Post-translational modification of therapeutic peptides by NisB, the dehydratase of the lantibiotic nisin

Post-translationally introduced dehydroamino acids often play an important role in the activity and receptor specificity of biologically active peptides. In addition, a dehydroamino acid can be coupled to a cysteine to yield a cyclized peptide with increased biostability and resistance against proteolytic degradation and/or modified specificity. The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. Its post-translational enzymatic modification involves NisB-mediated dehydration of serines and threonines and NisC-catalyzed coupling of cysteines to dehydroresidues, followed by NisT-mediated secretion. Here, we demonstrate that a L. lactis strain containing the nisBTC genes effectively dehydrates and secretes a wide range of medically relevant nonlantibiotic peptides among which variants of adrenocorticotropic hormone, vasopressin, an inhibitor of tripeptidyl peptidase II, enkephalin, luteinizing hormone-releasing hormone, angiotensin, and erythropoietin. For most of these peptides, ring formation was demonstrated. These data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/or modulated activities.     

Sec-Mediated Transport of Posttranslationally Dehydrated Peptides in Lactococcus lactis

Nisin is a lanthionine-containing antimicrobial peptide produced by Lactococcus lactis. Its (methyl)lanthionines are introduced by two posttranslational enzymatic steps involving the dehydratase NisB, which dehydrates serine and threonine residues, and the cyclase NisC, which couples these dehydrated residues to cysteines, yielding thioether-bridged amino acids called lanthionines. The prenisin is subsequently exported by the ABC transporter NisT and extracellularly processed by the peptidase NisP. L. lactis expressing the nisBTC genes can modify and secrete a wide range of nonlantibiotic peptides. Here we demonstrate that in the absence of NisT and NisC, the Sec pathway of L. lactis can be exploited for the secretion of dehydrated variants of therapeutic peptides. Furthermore, posttranslational modifications by NisB and NisC still occur even when the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively. However, transport of fully modified prenisin via the Sec pathway is impaired. The extent of NisB-mediated dehydration could be improved by raising the intracellular concentration NisB or by modulating the export efficiency through altering the signal sequence. These data demonstrate that besides the traditional lantibiotic transporter NisT, the Sec pathway with an established broad substrate range can be utilized for the improved export of lantibiotic enzyme-modified (poly)peptides.     

Sec-mediated transport of posttranslationally dehydrated peptides in Lactococcus lactis

Nisin is a lanthionine-containing antimicrobial peptide produced by Lactococcus lactis. Its (methyl)lanthionines are introduced by two posttranslational enzymatic steps involving the dehydratase NisB, which dehydrates serine and threonine residues, and the cyclase NisC, which couples these dehydrated residues to cysteines, yielding thioether-bridged amino acids called lanthionines. The prenisin is subsequently exported by the ABC transporter NisT and extracellularly processed by the peptidase NisP. L. lactis expressing the nisBTC genes can modify and secrete a wide range of nonlantibiotic peptides. Here we demonstrate that in the absence of NisT and NisC, the Sec pathway of L. lactis can be exploited for the secretion of dehydrated variants of therapeutic peptides. Furthermore, posttranslational modifications by NisB and NisC still occur even when the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively. However, transport of fully modified prenisin via the Sec pathway is impaired. The extent of NisB-mediated dehydration could be improved by raising the intracellular concentration NisB or by modulating the export efficiency through altering the signal sequence. These data demonstrate that besides the traditional lantibiotic transporter NisT, the Sec pathway with an established broad substrate range can be utilized for the improved export of lantibiotic enzyme-modified (poly)peptides.     

Supra-agonist peptides enhance the reactivation of memory CTL responses

Single amino acid substitutions at TCR contacts may transform a natural peptide Ag in CTL ligands with partial agonist, antagonist, or null activity. We obtained peptide variants by changing nonanchor amino acid residues involved in MHC class I binding. These peptides were derived from a subdominant HLA-A2-presented, latent membrane protein 2-derived epitope expressed in EBV-infected cells and in EBV-associated tumors. We found that small structural changes produced ligands with vastly different activities. In particular, the variants that associated more stably to HLA-A2/molecules did not activate any CTL function, behaving as null ligands. Interestingly, T cell stimulations performed with the combination of null ligands and the natural epitope produced significantly higher specific CTL reactivation than reactivation of CTLs induced by the wild-type epitope alone. In addition, these particular variants activated memory CTL responses in the presence of concentrations of natural epitope that per se did not induce T cell responses. We show here that null ligands increased ZAP-70 tyrosine kinase activation induced by the natural epitope. Our results demonstrate for the first time that particular peptide variants, apparently behaving as null ligands, interact with the TCR, showing a supra-agonist activity. These variant peptides did not affect the effector T cell functions activated by the natural epitope. Supra-agonist peptides represent the counterpart of antagonists and may have important applications in the development of therapeutic peptides.     

