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The majority of hepatitis C virus (HCV)-infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV-specific CTL responses directed to different HCV-derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA-A2-presented, HCV-derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon-α, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4-derived peptide antigen (amino acids 1789–1797). Treated patients presented stronger HCV-specific CTL responses and therapy-induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3-derived epitope (amino acids 1073–1081). By longitudinal analysis we show that five individuals responding to IFN-α therapy with decreases in alanine aminotransfrase levels presented a strong CTL activity directed to the NS3-derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV-derived epitopes with a dominant response to the NS3-derived peptide antigen. This suggests that CTL responses directed to this NS3-derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.

Syntheses are described of new dermorphin and [D-Ala2]deltorphin I analogues in which the phenylalanine, the tyrosine or the valine residues have been substituted by the corresponding N-alkylglycine residues. Structural investigations by CD measurements in different solvents and preliminary pharmacological experiments were carried out on the resulting peptide-peptoid hybrids. The contribution from aromatic side chain residues is prominent in the CD spectra of dermorphin analogues and the assignment of a prevailing secondary structure could be questionable. In the CD spectra of deltorphin analogues the aromatic contribution is lower and the dichroic curves indicate the predominance of random conformer populations. The disappearance of the aromatic contribution in the [Ntyr1,D-Ala2]-deltorphin spectrum could be explained in terms of high conformational freedom of the N-terminal residue. The kinetics of degradation of the synthetic peptoids digestion by rat and human plasma enzymes were compared with that of [Leu5]-enkephalin. The binding to opioid receptors was tested on crude membrane preparations from CHO cells stably transfected with the mu- and delta-opioid receptors. The biological potency of peptoids was compared with that of dermorphin in GPI preparations and with that of deltorphin I in MVD preparations. All the substitutions produced a dramatic decrease in the affinity of the peptide-peptoid hybrids for both the mu- and delta-opioid receptors. Nval5 and/or Nval6 containing hybrids behaved as mu-opioid receptor agonists and elicit a dose-dependent analgesia (tail-flick test) when injected i.c.v. in rats.

Biotechnology Center, University of Ferrara, Ferrara, ItalyThe latent membrane protein 2 is an immunogenic antigen expressed in Epstein-Barr virus(EBV)-associated tumors and consequently it may represent a target for specific cytotoxic Tlymphocyte (CTL)-based immunotherapies. However, the efficacy of such a therapy is lim-ited by the poor immunogenicity of the protein that induces weak CTL responses directed tothe CLGGLLTMV (CLG) epitope only in the minority of EBV-seropositive donors. We havenow demonstrated that selective peptide stimulation of peripheral blood lymphocytesinduced CLG-specific CTL in all donors, suggesting that this epitope can be a suitable targetfor specific immunotherapies. We found that the CLG peptide has a low affinity for HLA-A*0201 and does not produce stable complexes, both factors that are likely to determine thestrength of CTL responses to this epitope. Therefore, we synthesized and tested CLG analo-gues carrying single or combined amino acid substitutions to increase HLA/peptide stability.Among the analogues tested we identified two peptides which, compared to the natural epi-tope, showed higher affinity for HLA-A*0201 molecules, and produced stable complexes.These peptides demonstrated a potent, specific stimulatory capacity and could be used forselective CTL-based therapies.

