Conjugation of a peptide autoantigen to gold nanoparticles for intradermally administered antigen specific immunotherapy.

Antigen specific immunotherapy aims to tolerise patients to specific autoantigens that are responsible for the pathology of an autoimmune disease. Immune tolerance is generated in conditions where the immune response is suppressed and thus gold nanoparticles (AuNPs) are an attractive drug delivery platform due to their anti-inflammatory effects and their potential to facilitate temporal and spatial delivery of a peptide autoantigen in conjunction with pro-tolerogenic elements. In this study we have covalently attached an autoantigen, currently under clinical evaluation for the treatment of type 1 diabetes (PIC19-A3 peptide), to AuNPs to create nanoscale (<5 nm), negatively charged (-40 to -60 mV) AuNP-peptide complexes for immunotherapy. We also employ a clinically approved microneedle delivery system, MicronJet600, to facilitate minimally-invasive intradermal delivery of the nanoparticle constructs to target skin-resident antigen presenting cells, which are known to be apposite target cells for immunotherapy. The AuNP-peptide complexes remain physically stable upon extrusion through microneedles and when delivered into ex vivo human skin they are able to diffuse rapidly and widely throughout the dermis (their site of deposition) and, perhaps more surprisingly, the overlying epidermal layer. Intracellular uptake was extensive, with Langerhans cells proving to be the most efficient cells at internalising the AuNP-peptide complex (94% of the local population within the treated region of skin). In vitro studies showed that uptake of the AuNP-peptide complexes by dendritic cells reduced the capacity of these cells to activate naïve T cells. This indicator of biological functionality encourages further development of the AuNP-peptide formulation, which is now being evaluated in clinical trials.

Transcript imaging of the development of human T helper cells using oligonucleotide arrays.

Many pathological processes, including those causing allergies and autoimmune diseases, are associated with the presence of specialized subsets of T helper cells at the site of inflammation. Understanding the genetic program that controls the functional properties of T helper type 1 (Th1) versus T helper type 2 (Th2) cells may provide insight into the pathophysiology of inflammatory diseases. We compared the gene-expression profiles of human Th1 and Th2 cells using high-density oligonucleotide arrays with the capacity to display transcript levels of 6,000 human genes. Here we analyse the data sets derived from five independent experiments using statistical algorithms. This approach resulted in the identification of 215 differentially expressed genes, encoding proteins involved in transcriptional regulation, apoptosis, proteolysis, and cell adhesion and migration. A subset of these genes was further upregulated by exposure of differentiated Th1 cells to interleukin-12 (IL-12), as confirmed by kinetic PCR analysis, indicating that IL-12 modulates the effector functions of Th1 cells in the absence of antigenic stimulation. Functional assays and in vivo expression of selected genes have validated the biological relevance of our study. Our results provide new insight into the transcriptional program controlling the functional diversity of subsets of T helper cells.

Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays.

We have used high-density oligonucleotide probe arrays (chips) for bacterial transcript imaging. We designed a chip containing probes representing 106 Hemophilus influenzae genes and 100 Streptococcus pneumoniae genes. The apparent lack of polyadenylated transcripts excludes enrichment of mRNA by affinity purification and we thus used total, chemically biotinylated RNA as hybridization probe. We show that hybridization of Streptococcus RNA to a chip allows simultaneous quantification of the transcript levels. The sensitivity was found to be in the range of one to five transcripts per cell. The quantitative chip results were in good agreement with conventional Northern blot analysis of selected genes. This technology allows simultaneous and quantitative measurement of the transcriptional activity of entire bacterial genomes on a single oligonucleotide probe array.

Presenilins are processed by caspase-type proteases.