Identification of CTL epitopes in hepatitis C virus by a genome-wide computational scanning and a rational design of peptide vaccine

Developing a peptide-based vaccine for the highly variable hepatitis C virus (HCV) remains a challenging task. Variant viruses not only escape antigen presentation but also persist in a patient as quasi-species. Such variants are often antagonistic to the responding T cell repertoire. To overcome these problems, we herein propose a cocktail vaccine consisting of a few epitope peptides, which make it possible to outpace the emergence of variant viruses. To design such a vaccine, we developed a way to identify HLA-A*2402-binding peptides efficiently by means of the computational scanning of the whole genome of the pathogen. Most of the predicted peptides exhibited strong binding to the HLA-A*2402 molecule, while also inducing CD8 T cell responses from the patients’ peripheral blood mononuclear cells (PBMCs). Peptide-induced T cells were capable of lysing HCV-expressing HepG2 cells which process antigens endogenously. The amount of HCV core antigen in the patients’ livers suggested that the lytic activity of the peptide-induced T cells was clearly in a range suitable for therapeutic use. If T cells were activated under optimal conditions by high density peptides, then they tended to be relatively tolerant of single amino acid variations for cytolysis. Finally, an analysis of the viral population isolated in Japan suggested no obvious changes due to immune evasion in the viral genome even in a host population highly biased toward HLA-A*2402. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Inhibition of human immunodeficiency virus type 1 entry in cells expressing gp41-derived peptides

As the limitations of antiretroviral drug therapy, such as toxicity and resistance, become evident, interest in alternative therapeutic approaches for human immunodeficiency virus (HIV) infection is growing. We developed the first gene therapeutic strategy targeting entry of a broad range of HIV type 1 (HIV-1) variants. Infection was inhibited at the level of membrane fusion by retroviral expression of a membrane-anchored peptide derived from the second heptad repeat of the HIV-1 gp41 transmembrane glycoprotein. To achieve maximal expression and antiviral activity, the peptide itself, the scaffold for presentation of the peptide on the cell surface, and the retroviral vector backbone were optimized. This optimized construct effectively inhibited virus replication in cell lines and primary blood lymphocytes. The membrane-anchored C-peptide was also shown to bind to free gp41 N peptides, suggesting that membrane-anchored antiviral C peptides have a mode of action similar to that of free gp41 C peptides. Preclinical toxicity and efficacy studies of this antiviral vector have been completed, and clinical trials are in preparation.     

Naturally occurring peptidoglycan variants of Streptococcus pneumoniae.

Analysis by high-performance liquid chromatography of the stem peptide composition of cell walls purified from a large number of pneumococcal strains indicates that these bacteria produce a highly conserved species-specific peptidoglycan independent of serotype, isolation date, and geographic origin. Characteristic features of this highly reproducible peptide pattern are the dominance of linear stem peptides with a monomeric tripeptide, a tri-tetra linear dimer, and two indirectly cross-linked tri-tetra dimers being the most abundant components. Screening of strains with the high-performance liquid chromatography technique has identified two naturally occurring peptidoglycan variants in which the species-specific stem peptide composition was replaced by two drastically different and distinct stem peptide patterns, each unique to the particular clone of pneumococci producing it. Both isolates were multidrug resistant, including resistance to penicillin. In one of these clones--defined by multilocus enzyme analysis and pulsed-field gel electrophoresis of the chromosomal DNAs--the linear stem peptides were replaced by branched peptides that most frequently carried an alanyl-alanine substituent on the epsilon amino group of the diamino acid residue. In the second clone, the predominant stem peptide species replacing the linear stem peptides carried a seryl-alanine substituent. The abnormal peptidoglycans may be related to the altered substrate preference of transpeptidases (penicillin-binding proteins) in the pneumococcal variants.     

Structural and Thermodynamic Analysis of HIV-1 Fusion Inhibition Using Small gp41 Mimetic Proteins

Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed single-chain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated with resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR–NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches.     