E. REALI, R. GUERRINI*, B. GIORI, M. BORGHI, M. MARASTONI*, R. TOMATIS*, S. TRANIELLO,M. G. MASUCCI† & R. GAVIOLIDepartment of Biochemistry and Molecular Biology and *Department of PharmaceuticalSciences, University of Ferrara, Italy and † Microbiology and Tumour Biology Centre, Karolinska Institute, Stockholm, Sweden(Accepted for publication 19 March 1996)SUMMARYCytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by theirability to bind to MHC class I molecules. Polyclonal Epstein–Barr virus (EBV)-specific memory CTLresponses, reactivated from blood lymphocytes of HLA-A11-positive individuals by stimulation withthe autologous EBV-transformed lymphoblastoid cell line (LCL), are often dominated by reactivitiesdirected to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416–424 of EBVnuclear antigen-4 (EBNA4). We now report the selective activation of IVT-specific CTL by stimulationof lymphocytes with the corresponding synthetic peptide. A more than 10-fold increase in frequency ofCTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented byHLA-A11-transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstratedthat clones activated under these conditions are as efficient as clones activated by conventional LCLstimulations. Induction of memory CTL responses required low surface density of MHC:peptidecomplexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentra-tions of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealedthe presence of IVT-specific CTL precursors in a donor that failed to mount an IVT-specific responseupon stimulation with the autologous B95.8 virus-transformed LCL. The results suggest that stimulationwith synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and maybe particularly helpful for the selective expansion of subdominant CTL specificities.Keywords cytotoxic T lymphocytes immunogenic peptides HLA-A11 Epstein–Barr virusINTRODUCTIONCytotoxic T lymphocytes (CTL) play an important role in thecontrol of viral, bacterial and parasitic infections, and can recog-nize and destroy tumour cells [1–6]. The molecular targets of theirattack are peptide fragments, derived from processed antigenicproteins, selected for presentation at the surface of target cells bytheir ability to bind to the antigen-presenting groove of MHC classI molecules [7,8].Vaccination with synthetic peptides or with peptide-pulsedcells was shown to induce specific CTL against immunogenicviral or tumour-derived epitopes in vivo [9–12]. In addition,passive transfer of these CTL has been successfully employed inthe management of these diseases [2,4,13].Autologous virus-infected or malignant cells are commonlyused for in vitro induction and expansion of virus- or tumour-specific CTL. These protocols of stimulation lead to the activationof polyclonal CTL responses where only a fraction of the effectorsis likely to bear the desired specificities. In addition, most CTL inpolyclonal cultures are directed to immunodominant epitopes,while reactivities to subdominant epitopes are often lost duringin vitro propagation [14,15]. This is a potential drawback of thesestrategies, since immunodominant epitopes are often selected formutations in vivo, hampering their effectiveness as therapeutictargets [16–21]. Hence, alternative protocols of T cell activationwould allow triggering of CTL responses with unique specificities,and the selective activation of responses to subdominant epitopeswhich are not detected in polyclonal CTL cultures obtained byconventional stimulations.In this study we have exploited the presence of Epstein–Barrvirus (EBV)-specific memory CTL precursors in blood lympho-cytes from EBV

The highly hydrophobic C60 (buckminsterfullerene) was water solubilized by covalently linking the synthon 1,2-dihydro-1,2-methanofullerene [60]-61-carboxylic acid to the alpha-amino group of the hydrophilic 4-8 sequence of peptide T, known to display potent human monocyte chemotaxis. The resulting compound, characterized by a variety of analytical techniques, including a UV spectrum in aqueous solution, exhibits remarkable chemotactic potency, comparable to that of the parent pentapeptide. Furthermore, this fullerene-peptide conjugate inhibits, albeit weakly, HIV-1 protease.

1H spectra at 270 MHz of the Met-enkephalin pentapeptide (Tyr-Gly-Gly-Phe-Met) in aqueous solution, as a function of pH and of temperature, are reported. The analysis of the chemical shifts, line widths, coupling constants and rotamer populations around χ1 and χ2, suggests the existence of three conformational regions in the pH range 1–10, due to some rearrangements of the first two amino acids, Tyr-1 and Gly-2. A first conformational transition occurs corresponding to the N-terminus titration, with an exchange rate between conformational states which is not too fast. The second transition occurs concomitantly with the phenolic group titration. The conformational states of the cationic and zwitterionic forms are probably very similar and are characterized by the absence of intramolecular hydrogen bonds and of head-to-tail interactions. A bent structure, with the Tyr-1 ring Facing the Gly-2 could contribute to the equilibrium of the conformational states of Met-enkephalin in the pH range 8–10. This structure could be destabilized as a consequence of the phenolic group deprotonation. The Met-5 and Phe-4 residues appear to have little sensitivity to the conformational transitions of the backbone and both show a settling of the side chains corresponding to the carboxyl group titration, thus showing a reciprocal steric hindrance. The temperature coefficients of the chemical shifts of amide protons at acidic pH are in the range of solvated peptides. In the temperature range 0–75°C, the 3JαNH coupling constants were independent of temperature.

The purpose of this study was to evaluate the effects of dermorphin, a new synthetic powerful opiate-like heptapeptide, on plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in fertile and postmenopausal women. In fertile subjects, dermorphin (5.5 micrograms/kg min for 30 min) decreases plasma LH (p less than 0.01 vs. baseline and placebo values), but not plasma FSH. The area under the curve during dermorphin infusion was significantly lower than during placebo infusion (p less than 0.01). Pretreatment with the opioid receptor antagonist naloxone, blocked the decrease of plasma LH levels. In postmenopausal women not subjected to any treatment, dermorphin infusion did not significantly modify plasma LH and FSH levels. On the contrary, its administration to postmenopausal subjects treated with conjugated estrogens and medroxyprogesterone acetate significantly decreased plasma LH levels (p less than 0.01, vs. baseline, placebo and area under the curve). Considering the modulatory role exerted by ovarian steroids on the activity of such receptors, these data also indicate that opioid systems play a very important part in the hypothalamus-pituitary-ovarian axis.