Presenilin 1 (PS1) and presenilin 2 (PS2) are endoproteolytically processed in vivo and in cell transfectants to yield 27-35-kDa N-terminal and 15-24-kDa C-terminal fragments. We have studied the cleavage of PS1 and PS2 in transiently and stably transfected hamster kidney and mouse and human neuroblastoma cells by immunoblot and pulse-chase experiments. C-terminal fragments were isolated by affinity chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. The processing sites identified in PS1 and PS2 (Asp345/Ser346 and Asp329/Ser330, respectively) are typical for caspase-type proteases. Specific caspase inhibitors and cleavage site mutations confirmed the involvement of caspase(s) in PS1 and PS2 processing in cell transfectants. Fluorescent peptide substrates carrying the PS-identified cleavage sites were hydrolyzed by proteolytic activity from mouse brain. The PS2-derived peptide substrate was also cleaved by recombinant human caspase-3. Additional processing of PS2 by non-caspase-type proteases was also observed.

Emergence of resistant variants of HIV in vivo during monotherapy with the proteinase inhibitor saquinavir.

We examined the phenotypic and genotypic properties of virus from the peripheral blood mononuclear cells (PBMC) and plasma of eight HIV-1-infected asymptomatic patients before and during monotherapy with the proteinase inhibitor saquinavir. Susceptibility of primary isolates to drug was assessed in PBMC culture by deriving IC50 and IC90 values. The observed increases in IC50 and IC90 after approximately one year of therapy with a dosage of 600 mg tds suggests the presence of virus resistant to saquinavir in vivo. The magnitude of this altered susceptibility ranged from three-fold to in one case 100-fold. In two patients a greater than eight-fold decrease in susceptibility to saquinavir was observed. Sequencing of the proteinase genes in viral RNA obtained from patient plasma and/or PBMC was carried out by PCR in parallel with sensitivity testing. In each case between nine and 12 clones were analysed. In the two patients from whom virus had greater than eight-fold reduction in susceptibility, a point mutation was observed in the viral proteinase (Leu90--> Met/Ile). Further mutations were observed at residues 36, 71 and 84 in these subjects. In a third patient, in whom an eight-fold increase in HIV IC50 of saquinavir was observed, no mutations were detected in the proteinase; sequencing of proteinase cleavage sites in viral gag-pol revealed no significant mutations. In no patient was a Gly48-->Val mutation observed, although this has been associated with resistance in vitro. The Leu90-->Met mutation was observed in five subjects, but a greater than eight-fold phenotypic change in antiviral susceptibility was seen in only two of these. Hence, in vivo, the Leu90-->Met but not the Gly48-->Val mutation is necessary, but not sufficient, for phenotypic resistance to saquinavir in HIV.

Evaluation of hepatitis C virus envelope proteins expressed in E. coli and insect cells for use as tools for antibody screening.

BACKGROUND/METHODS: The two envelope proteins of hepatitis C virus, E1 and E2, were expressed in E. coli and, as secretory proteins, in Sf9 insect cells using recombinant baculoviruses. Co-infection of insect cells with E1 and E2-recombinant baculoviruses was performed, which has been shown to result in formation of E1-E2 dimers. All envelope proteins were purified by Ni2+-NTA chromatography and used for screening of serum samples in a HCV EIA assay. Serum samples of normal blood donors, chronically HCV-infected patients, a mixed titer panel and several seroconversion panels were screened and compared to test results with Cobas Core Anti-HCV EIA. RESULTS: Screening of the sera of chronically HCV-infected patients (100% positive in Cobas Core Anti-HCV EIA) revealed 10-40% anti-E1 positive sera using different Sf9-expressed, glycosylated proteins and 93% using E. coli-expressed, non-glycosylated E1 protein. When the same sera were tested with different E2 proteins expressed in Sf9 cells and in E. coli, about 70-73% showed anti-E2 reactivity. When the proteins from Sf9 cells co-infected with E1- and E2-recombinant baculoviruses were tested, 70-80% of the same sera showed anti-envelope reactivity. CONCLUSIONS: Testing of these patient antisera, and those from the well-characterized mixed titer panel BBI-PHV203, showed that recombinant E1 expressed in E. coli and co-expressed E1 and E2 proteins from Sf9 cells could be used as additional tools for anti-HCV antibody screening.