Circular dichroism studies on synthetic signal peptides indicate beta-conformation as a common structural feature in highly hydrophobic environment

The conformations of synthetic peptides corresponding to signal sequences of chicken lysozyme and Escherichia coli proteins alkaline phosphatase and lipoprotein (wild-type) and their "variants" with a charged amino acid in the hydrophobic region, have been studied by circular dichroism spectroscopy in trifluoroethanol and micelles of sodium dodecyl sulfate, Brij 35, and sodium deoxycholate. In trifluoroethanol and aqueous mixtures of trifluoroethanol, the "wild-type" and variant signal sequences show similar conformational behavior. The wild-type signal peptides show increasing amounts of beta-structure going from sodium dodecyl sulfate to deoxycholate micelles (i.e. increasing order of hydrophobicity). The variant signal sequences, however, are largely unordered in micelles. The absence of beta-structure in variant signal sequences which do not initiate protein translocation across membranes, strongly suggests that the ability of signal sequences to adopt beta-structure in a highly hydrophobic environment is important for function.     

An antiviral peptide targets a coiled-coil domain of the human T-cell leukemia virus envelope glycoprotein

Retrovirus entry into cells is mediated by the viral envelope glycoproteins which, through a cascade of conformational changes, orchestrate fusion of the viral and cellular membranes. In the absence of membrane fusion, viral entry into the host cell cannot occur. For human T-cell leukemia virus type 1 (HTLV-1), synthetic peptides that mimic a carboxy-terminal region of the transmembrane glycoprotein (TM) ectodomain are potent inhibitors of membrane fusion and virus entry. Here, we demonstrate that this class of inhibitor targets a fusion-active structure of HTLV-1 envelope. In particular, the peptides bind specifically to a core coiled-coil domain of envelope, and peptide variants that fail to bind the coiled-coil lack inhibitory activity. Our data indicate that the inhibitory peptides likely function by disrupting the formation of a trimer-of-hairpins structure that is required for membrane fusion. Importantly, we also show that peptides exhibiting dramatically increased potency can be readily obtained. We suggest that peptides or peptide mimetics targeting the fusion-active structures of envelope may be of therapeutic value in the treatment of HTLV-1 infections.     

Molecular characterization of anti-idiotype antibody-resistant variants of a murine B cell lymphoma

We have previously identified Id- tumor vaiants that emerge after anti-Id mAb therapy of the murine B cell lymphoma 38C13. This report characterizes the molecular basis for these variants. By using a modification of the polymerase chain reaction (PCR), mu and kappa Ig loci were sequenced from nine Id- variants derived directly by anti-Id immunoselection in vivo. Ig kappa loci sequence analysis was also performed from 10 additional variants amplified directly from tumor cells in vitro without immunoselection. We demonstrate that the molecular mechanism underlying tumor cell escape in this model is the spontaneous emergence of variants that have undergone kappa L chain gene "re-rearrangement" before positive selection by the anti-Id antibody. A second round of re-rearrangement was also demonstrated to occur within primary tumor variants. Re-rearrangement of the 38C13 tumor cell Ig kappa locus is strongly biased toward use of variable kappa genes within the conserved V kappa-Ox1 gene family, although their use is not exclusive. With the use of RNA PCR re-rearrangement was documented to occur in vitro at a frequency of approximately 1.0 x 10(-5)/cell. These findings may have important implications for the application of anti-Id antibodies as a therapeutic approach for human lymphomas and for understanding of the Ig gene rearrangement process.     

Activity and stability of human kallikrein-2-specific linear and cyclic peptide inhibitors.

Human glandular kallikrein (KLK2) is a highly prostate-specific serine protease, which is mainly excreted into the seminal fluid, but part of which is also secreted into circulation from prostatic tumors. Since the expression level of KLK2 is elevated in aggressive tumors and it has been suggested to mediate the metastasis of prostate cancer, inhibition of the proteolytic activity of KLK2 is of potential therapeutic value. We have previously identified several KLK2-specific linear peptides by phage display technology. Two of its synthetic analogs, A R R P A P A P G (KLK2a) and G A A R F K V W W A A G (KLK2b), show specific inhibition of KLK2 but their sensitivity to proteolysis in vivo may restrict their potential use as therapeutic agents. In order to improve the stability of the linear peptides for in vivo use, we have prepared cyclic analogs and compared their biological activity and their structural stability. A series of cyclic variants with cysteine bridges were synthesized. Cyclization inactivated one peptide (KLK2a) and its derivatives, while the other peptide (KLK2b) and its derivatives remained active. Furthermore, backbone cyclization of KLK2b improved significantly the resistance against proteolysis by trypsin and human plasma. Nuclear magnetic resonance studies showed that cyclization of the KLK2b peptides does not make the structures more rigid. In conclusion, we have shown that backbone cyclization of KLK2 inhibitory peptides can be used to increase stability without losing biological activity. This should render the peptides more useful for in vivo applications, such as tumor imaging and prostate cancer targeting.     