Peptides binding to HLA-A11 contain a hydrophobic or a small polar amino acid at position 2 and a lysine at the carboxy terminus. Synthetic peptides carrying natural and unnatural amino acids in position 2 were used to determine the requirements for formation of stable HLA-A11/peptide complexes. By kinetic analysis we demonstrate that a stereospecific interaction between the side chain residue in position 2 and a subsite of pocket B is required to obtain stable HLA/peptide complexes. This specific interaction is mediated by a methyl group or by an ethyl group bound to the asymmetric Cβ atom with the correct configuration. Experiments performed with different peptide sequences suggest that the presence of adequate anchor residues may be sufficient to produce stable HLA/peptide complexes.

It has been proposed [ 1 ] that the folding of a protein molecule begins at the amino end even before the synthesis is complete. Such a sequential mechanism presumes that the protei’n conformation is primarily due to interactions between amino acid residues close in the sequence and implies that parts of the polypeptide chain, particularly those near the terminal amino end, may fold into stable conformations that can still be recognized in the complete molecule and act as ‘internal templates’ around which the rest of the chain is folded. The results obtained by studying the conformational properties of the N-terminal eicosapeptide (S-peptide) [2, 31 of bovine pancreatic ribonuclease and of several S-peptide synthetic analogs [4] in aqueous solution and in the presence of helicogenic solvent, do not agree with the hypothetical sequential mechanism mentioned above. The hen egg-white lysozyme was shown by X-ray analysis [5] to contain three runs of helix (residues 515,24-34 and 88-96) for which the axial translation per residue and the number of residues per turn fall close to the a-helix values. The aim of the present paper is to report some preliminary conformational studies on the synthetic tetradecapeptide corresponding to the 2-l 5 N-terminal fragment of hen egg-white lysozyme. The peptide conformation has been investigated by circular dichroism in aqueous solution and in the presence of trifluoroethanol which is known to cause a transition from the random to the helical form in polypeptide systems.

Peptide T, an octapeptide of sequence ASTTTNYT that binds to human T cells, was studied as a zwitterion in DMSOd6 solution by means of proton NMR spectroscopy at 500 MHz. The unusual dispersion of the resonances of residues of the same type (T) makes it possible to assign all resonances to specific residues by means of several 2D techniques. The non-random nature of the conformation is substantiated by the observation of sequential nuclear Overhauser enhancements (NOEs). The low value of the temperature coefficient of the chemical shift of the NH of T8 and a diagnostic NOE between the NHs of T7 and T8 hint that a β-turn including T5, N6, Y7 and T8 is a prominent conformational feature in solution. The ring current high field shifts of the methyl group and of the NH of T8 are consistent with an interaction with the side-chain of Y7, favoured by the β-turn.

The aim of this study was to investigate further the influence of dermorphin (D), a new potent opioid peptide (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), on the functional activity of the pituitary-adrenocortical system in man. Six normal men were treated with oral metyrapone to stimulate the secretion of ACTH, beta-lipotropin, and beta-endorphin. In these subjects, significant suppression of metyrapone-evoked release of ACTH and related peptides occurred during D infusion (5.5 micrograms/kg X min for 30 min) compared with that during saline infusion. These results indicate that D can induce a significant decline in plasma levels of ACTH, beta-lipotropin, and beta-endorphin, the major circulating peptides from the C-terminal part of proopiocortin, and suggest that opioid peptides may be involved in the control of the functional activity of pituitary-adrenocortical activity in man.

Deltorphin is an opioid peptide with the sequence H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, recently isolated from the skin of Phyllomedusa sauvagei. Its enormous selectivity towards the δ-opioid receptor and the similarity of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), a μ selective peptide isolated from the same natural source, prompted a comparative conformational study. A 1H-NMR study in two different solvent systems showed that the conformational preferences of the N-terminal sequences of the two peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects. Besides a general trend consistent with the role of the membrane in the preselection of the peptides, the present study demonstrates the crucial role played by charged residues in the interaction inside the receptors.