliphatic hydrophobic residues at positions 3, 5, and 8 of 10-mers and position 3 of 11-mers significantly enhanced HLA-B*3501 binding. Similar effects rendered aromatic, bulky residues, acidic or polar residues of 11-mers at position 1 as well as at positions 4, 8, and 10, respectively. Negative effects were observed for residues carrying positively charged side-chains at position 7 of 11-mers. The refined HLA-B*3501 peptide binding motifs enhanced the identification of potential T-cell epitopes. The disparity between positive effects at the middle and C-terminal part (positions 5 – 8 and 10) of 11-mers and shorter peptides supports the extrusion of 11-mer residues at positions 5, 6, and 7, away from the HLA-B*3501 binding cleft.