Structure-guided design of peptidic ligand for human prostate specific antigen.

Prostate specific antigen (PSA) is a member of kallikrein family having serine protease-like activity and acts as a prognostic marker of prostate carcinoma. Various studies have been performed on inhibition of PSA and such targeting requires the identification of highly selective peptide inhibitors. PSA was purified from human seminal plasma by rapid and efficient methods, and binding studies for various peptides were carried out by fluorescence spectroscopy and SPR. The 'S' of PSA is predominated by hydrophobic residues, and hence many hydrophobic peptides were used to determine their binding affinity to PSA by fluorescence spectroscopy. We observed that LLFW, FFKW, and KFW binds strongly to PSA, among them LLFW showed strong binding. SPR also showed strong binding affinity of PSA toward peptides with hydrophobic and basic residues. Among the peptides used, FWYS showed dramatic increase in binding affinity (10(-10) M). The peptides analyzed for binding studies, suggests that peptide with Trp residue along with basic or hydrophobic amino acids may be useful for designing specific inhibitors for PSA. The strong affinities of designed peptides for PSA can be a valuable tool for designing therapeutic agents for prostate carcinomas.     

Structures of the dimeric and monomeric variants of magainin antimicrobial peptides (MSI-78 and MSI-594) in micelles and bilayers, determined by NMR spectroscopy

Magainins are antimicrobial peptides that selectively disrupt bacterial cell membranes. In an effort to determine the propensity for oligomerization of specific highly active magainin analogues in membrane mimetic systems, we studied the structures and lipid interactions of two synthetic variants of magainins (MSI-78 and MSI-594) originally designed by Genaera Corp. Using NMR experiments on these peptides solubilized in dodecylphosphocholine (DPC) micelles, we found that the first analogue, MSI-78, forms an antiparallel dimer with a "phenylalanine zipper" holding together two highly helical protomers, whereas the second analogue, MSI-594, whose phenylalanines 12 and 16 were changed into glycine and valine, respectively, does not dimerize under our experimental conditions. In addition, magic angle spinning solid-state NMR experiments carried out on multilamellar vesicles were used to corroborate the helical conformation of the peptides found in detergent micelles and support the existence of a more compact structure for MSI-78 and a pronounced conformational heterogeneity for MSI-594. Since magainin activity is modulated by oligomerization within the membrane bilayers, this study represents a step forward in understanding the role of self-association in determining magainin function.     

Inducing tolerance by intranasal administration of long peptides in naive and primed CBA/J mice

To assess the capacity of a peptide-based immunotherapy to induce systemic tolerance via the nasal route, we designed three long overlapping peptides of 44-60 aa covering the entire sequence of phospholipase A2 (PLA2), a major bee venom allergen. Both prophylactic and therapeutic intranasal administrations of long peptides to PLA2-hypersensitive CBA/J mice induced specific T cell tolerance to the native allergen. In prophylactic conditions, this tolerance was marked by a suppression of subsequent specific IgE response, whereas the therapeutic approach in presensitized mice induced a more than 60% decrease in PLA2-specific IgE. This decline was associated with a shift in the cytokine response toward a Th1 profile, as demonstrated by decreased PLA2-specific IgG1 and enhanced IgG2a levels, and by a decline in the specific IL-4/IFN-gamma ratios. T cell transfer from long peptide-tolerized mice to naive animals abrogated the expected anti-PLA2 IgE and IgG1 Ab response, as well as specific T cell proliferation, but enhanced specific IgG2a response upon sensitization with PLA2. These events were strongly suggestive of a clonal anergy affecting more profoundly Th2 than the Th1 subsets. In conclusion, these results demonstrate that allergen-derived long peptides delivered via the nasal mucosa may offer an alternative to immunotherapy with native allergens without the inherent risk of systemic anaphylactic reactions. Moreover, long peptides, in contrast to immunotherapy strategies based on short peptides, have the advantage of covering all potential T cell epitopes, and may represent novel and safe tools for the therapy of allergic diseases.     

Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein

Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.     

