Biomedical subjects

David C Jackson

Publications and source records attributed to David C Jackson.

26 records · Page 2Linked to original sources

PubMed2003

Maturation of dendritic cells with lipopeptides that represent vaccine candidates for hepatitis C virus.

The ability of antigens to elicit immune responses depends upon their initial recognition, uptake, processing and presentation by dendritic cells. This fact has been recognized by many workers and dendritic cells are now regarded as natural 'adjuvants' in the business of vaccine design. One way of persuading dendritic cells to become interested in foreign material is to decorate it with lipid moieties found in bacteria. This approach has been used in the context of synthetic peptide-based immunogens and depending on the nature of the epitopes included, can provide highly immunogenic structures capable of eliciting antibody or cytotoxic T cell responses. In this paper we describe the results of experiments in which the stimulatory effects of peptide-based vaccine candidates on human dendritic cells are examined. Our findings indicate that lipidated structures comprising vaccine target sequences of viral origin coupled to the synthetic lipid groups of bacteria are able to induce the maturation of dendritic cells, as measured by the expression of cell surface MHC class II molecules.

Cell Differentiation↗

PubMed2003

Disabling an integral CTL epitope allows suppression of autoimmune diabetes by intranasal proinsulin peptide.

Insulin is a major target of the autoimmune response associated with destruction of pancreatic beta cells in type 1 diabetes. A peptide that spans the junction of the insulin B chain and the connecting (C) peptide in proinsulin has been reported to stimulate T cells from humans at risk for type 1 diabetes and autoimmune diabetes-prone NOD mice. Here we show that proinsulin B24-C36 peptide binds to I-A(g7), the MHC class II molecule of the NOD mouse, and, after intranasal administration, induces regulatory CD4(+) T cells that, in the absence of CD8(+) T cells, block the adoptive transfer of diabetes. Curiously, however, intranasal B24-C36 did not inhibit development of spontaneous diabetes in treated mice. We then determined that B24-C36, and its core sequence B25-C34, bind to K(d), the NOD mouse MHC class I molecule, and elicit CD8(+) CTLs. When the CD8(+) T lymphocyte epitope was truncated at the C34 valine anchor residue for binding to K(d), the residual CD4(+) T cell epitope, B24-C32/33, significantly inhibited diabetes development after a single intranasal dose. This study identifies a novel CTL epitope in proinsulin and demonstrates that the therapeutic potential of a "tolerogenic" autoantigen peptide can be compromised by the presence of an integral CTL epitope.

Administration, Intranasal↗

PubMed2002

Highly immunogenic and totally synthetic lipopeptides as self-adjuvanting immunocontraceptive vaccines.

In this study, we describe the synthesis of various lipopeptides based on the sequence of luteinizing hormone-releasing hormone (LHRH) and report on their abilities to induce Abs against this "self" hormone when inoculated into mice in the absence of additional adjuvant. The peptides consisted of a colinear CD4(+) T helper cell epitope from the L chain of influenza virus hemagglutinin and LHRH, which has B cell epitopes but no T cell epitopes present in its sequence. Lipids were attached either at the N terminus or between the T cell epitope and LHRH, in the approximate center of the peptide. The lipopeptide constructs displayed different solubilities and immunological properties that depended not only on the lipid content but also on the position of attachment of the lipids. Some of these constructs were highly immunogenic, inducing high titers of Ab, which were capable of efficiently sterilizing female mice when administered in saline by s.c. or intranasal routes. The most effective vaccines were highly soluble, contained the dipalmitoyl-S-glyceryl cysteine moiety, and had this lipid attached at the center of the molecule. The relative ability of the lipopeptides to induce an Ab response in the absence of external adjuvant was reflected by their ability to up-regulate the surface expression of MHC class II molecules on immature dendritic cells. These results demonstrate that the composition and position within peptide vaccines of self-adjuvanting lipid groups can influence the ability to induce the maturation of dendritic cells and, in turn, the magnitude of the resulting Ab response.

Adjuvants, Immunologic↗

PubMed2002

Identification of antigenically active tryptic fragments of apical membrane antigen-1 (AMA1) of Plasmodium chabaudi malaria: strategies for assembly of immunologically active peptides.