Tetracycline-responsive gene expression in mouse brain after amplicon-mediated gene transfer.

Amplicon vectors incorporate genetic elements from Herpes simplex virus (HSV) in a plasmid form which is packaged into virions in the presence of a replication-defective helper virus. We constructed a new amplicon vector, pHermes-tet-lacZ, that carries the bacterial beta-galactosidase (lacZ) gene under the control of a minimal promoter preceded by a heptameric tetracycline operator. The minimal promoter element is activated by a tetracycline-responsive hybrid protein, the gene for which is also present in the vector. This amplicon was propagated in parallel in two different permissive cell lines, E5 and 2-2, in the presence of two helper viruses, d120 and 5dl1.2, respectively. The viral stocks produced were injected into the hippocampal region of the mouse brain, where strong localized expression of the transgene developed in the granular cell layer of the dentate gyrus with limited cytotoxicity. The transgene expression could be repressed by a factor of 10 after administration of tetracyclines. The repression level depended on the helper virus present in the injected viral stock. The in vivo regulation of transgene expression conferred by the tetracycline responsive element improves the flexibility of amplicon vectors as tools for gene transfer into the brain.

Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells.

The putative envelope glycoproteins of hepatitis C virus (HCV), E1 and E2, were expressed as recombinant, secretory proteins in Sf9 insect cells through infection with recombinant baculoviruses. The influenza virus hemagglutinin signal sequence (HASS) was inserted upstream of the HCV-cDNAs in order to effect secretion. Furthermore, a hexa-histidine tag for purification on a Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) column and a protein kinase A (PKA) recognition sequence for in vitro-phospholabeling were fused upstream of the HCV-cDNA. E1- and E2 proteins lacking their carboxy-terminal, hydrophobic sequence were produced by baculovirus-infected insect cells in bioreactors of 23 1. The medium was concentrated and proteins were purified under native conditions on Ni(2+)-NTA columns. Purified proteins could be phospholabeled in vitro using the catalytic subunit of protein kinase. A isolated from bovine heart and gamma-[32P]ATP. Labeled E1 and E2 proteins expressed in insect cells could be immunoprecipitated with sera from HCV-infected patients. Co-expression of these E1 and E2 proteins led to the formation of E1-E2 complexes within the insect cell and to secretion of these complexes into the medium.

Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase.

The expression and processing of hepatitis C virus core protein was analyzed. Two protein bands, 21 kDa (P21), corresponding to the full-length core, and 19 kDa (P19), were detected as major products when core protein was expressed in the standard rabbit reticulocyte lysate system or in Sf9 insect cells. Core proteins with amino-terminal hexa-histidine tags were expressed which allowed the purification of the hexa-histidine P19 core with NI(2+)-NTA columns. With the help of mass spectrometry, the molecular weight of hexa-histidine-P19 was analyzed and its carboxy-terminus could be calculated. Fusion proteins of truncated core/core-E1 species fused to mouse dihydrofolate reductase (mDHFR) showed cleavage in the expected region. Cleavage sites could be determined by amino-terminal protein sequencing of the DHFR-fusion partner. Our data show that there are not one but two core products with an apparent molecular weight of about 19 kDa, ending either at amino acid leucine 179 or leucine 182, respectively. These cleavages in the hydrophobic, carboxy-terminal region of HCV core suggest processing by (a) recently proposed eucaryotic signal peptide peptidase(s) (F. Lyko et al. (1995) J. Biol. Chem. 270, 19873-19878). Furthermore, our results demonstrate that cleavage at these sites and the formation of the P19 species does not require previous processing at the signalase site (position 191/192) of the HCV-polyprotein.

In vivo resistance to a human immunodeficiency virus type 1 proteinase inhibitor: mutations, kinetics, and frequencies.