Expression of PNA-binding sites on specific glycoproteins by human melanoma cells is associated with a high metastatic potential

Lectin-binding patterns of seven human melanoma clones and variants selected from the same parental cell line and differing in their spontaneous metastatic potential in an animal model were compared by flow cytometry and Scatchard analysis. Human melanoma clones and variants with high and low metastatic potential could be distinguished by their peanut agglutinin (PNA)–binding patterns, but not by their wheat germ agglutinin (WGA)–, Ulex europaeus agglutinin I (UEA I)–, and soybean agglutinin (SBA)–binding patterns. Low metastatatic clones and variants proved to be made up of a single poorly peanut agglutinin–binding cell population (2.20–3.52 × 10⁶ sites/cell, Ka = 2.48–2.75 × 10⁶ M^?1). By contrast, highly metastatic variants were found to be constituted by two cellular subpopulations, exhibiting respectively a moderate (2.62–3.72 × 10⁶ sites/cell) and a high peanut agglutinin staining (17.68–18.76 × 10⁶ sites/cell). One highly metastatic clone was found to be homogeneously constituted by a single population of cells strongly binding this lectin (18.86 × 10⁶ sites/cell) with an association constant of 4.06 × 10⁶ M^?1. Using an EPICS V cytometer, these two subpopulations were sorted from a highly metastatic variant and tested for their metastatic abilities: cells with high PNA binding generated a higher frequency of metastases than did moderately PNA-binding cells. Following treatment with Vibrio cholerae neuraminidase, all cells from all variants and clones were brightly labeled by PNA, collecting in a single peak with similar fluorescence intensities. Electrophoresis of total cellular proteins and subsequent detection with labeled PNA on Western blots show two major PNA-reactive glycoproteins with apparent molecular weights of 140 and 110 kDa (MAGP1 and MAGP2), expressed only in highly metastatic cells, but which can be strongly labeled by PNA in slightly metastatic cells following a treatment with neuraminidase. These results provide evidence that the expression of terminal galactose (β1–3)N-acetyl galactosamine structures, positioned on MAGP1 and MAGP2 glycoproteins, is associated with the metastatic potential of human melanoma cells.     

The hypervariable region of Anaplasma marginale major surface protein 2 (MSP2) contains multiple immunodominant CD4+ T lymphocyte epitopes that elicit variant-specific proliferative and IFN-gamma responses in MSP2 vaccinates

Major surface protein 2 (MSP2) is an immunodominant outer membrane protein of Anaplasma marginale and Anaplasma phagocytophilum pathogens that cause bovine anaplasmosis and human granulocytic ehrlichiosis, respectively. MSP2 has a central hypervariable region (HVR) flanked by highly conserved amino and carboxyl termini. During A. marginale infection, dynamic and extensive amino acid sequence variation in MSP2 occurs through recombination of msp2 pseudogenes into the msp2 expression site, followed by sequential segmental gene conversions to generate additional variants. We hypothesized that MSP2 variation leads to significant changes in Th cell recognition of epitopes in the HVR. T cell epitopes were mapped using T cells from native MSP2-immunized cattle and overlapping peptides spanning the most abundant of five different MSP2 HVRs in the immunogen. Several epitopes elicited potent effector/memory Th cell proliferative and IFN-gamma responses, including those in three discreet blocks of sequence that undergo segmental gene conversion. Th cell clones specific for an epitope in the block 1 region of the predominant MSP2 variant type failed to respond to naturally occurring variants. However, some of these variants were recognized by oligoclonal T cell lines from MSP2 vaccinates, indicating that the variant sequences contain immunogenic CD4(+) T cell epitopes. In competition/antagonism assays, the nonstimulatory variants were not inhibitory for CD4(+) T cells specific for the agonist peptide. Dynamic amino acid sequence variation in MSP2 results in escape from recognition by some effector/memory MSP2-specific Th cells. Antigenic variation in MSP2 Th cell and B cell epitopes may contribute to immune evasion that allows long-term persistence of A. marginale in the mammalian reservoir.     

Oncolytic adenoviruses in anticancer therapy: Current status and prospects

Lytic virus infection results in production of a virus progeny and lysis of the infected cell. Tumor cells are usually more sensitive to virus infection. Studies indicate that viral oncolysis provides a promising alternative approach to cancer therapy. The ability of viruses to selectively kill cancer cells is long known, but construction of virus variants with an improved therapeutic potential was impossible until recent advances in virus and cell molecular biology and the development of modern methods for directed modification of viruses. Adenoviruses are one of the best studied models of oncolytic viruses. These DNA viruses are convenient for genetic manipulation and show minimal pathogenicity. The review summarizes the data on the directions and approaches to generation of highly efficient variants of oncolytic adenoviruses. The approaches include introduction of directed genetic modifications into the virus genome, accelerated selection of oncolytic virus variants following treatment with mutagens, the use of adenoviruses as vectors to introduce therapeutic gene products, optimization of viral delivery systems, minimization of the negative effects from the host immune system, etc. The dynamic development of studies in the field holds promise that many variants of oncolytic adenoviruses will find clinical application in the nearest future.     

Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication

It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.