Apical membrane antigen-1 (AMA1) is a prime vaccine candidate for inclusion in a vaccine against malaria. It is known that the disulphide bond stabilised conformation of this antigen is important for eliciting a protective antibody response, however little is known about the epitopes within this molecule that are targeted by the immune response. We have used a peptide approach for the identification and characterisation of such regions. In this study, the in vitro refolded, recombinant ectodomain of AMA1 from the D strain of Plasmodium chabaudi adami, was digested with trypsin and individual peptide fragments examined for antigenic activity. We found that a tryptic fragment, which was derived from a loop-like structure within the putative domain I of the intact AMA1 molecule, was highly reactive with antibodies from the sera of hyperimmune mice. Two different synthetic peptide constructs incorporating this antigenically active fragment were assembled. The first consisted of two separate peptide chains which were linked through a disulphide bond formed using chemo-selective chemistry. A larger 45-mer loop peptide, generated by the oxidation of two cysteine residues close to the N- and C-termini of the 45-mer, represented the complete loop structure and incorporated the tryptic fragment. Each peptide construct was also able to elicit production of high titres of antibodies in mice and furthermore, the 45-residue loop peptide elicited antibodies capable of binding to AMA1 with titres comparable to those present in a mouse which had recovered from multiple exposures to P. chabaudi adami parasites. Passive immunisation with anti-loop antibodies did not suppress the development of parasitaemia in mice challenged with P. chabaudi adami suggesting that although highly immunogenic, the peptides represented inadequate or inappropriate epitopes for vaccination purposes.

Amino Acid Sequence↗

PubMed2002

Chicken anemia virus VP2 is a novel dual specificity protein phosphatase.

The function of viral protein 2 (VP2) of the immunosuppressive circovirus chicken anemia virus (CAV) has not yet been established. We show that the CAV VP2 amino acid sequence has some similarity to a number of eukaryotic, receptor, protein-tyrosine phosphatase (PTPase) alpha proteins as well as to a cluster of human TT viruses within the Sanban group. To investigate if CAV VP2 functions as a PTPase, purified glutathione S-transferase (GST)-VP2 fusion protein was assayed for PTPase activity using the generalized peptide substrates ENDpYINASL and DADEpYLIPQQG (where pY represents phosphotyrosine), with free phosphate detected using the malachite green colorimetric assay. CAV GST-VP2 was shown to catalyze dephosphorylation of both substrates. CAV GST-VP2 PTPase activity for the ENDpYINASL substrate had a V(max) of 14,925 units/mg.min and a K(m) of 18.88 microm. Optimal activity was observed between pH 6 and 7, and activity was specifically inhibited by 0.01 mm orthovanadate. We also show that the ORF2 sequence of the CAV-related human virus TT-like minivirus (TLMV) possessed PTPase activity and steady state kinetics equivalent to CAV GST-VP2 when expressed as a GST fusion protein. To establish whether these viral proteins were dual specificity protein phosphatases, the CAV GST-VP2 and TLMV GST-ORF2 fusion proteins were also assayed for serine/threonine phosphatase (S/T PPase) activity using the generalized peptide substrate RRApTVA, with free phosphate detected using the malachite green colorimetric assay. Both CAV GST-VP2 and TLMV GST-ORF2 fusion proteins possessed S/T PPase activity, which was specifically inhibited by 50 mm sodium fluoride. CAV GST-VP2 exhibited S/T PPase activity with a V(max) of 28,600 units/mg.min and a K(m) of 76 microm. Mutagenesis of residue Cys(95) to serine in CAV GST-VP2 abrogated both PTPase and S/T PPase activity, identifying it as the catalytic cysteine within the proposed signature motif. These studies thus show that the circoviruses CAV and TLMV encode dual specificity protein phosphatases (DSP) with an unusual signature motif that may play a role in intracellular signaling during viral replication. This is the first DSP gene to be identified in a small viral genome.

Amino Acid Motifs↗

PubMed2002

Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy.