Resistance to saquinavir (Ro 31-8959), an inhibitor of human immunodeficiency virus type I proteinase, was studied in peripheral blood mononuclear cell-derived proviral DNA from patients undergoing prolonged treatment. A Leu90-->Met exchange was the predominant resistance mutation in vivo; Gly48-->Val or doubly mutant virus was rarely observed. After 8-12 months of treatment with saquinavir alone (600 mg, 3 times/day) or in combination with zidovudine (200 mg, 3 times/day), approximately 45% of all patients carried provirus with mutant proteinase; the incidence was lower (22%) in patients treated with a combination of saquinavir, zidovudine, and dideoxycytidine. There was a good relationship between genotypic analysis of saquinavir resistance and data from virus assays, confirming that Leu90-->Met and Gly48-->Val are the essential exchanges in the proteinase that determine loss of sensitivity to this inhibitor. Absence of genotypic resistance correlated with a sustained decrease in plasma viral RNA. There was a positive correlation between a Met90 mutation and some residues at natural polymorphic sites (positions 10, 36, 63, and 71).

Tumor necrosis factor receptor p55 mediates induction of HIV type 1 expression in chronically infected U1 cells.

Tumor necrosis factor alpha (TNF-alpha) is a potent inducer of human immunodeficiency virus type 1 (HIV-1) expression in chronically infected cells. The aim of this study was to investigate the role played by the two known TNF-alpha receptors, TNFR-p55 and TNFR-p75, in the activation of HIV-1 expression. As a model system the latently infected human promonocytic cell line U1 was stimulated with wild-type TNF-alpha, with TNF-alpha muteins that specifically bind to one or the other receptor or with receptor-specific monoclonal antibodies. Induction of HIV-1 expression, measured by p24 core antigen capture enzyme-linked immunosorbent assay (ELISA), was found to be exclusively triggered by TNFR-p55 stimulation. However, our results also showed that the addition of TNFR-p75-specific ligands negatively modulated the HIV-1 expression induced via TNFR-p55.

Reduced sensitivity to saquinavir: an update on genotyping from phase I/II trials.

A genotypic analysis of the HIV-1 proteinase was performed on clinical specimen obtained from patients after different periods of Saquinavir (SQV) treatment. Proteinase genes of integrated proviral DNA from PBMC were isolated by PCR, cloned and individual sequences were obtained. Genotypic resistance was defined by the Gly48-->Val and Leu90-->Met exchanges. Frequencies and kinetics of resistance development will be reported for phase I/II trials V13330. V13329, O13328 and ACTG229 in patients on monotherapy or combination therapy with RT inhibitors. Data from V13330 have been analysed in more detail for correspondence of genotypic and phenotypic resistance and any correlation between resistance and changes in plasma viral RNA load. Furthermore, we will discuss the data from our extensive proteinase gene sequence collection with respect to mutational changes which would be indicative of resistance to other inhibitors of HIV-1 proteinase.

An in vitro assay for hepatitis C virus NS3 serine proteinase.

Hepatitis C virus (HCV) encodes a polyprotein of which the majority of nonstructural proteins are matured by the viral serine proteinase located in the N terminus of NS3. Intracellular studies using recombinant vaccinia virus have shown that both NS3 and its cofactor NS4A are required to enhance processing at the NS3-dependent cleavage sites. We developed an in vitro (cell-free) assay in which the HCV serine proteinase was shown to be enzymatically active, by mixing lysates of cells expressing either the serine proteinase or a nonstructural protein substrate. NS3 cleaved in a highly reproducible manner at the NS5A/5B site in the presence of NS4A, whereas NS3 alone was enzymatically inactive. NS4A could be provided either linked to NS3 or as part of the substrate. In contrast, irrespective of the presence or absence of NS4A, no NS3-mediated processing was observed at the NS3/4A, NS4A/4B, and NS4B/5A sites in this assay. In vitro cleavage at the NS5A/5B site occurred rapidly, within 1 min at temperatures ranging from 0 to 20 degrees, but was incomplete and required detergent-solubilized lysates. General serine proteinase inhibitors did not decrease processing activity. The in vitro model described in this study is a new tool: (1) to study the structure and the function of HCV serine proteinase and NS5A/5B cleavage site, and (2) to test NS3 serine proteinase inhibitors.

Substrate determinants for cleavage in cis and in trans by the hepatitis C virus NS3 proteinase.

Processing of the hepatitis C virus polyprotein is accomplished by a series of cotranslational and posttranslational cleavages mediated by host cell signalases and two virally encoded proteinases. Of these the NS3 proteinase is essential for processing at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions. Processing between NS3 and NS4A occurs in cis, implying an intramolecular reaction mechanism, whereas cleavage at the other sites can also be mediated in trans. Sequence analysis of the amino termini of mature cleavage products and comparisons of amino acid residues around the scissile bonds of various hepatitis C virus isolates identified amino acid residues which might contribute to substrate specificity and processing efficiency: an acidic amino acid at the P6 position, a Thr or Cys at the P1 position, and a Ser or Ala at the P1' position. To study the importance of these residues for NS3-mediated cleavage we have undertaken a mutational analysis using an NS3'-5B polyprotein expressed by recombinant vaccinia viruses in mammalian cells. For all NS3-dependent cleavage sites P1 substitutions had the most drastic effects on cleavage efficiency, showing that amino acid residues at this position are the most critical substrate determinants. Since less drastic effects were found for substitutions at the P1' position, these residues appear to be less important for proper cleavage. For all cleavage sites the P6 acidic residue was dispensable, suggesting that it is not essential for substrate recognition and subsequent cleavage. Analysis of a series of mutations at the NS3/4A site revealed great flexibility for substitutions compared with more stringent requirements at the trans cleavage sites. On the basis of these results we propose a model in which processing in cis is determined primarily by polyprotein folding, whereas cleavage in trans is governed not only by the structure of the polyprotein but also by specific interactions between the proteinase and the polyprotein substrate at or around the scissile bond.

Characterization of human immunodeficiency virus type 1 mutants with decreased sensitivity to proteinase inhibitor Ro 31-8959.

A human immunodeficiency virus type 1 (HIV-1) variant with highly reduced susceptibility to Ro 31-8959, an inhibitor of the viral proteinase, has been selected by repeated passage of wild-type virus in CEM cells in the presence of increasing concentrations of the inhibitor. Peptide sequences of the proteinase of selected virus were obtained from proviral DNA. Sequence comparison to wild-type (wt) proteinase demonstrated two amino acid substitutions in the resistant virus, a Gly to Val exchange at position 48 and a Leu to Met exchange at position 90. Furthermore, sequences of intermediate passage virus suggest contributions from positions 12, 36, 57, and 63 in early steps of resistance development. The selected virus showed a ca. 40-fold increase in 50% inhibitory concentration of Ro 31-8959. Growth kinetics of resistant virus were comparable to wild-type virus and the resistant genotype proved to be stable in the absence of inhibitor. Directed mutagenesis of the HIV-1 HXB2 proteinase at positions 48 and 90 suggested that each mutation alone led to a moderate decrease in sensitivity of the recombinant virus to proteinase inhibitor. However, a recombinant virus carrying both mutations in the proteinase gene showed a significant reduction in its sensitivity to Ro 31-8959 thus proving the importance of these exchanges for the resistance phenotype.

Identification of an amino acid substitution involved in the reduction of sensitivity of HIV-1 to an inhibitor of viral proteinase.

Clones derived from HIV variants previously characterized as resistant to Ro31-8959, an inhibitor of viral proteinase (PR), were sequenced. Substitution of glycine by valine at position 48 of the PR protein was found. None of the 20 clones derived from wild type HTLV-IIIB contain this mutation. Since such a position is located in a conserved region of PR, it is possible that the substitution can affect the interaction of the enzyme with the inhibitor.

Kinetic and structural analyses of hepatitis C virus polyprotein processing.