The prevalence of hepatitis B virus vaccine escape mutants has increased as a consequence of the introduction of global vaccination programs. Furthermore and as a consequence of the organization of the genome of hepatitis B virus (HBV) into overlapping reading frames, the selection of polymerase mutants during long-term lamivudine therapy can select viruses with changes in the overlapping S gene coding for the hepatitis B small antigen (HBsAg). We have investigated the role of lamivudine in selecting HBV mutants with antigenically altered HBsAg protein using pooled human vaccine sera in enzyme immunosorbent assays and radioimmunoassays. HBsAg proteins containing the vaccine escape mutations G145R and D144E/G145R demonstrated markedly reduced binding to anti-HBs antibody. HBsAg mutants including E164D, W196S, I195M, M198I, and E164D/I195M (corresponding to the polymerase protein changes of V519L, M550I, L526M/M550V V553I, and V519L/L526M/M550V) selected during lamivudine treatment also demonstrated reduced binding to anti-HBs antibody. These findings raise the possibility of lamivudine-resistant mutants arising that possess antigenically distinct HBsAg proteins.

DNA-Directed DNA Polymerase↗

PubMed2002

Induction of long-term memory CD8(+) T cells for recall of viral clearing responses against influenza virus.

Induction of cytotoxic T-cell-mediated virus-clearing responses by influenza virus T cell determinant-containing peptide immunogens was examined. The most potent synthetic immunogens for eliciting pulmonary viral-clearing responses contained peptides representing determinants for CD4 and CD8 T cells (TH and CTL peptides, respectively) together with two or four palmitic acid (Pal) groups. Inoculated in adjuvant, these Pal2- or Pal4-CTL-TH lipopeptides and the nonlipidated CTL peptide induced equivalent levels of cytolytic activity in the primary effector phase of the response. The ability to recall lytic responses, however, diminished much more rapidly in CTL peptide-primed than in lipopeptide-primed mice. By 15 months postpriming, the recalled lytic activity in lipopeptide-inoculated mice remained potent, but the response induced by the CTL peptide was weak. Enumeration of specific gamma interferon-secreting CD8 T cells revealed that a greater number of these T cells had entered or remained in the memory pool in lipopeptide-primed mice, arguing for a quantitative rather than qualitative enhancement of the response on recall. Addition of either the lipid or the TH peptide to the CTL peptide was not sufficient to provide these long-lived antiviral responses, but inclusion of both components augmented the response. CD4 T cells elicited by the lipopeptides did not influence the rate of viral clearance upon challenge and most likely had a role in induction or maintenance of the memory response. It therefore appears that the lipopeptide immunogens, although not significantly superior at inducing primary effector CD8 T cells, elicit a much more effective memory population, the recall of which may account for their superiority in inducing pulmonary protection after viral challenge.

Animals↗

PubMedDate not supplied

An insect antibacterial peptide-based drug delivery system.

The ability of the short, proline-rich native antibacterial peptides to penetrate bacterial and host cells suggests the utility of these transport systems in delivering peptidic cargo into cells. We studied the uptake of pyrrhocoricin and its most potent dimeric analogue by bacteria as well as human dendritic cells and fibroblasts. Native pyrrhocoricin entered the susceptible organism Escherichia coli very efficiently and the nonsusceptible bacterium Staphylococcus aureus to a significant degree. The antibacterial peptide also penetrated human monocyte-derived dendritic cells. It failed, however, to enter fibroblasts, whereas the designer analogue Pip-pyrr-MeArg dimer penetrated all the cell types that were studied. When glucoincretin hormone Glp-1 fragment 7-36 was cosynthesized with the dimer, the antibacterial peptide derivative lost its ability to cross the bacterial membrane layer. In contrast, a chimera of the Pip-pyrr-MeArg dimer and two copies of a shorter (nine residues) class I major histocompatibility complex epitope successfully entered bacterial and mammalian cells. While the Pip-pyrr-MeArg dimer was not immunogenic when inoculated into mice, the chimera elicited a strong cytotoxic T-cell response, indicating the maintenance of the antigenic integrity of the cargo in the peptide conjugate. The chimera when tested for its immunological properties activated human dendritic cells significantly more strongly than any of the two independent fragments alone, yet lacked mammalian cell toxicity. These results confirm the utility of designed pyrrhocoricin analogues for delivery of peptidic cargo across cell membranes in general, and their potential as carriers for epitope-based vaccines in particular.

Amino Acid Sequence↗