Recombinant vaccinia viruses were used to study the processing of hepatitis C virus (HCV) nonstructural polyprotein precursor. HCV-specific proteins and cleavage products were identified by size and by immunoprecipitation with region-specific antisera. A polyprotein beginning with 20 amino acids derived from the carboxy terminus of NS2 and ending with the NS5B stop codon (amino acids 1007 to 3011) was cleaved at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B sites, whereas a polyprotein in which the putative active site serine residue was replaced by an alanine remained unprocessed, demonstrating that the NS3-encoded serine-type proteinase is essential for cleavage at these sites. Processing of the NS3'-5B polyprotein was complex and occurred rapidly. Discrete polypeptide species corresponding to various processing intermediates were detected. With the exception of NS4AB-5A/NS5A, no clear precursor-product relationships were detected. Using double infection of cells with vaccinia virus recombinants expressing either a proteolytically inactive NS3'-5B polyprotein or an active NS3 proteinase, we found that cleavage at the NS4A/4B, NS4B/5A, and NS5A/5B sites could be mediated in trans. Absence of trans cleavage at the NS3/4A junction together with the finding that processing at this site was insensitive to dilution of the enzyme suggested that cleavage at this site is an intramolecular reaction. The trans-cleavage assay was also used to show that (i) the first 211 amino acids of NS3 were sufficient for processing at all trans sites and (ii) small deletions from the amino terminus of NS3 selectively affected cleavage at the NS4B/5A site, whereas more extensive deletions also decreased processing efficiencies at the other sites. Using a series of amino-terminally truncated substrate polyproteins in the trans-cleavage assay, we found that NS4A is essential for cleavage at the NS4B/5A site and that processing at this site could be restored by NS4A provided in cis (i.e., together with the substrate) or in trans (i.e., together with the proteinase). These results suggest that in addition to the NS3 proteinase, NS4A sequences play an important role in HCV polyprotein processing.

Severe immunodeficiency associated with a human immunodeficiency virus 1 NEF/3'-long terminal repeat transgene.

We have generated several transgenic mouse strains carrying a human immunodeficiency virus 1 (HIV-1) NEF/3' long terminal repeat (LTR) transgene under control of a T cell-specific promoter-enhancer element, showing a depletion of CD4+ T cells in the thymus and periphery. The immunological functions of the line with the most dramatic changes in lymphocyte populations, B6/338L, were analyzed in greater detail. The presence of the transgene in the heterozygous animal is associated with a dominant severe immunodeficiency. Older animals develop lymph-adenopathy and splenomegaly. CD4+CD8+ and CD4+CD8- single positive thymocytes already are depleted in these mice at the earliest stages in ontogeny, and peripheral T cells are reduced in frequency and present cell surface marker expression, which is characteristic for memory and activated T cells. The immunological response of B6/338L mice to several viral infections is also greatly impaired. Thus, the HIV-1 NEF/3' LTR as transgene in T cells can cause immunodeficiency and disease with striking similarities to a known retrovirus-induced immunodeficiency called murine AIDS (H. C. Morse III, S. K. Chattopadhyay, M. Makino, T. N. Frederickson, A. W. Hügin, and J. W. Hartley. 1992. AIDS. 6:607).

Conserved residues Pro-109 and Asp-116 are required for interaction of the human immunodeficiency virus type 1 integrase protein with its viral DNA substrate.

The human immunodeficiency virus type 1 integrase protein can be specifically cross-linked to viral long terminal repeat substrate oligonucleotides in vitro by using UV light. Site-directed mutagenesis and deletion analyses were used to define the domains involved in the interaction of integrase with the viral DNA substrate. Our results showed that mutation of conserved residues Pro-109 and Asp-116, which are found to be critical for the endonuclease and integration activities of IN protein, abolished the ability of the protein to cross-link to its DNA substrate. Furthermore, deletion analysis experiments showed that removal of 39 amino acids from the amino terminus and deletion of 15 amino acids from the carboxyl terminus abolished DNA cross-linking.